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1
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Dalin S, Webster S, Sugawara N, Wu Q, Zhang S, Macias C, Sapède E, Cui T, Liang V, Tran L, Beroukhim R, Haber JE. Mutations and structural variants arising during double-strand break repair. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2025:2025.02.28.640809. [PMID: 40093125 PMCID: PMC11908181 DOI: 10.1101/2025.02.28.640809] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 03/19/2025]
Abstract
Double-strand break (DSB) repair is highly mutagenic compared to normal replication. In budding yeast, repair of an HO endonuclease-induced DSB at MATα can be repaired by using a transcriptionally silent HMR::Kl-URA3 donor. During repair, -1 deletions in homonucleotide runs are strongly favored over +1 insertions, whereas during replication, spontaneous +1 and -1 events are equal. Microhomology-bounded, repair-associated intragenic deletions (IDs) are recovered 12 times more frequently than tandem duplications (TDs). IDs have a mean length of 56 bp, while TDs average 22 bp. These data suggest a picture of the structure of the repair replication fork: IDs and TDs occur within the open structure of a migrating D-loop, where the 3' end of a partly copied new DNA strand can dissociate and anneal with a single-stranded region of microhomology that lies either ~80 bp ahead or ~40 bp behind the 3' end. Another major class of repair-associated mutations (~10%) are interchromosomal template switches (ICTS), even though the K. lactis URA3 sequence in HMR is only 72% identical (homeologous) with S. cerevisiae ura3-52. ICTS events begin and end at regions of short (~7 bp) microhomology; however, ICTS events are constrained to the middle of the copied sequence. Whereas microhomology usage in intragenic deletions is not influenced by adjacent homeology, we show that extensive pairing of adjacent homeology plays a critical role in ICTS. Thus, although by convention, structural variants are characterized by the precise base pairs at their junction, microhomology-mediated template switching actually requires alignment of extensive adjacent homeology.
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Affiliation(s)
- Simona Dalin
- Cancer Program, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
- Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA
| | - Sophie Webster
- Cancer Program, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
- Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA
| | - Neal Sugawara
- Rosenstiel Basic Medical Sciences Research Center and Department of Biology, Brandeis University, Waltham, Massachusetts 02454
| | - Qiuqin Wu
- Rosenstiel Basic Medical Sciences Research Center and Department of Biology, Brandeis University, Waltham, Massachusetts 02454
| | - Shu Zhang
- Cancer Program, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
- Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA
| | - Carmen Macias
- Rosenstiel Basic Medical Sciences Research Center and Department of Biology, Brandeis University, Waltham, Massachusetts 02454
| | - Elena Sapède
- Rosenstiel Basic Medical Sciences Research Center and Department of Biology, Brandeis University, Waltham, Massachusetts 02454
| | - Tracy Cui
- Cancer Program, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
- Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA
- Rosenstiel Basic Medical Sciences Research Center and Department of Biology, Brandeis University, Waltham, Massachusetts 02454
| | - Victoria Liang
- Rosenstiel Basic Medical Sciences Research Center and Department of Biology, Brandeis University, Waltham, Massachusetts 02454
| | - Laura Tran
- Rosenstiel Basic Medical Sciences Research Center and Department of Biology, Brandeis University, Waltham, Massachusetts 02454
| | - Rameen Beroukhim
- Cancer Program, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
- Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA
- Harvard Medical School, Boston, Massachusetts 02115, USA
| | - James E. Haber
- Rosenstiel Basic Medical Sciences Research Center and Department of Biology, Brandeis University, Waltham, Massachusetts 02454
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2
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Yu Y, Wang X, Fox J, Li Q, Yu Y, Hastings PJ, Chen K, Ira G. RPA and Rad27 limit templated and inverted insertions at DNA breaks. Nucleic Acids Res 2025; 53:gkae1159. [PMID: 39673516 PMCID: PMC11724301 DOI: 10.1093/nar/gkae1159] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/06/2024] [Revised: 10/26/2024] [Accepted: 11/06/2024] [Indexed: 12/16/2024] Open
Abstract
Formation of templated insertions at DNA double-strand breaks (DSBs) is very common in cancer cells. The mechanisms and enzymes regulating these events are largely unknown. Here, we investigated templated insertions in yeast at DSBs using amplicon sequencing across a repaired locus. We document very short (most ∼5-34 bp), templated inverted duplications at DSBs. They are generated through a foldback mechanism that utilizes microhomologies adjacent to the DSB. Enzymatic requirements suggest a hybrid mechanism wherein one end requires Polδ-mediated synthesis while the other end is captured by nonhomologous end joining (NHEJ) or by alternative end joining (Alt-EJ). This process is exacerbated in mutants with low levels or mutated RPA (rtt105Δ; rfa1-t33) or extensive resection deficiency (sgs1Δ exo1Δ). Templated insertions from various distant genomic locations also increase in RPA mutants as well as in rad27Δ and originate from fragile regions of the genome. Among complex insertions, common events are insertions of two sequences, originating from the same locus and with inverted orientation. We propose that these inversions are also formed by microhomology-mediated template switching. Together, we propose that a shortage of RPA, typical in cancer cells, may be a factor that stimulates the formation of templated insertions.
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Affiliation(s)
- Yang Yu
- Baylor College of Medicine, Department of Molecular and Human Genetics, One Baylor Plaza, Houston, TX 77030, USA
| | - Xin Wang
- Department of Cardiology, Boston Children's Hospital, 300 Longwood Avenue, Boston, MA 02115, USA
- Department of Pediatrics, Harvard Medical School, 25 Shattuck Street, Boston, MA 02115, USA
- Kidney and Urinary Tract Center, The Abigail Wexner Research Institute at Nationwide Children's hospital, Columbus, OH 43215, USA
| | - Jordan Fox
- Baylor College of Medicine, Department of Molecular and Human Genetics, One Baylor Plaza, Houston, TX 77030, USA
| | - Qian Li
- Baylor College of Medicine, Department of Molecular and Human Genetics, One Baylor Plaza, Houston, TX 77030, USA
| | - Yang Yu
- Department of Cardiology, Boston Children's Hospital, 300 Longwood Avenue, Boston, MA 02115, USA
- Department of Pediatrics, Harvard Medical School, 25 Shattuck Street, Boston, MA 02115, USA
| | - P J Hastings
- Baylor College of Medicine, Department of Molecular and Human Genetics, One Baylor Plaza, Houston, TX 77030, USA
| | - Kaifu Chen
- Department of Cardiology, Boston Children's Hospital, 300 Longwood Avenue, Boston, MA 02115, USA
- Department of Pediatrics, Harvard Medical School, 25 Shattuck Street, Boston, MA 02115, USA
| | - Grzegorz Ira
- Baylor College of Medicine, Department of Molecular and Human Genetics, One Baylor Plaza, Houston, TX 77030, USA
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3
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Sugawara N, Towne MJ, Lovett ST, Haber JE. Spontaneous and double-strand break repair-associated quasipalindrome and frameshift mutagenesis in budding yeast: role of mismatch repair. Genetics 2024; 227:iyae068. [PMID: 38691577 DOI: 10.1093/genetics/iyae068] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2023] [Revised: 08/09/2023] [Accepted: 03/06/2024] [Indexed: 05/03/2024] Open
Abstract
Although gene conversion (GC) in Saccharomyces cerevisiae is the most error-free way to repair double-strand breaks (DSBs), the mutation rate during homologous recombination is 1,000 times greater than during replication. Many mutations involve dissociating a partially copied strand from its repair template and re-aligning with the same or another template, leading to -1 frameshifts in homonucleotide runs, quasipalindrome (QP)-associated mutations and microhomology-mediated interchromosomal template switches. We studied GC induced by HO endonuclease cleavage at MATα, repaired by an HMR::KI-URA3 donor. We inserted into HMR::KI-URA3 an 18-bp inverted repeat where one arm had a 4-bp insertion. Most GCs yield MAT::KI-ura3::QP + 4 (Ura-) outcomes, but template-switching produces Ura+ colonies, losing the 4-bp insertion. If the QP arm without the insertion is first encountered by repair DNA polymerase and is then (mis)used as a template, the palindrome is perfected. When the QP + 4 arm is encountered first, Ura+ derivatives only occur after second-end capture and second-strand synthesis. QP + 4 mutations are suppressed by mismatch repair (MMR) proteins Msh2, Msh3, and Mlh1, but not Msh6. Deleting Rdh54 significantly reduces QP mutations only when events creating Ura+ occur in the context of a D-loop but not during second-strand synthesis. A similar bias is found with a proofreading-defective DNA polymerase mutation (poI3-01). DSB-induced mutations differed in several genetic requirements from spontaneous events. We also created a + 1 frameshift in the donor, expanding a run of 4 Cs to 5 Cs. Again, Ura+ recombinants markedly increased by disabling MMR, suggesting that MMR acts during GC but favors the unbroken, template strand.
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Affiliation(s)
- Neal Sugawara
- Department of Biology and Rosenstiel Basic Medical Sciences Research Center MS029, Brandeis University, Waltham, MA 02454-9110, USA
| | - Mason J Towne
- Department of Biology and Rosenstiel Basic Medical Sciences Research Center MS029, Brandeis University, Waltham, MA 02454-9110, USA
| | - Susan T Lovett
- Department of Biology and Rosenstiel Basic Medical Sciences Research Center MS029, Brandeis University, Waltham, MA 02454-9110, USA
| | - James E Haber
- Department of Biology and Rosenstiel Basic Medical Sciences Research Center MS029, Brandeis University, Waltham, MA 02454-9110, USA
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4
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Yu Y, Wang X, Fox J, Li Q, Yu Y, Hastings PJ, Chen K, Ira G. RPA and Rad27 limit templated and inverted insertions at DNA breaks. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.03.07.583931. [PMID: 38496432 PMCID: PMC10942419 DOI: 10.1101/2024.03.07.583931] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 03/19/2024]
Abstract
Formation of templated insertions at DNA double-strand breaks (DSBs) is very common in cancer cells. The mechanisms and enzymes regulating these events are largely unknown. Here, we investigated templated insertions in yeast at DSBs using amplicon sequencing across a repaired locus. We document very short (most ∼5-34 bp), templated inverted duplications at DSBs. They are generated through a foldback mechanism that utilizes microhomologies adjacent to the DSB. Enzymatic requirements suggest a hybrid mechanism wherein one end requires Polδ-mediated synthesis while the other end is captured by nonhomologous end joining (NHEJ). This process is exacerbated in mutants with low levels or mutated RPA ( rtt105 Δ; rfa1 -t33) or extensive resection mutant ( sgs1 Δ exo1 Δ). Templated insertions from various distant genomic locations also increase in these mutants as well as in rad27 Δ and originate from fragile regions of the genome. Among complex insertions, common events are insertions of two sequences, originating from the same locus and with inverted orientation. We propose that these inversions are also formed by microhomology-mediated template switching. Taken together, we propose that a shortage of RPA typical in cancer cells is one possible factor stimulating the formation of templated insertions.
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5
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Bader AS, Bushell M. iMUT-seq: high-resolution DSB-induced mutation profiling reveals prevalent homologous-recombination dependent mutagenesis. Nat Commun 2023; 14:8419. [PMID: 38110444 PMCID: PMC10728174 DOI: 10.1038/s41467-023-44167-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2022] [Accepted: 12/04/2023] [Indexed: 12/20/2023] Open
Abstract
DNA double-strand breaks (DSBs) are the most mutagenic form of DNA damage, and play a significant role in cancer biology, neurodegeneration and aging. However, studying DSB-induced mutagenesis is limited by our current approaches. Here, we describe iMUT-seq, a technique that profiles DSB-induced mutations at high-sensitivity and single-nucleotide resolution around endogenous DSBs. By depleting or inhibiting 20 DSB-repair factors we define their mutational signatures in detail, revealing insights into the mechanisms of DSB-induced mutagenesis. Notably, we find that homologous-recombination (HR) is more mutagenic than previously thought, inducing prevalent base substitutions and mononucleotide deletions at distance from the break due to DNA-polymerase errors. Simultaneously, HR reduces translocations, suggesting a primary role of HR is specifically the prevention of genomic rearrangements. The results presented here offer fundamental insights into DSB-induced mutagenesis and have significant implications for our understanding of cancer biology and the development of DDR-targeting chemotherapeutics.
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Affiliation(s)
- Aldo S Bader
- Cancer Research UK Beatson Institute, Glasgow, G61 1BD, UK.
- Cancer Research UK/CI, University of Cambridge, Li Ka Shing Centre, Cambridge, CB2 0RE, UK.
- The Gurdon Institute, University of Cambridge, Biochemistry, Cambridge, UK.
| | - Martin Bushell
- Cancer Research UK Beatson Institute, Glasgow, G61 1BD, UK.
- Institute of Cancer Sciences, University of Glasgow, Glasgow, G61 1QH, UK.
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6
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Frey B, Borgmann K, Jost T, Greve B, Oertel M, Micke O, Eckert F. DNA as the main target in radiotherapy-a historical overview from first isolation to anti-tumour immune response. Strahlenther Onkol 2023; 199:1080-1090. [PMID: 37620671 DOI: 10.1007/s00066-023-02122-5] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/03/2023] [Accepted: 07/10/2023] [Indexed: 08/26/2023]
Abstract
DNA damage is one of the foremost mechanisms of irradiation at the biological level. After the first isolation of DNA by Friedrich Miescher in the 19th century, the structure of DNA was described by Watson and Crick. Several Nobel Prizes have been awarded for DNA-related discoveries. This review aims to describe the historical perspective of DNA in radiation biology. Over the decades, DNA damage has been identified and quantified after irradiation. Depending on the type of sensing, different proteins are involved in sensing DNA damage and repairing the damage, if possible. For double-strand breaks, the main repair mechanisms are non-homologous end joining and homologous recombination. Additional mechanisms are the Fanconi anaemia pathway and base excision repair. Different methods have been developed for the detection of DNA double-strand breaks. Several drugs have been developed that interfere with different DNA repair mechanisms, e.g., PARP inhibitors. These drugs have been established in the standard treatment of different tumour entities and are being applied in several clinical trials in combination with radiotherapy. Over the past decades, it has become apparent that DNA damage mechanisms are also directly linked to the immune response in tumours. For example, cytosolic DNA fragments activate the innate immune system via the cGAS STING pathway.
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Affiliation(s)
- Benjamin Frey
- Translational Radiation Biology, Department of Radiation Oncology, Universitätsklinikum Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany
| | - Kerstin Borgmann
- Laboratory of Radiobiology and Radiation Oncology, Department of Radiotherapy and Radiation Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
| | - Tina Jost
- Translational Radiation Biology, Department of Radiation Oncology, Universitätsklinikum Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany
| | - Burkhard Greve
- Department of Radiation Oncology, University Hospital Muenster, Muenster, Germany
| | - Michael Oertel
- Department of Radiation Oncology, University Hospital Muenster, Muenster, Germany
| | - Oliver Micke
- Department of Radiotherapy and Radiation Oncology, Franziskus Hospital Bielefeld, Kiskerst. 26, 33615, Bielefeld, Germany.
| | - Franziska Eckert
- Department of Radiation Oncology, AKH, Comprehensive Cancer Center Vienna, Medical University Vienna, Vienna, Austria
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7
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Hinch R, Donnelly P, Hinch AG. Meiotic DNA breaks drive multifaceted mutagenesis in the human germ line. Science 2023; 382:eadh2531. [PMID: 38033082 PMCID: PMC7615360 DOI: 10.1126/science.adh2531] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2023] [Accepted: 09/29/2023] [Indexed: 12/02/2023]
Abstract
Meiotic recombination commences with hundreds of programmed DNA breaks; however, the degree to which they are accurately repaired remains poorly understood. We report that meiotic break repair is eightfold more mutagenic for single-base substitutions than was previously understood, leading to de novo mutation in one in four sperm and one in 12 eggs. Its impact on indels and structural variants is even higher, with 100- to 1300-fold increases in rates per break. We uncovered new mutational signatures and footprints relative to break sites, which implicate unexpected biochemical processes and error-prone DNA repair mechanisms, including translesion synthesis and end joining in meiotic break repair. We provide evidence that these mechanisms drive mutagenesis in human germ lines and lead to disruption of hundreds of genes genome wide.
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Affiliation(s)
- Robert Hinch
- Big Data Institute, University of Oxford; Oxford, UK
| | - Peter Donnelly
- Wellcome Centre for Human Genetics, University of Oxford; Oxford, UK
- Genomics plc; Oxford, UK
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8
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Al-Zain AM, Nester MR, Ahmed I, Symington LS. Double-strand breaks induce inverted duplication chromosome rearrangements by a DNA polymerase δ-dependent mechanism. Nat Commun 2023; 14:7020. [PMID: 37919272 PMCID: PMC10622511 DOI: 10.1038/s41467-023-42640-5] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2023] [Accepted: 10/17/2023] [Indexed: 11/04/2023] Open
Abstract
Inverted duplications, also known as foldback inversions, are commonly observed in cancers and are the major class of chromosome rearrangement recovered from yeast cells lacking Mre11 nuclease activity. Foldback priming at DNA double-strand breaks (DSBs) is one mechanism proposed for the generation of inverted duplications. However, the other pathway steps have not been fully elucidated. Here, we show that a DSB induced near natural inverted repeats drives high frequency inverted duplication in Sae2 and Mre11-deficient cells. We find that DNA polymerase δ proof-reading activity, but not Rad1 nuclease, trims the heterologous flaps formed after foldback annealing. Additionally, Pol32 is required for the generation of inverted duplications, suggesting that Pol δ catalyzes fill-in synthesis primed from the foldback to create a hairpin-capped chromosome that is subsequently replicated to form a dicentric inversion chromosome. Finally, we show that stabilization of the dicentric chromosome after breakage involves telomere capture by non-reciprocal translocation mediated by repeat sequences or by deletion of one centromere.
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Affiliation(s)
- Amr M Al-Zain
- Program in Biological Sciences, Columbia University, New York, NY, 10027, USA
- Department of Microbiology & Immunology, Columbia University Irving Medical Center, New York, NY, 10032, USA
| | - Mattie R Nester
- Department of Microbiology & Immunology, Columbia University Irving Medical Center, New York, NY, 10032, USA
| | - Iffat Ahmed
- Department of Microbiology & Immunology, Columbia University Irving Medical Center, New York, NY, 10032, USA
| | - Lorraine S Symington
- Department of Microbiology & Immunology, Columbia University Irving Medical Center, New York, NY, 10032, USA.
- Department of Genetics & Development, Columbia University Irving Medical Center, New York, NY, 10032, USA.
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9
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Dalin S, Webster S, Sugawara N, Zhang S, Wu Q, Cui T, Liang V, Beroukhim R, Haber JE. Double-strand break repair-associated intragenic deletions and tandem duplications suggest the architecture of the repair replication fork. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.10.09.561461. [PMID: 37873277 PMCID: PMC10592705 DOI: 10.1101/2023.10.09.561461] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 10/25/2023]
Abstract
Double-strand break (DSB) repair is associated with a 1000-fold increase in mutations compared to normal replication of the same sequences. In budding yeast, repair of an HO endonuclease-induced DSB at the MATα locus can be repaired by using a homologous, heterochromatic HMR::Kl-URA3 donor harboring a transcriptionally silenced URA3 gene, resulting in a MAT::URA3 (Ura+) repair product where URA3 is expressed. Repair-associated ura3- mutations can be selected by resistance to 5-fluoroorotic acid (FOA). Using this system, we find that a major class of mutations are -1 deletions, almost always in homonucleotide runs, but there are few +1 insertions. In contrast, +1 and -1 insertions in homonucleotide runs are nearly equal among spontaneous mutations. Approximately 10% of repair-associated mutations are interchromosomal template switches (ICTS), even though the K. lactis URA3 sequence embedded in HMR is only 72% identical with S. cerevisiae ura3-52 sequences on a different chromosome. ICTS events begin and end in regions of short microhomology, averaging 7 bp. Long microhomologies are favored, but some ICTS junctions are as short as 2 bp. Both repair-associated intragenic deletions (IDs) and tandem duplications (TDs) are recovered, with junctions sharing short stretches of, on average, 6 bp of microhomology. Intragenic deletions are more than 5 times more frequent than TDs. IDs have a mean length of 60 bp, but, surprisingly there are almost no deletions shorter than 25 bp. In contrast, TDs average only 12 bp. The usage of microhomologies among intragenic deletions is not strongly influenced by the degree of adjacent homeology. Together, these data provide a picture of the structure of the repair replication fork. We suggest that IDs and TDs occur within the migrating D-loop in which DNA polymerase δ copies the template, where the 3' end of a partly copied new DNA strand can dissociate and anneal with a single-stranded region of microhomology that lies either in front or behind the 3' end, within the open structure of a migrating D-loop. Our data suggest that ~100 bp ahead of the polymerase is "open," but that part of the repair replication apparatus remains bound in the 25 bp ahead of the newly copied DNA, preventing annealing. In contrast, the template region behind the polymerase appears to be rapidly reannealed, limiting template switching to a very short region.
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Affiliation(s)
- Simona Dalin
- Cancer Program, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
- Departments of Cancer Biology and Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA
| | - Sophie Webster
- Cancer Program, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
- Departments of Cancer Biology and Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA
| | - Neal Sugawara
- Rosenstiel Center and Department of Biology, Brandeis University, Waltham, Massachusetts 02454-9110
| | - Shu Zhang
- Cancer Program, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
- Departments of Cancer Biology and Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA
| | - Qiuqin Wu
- Rosenstiel Center and Department of Biology, Brandeis University, Waltham, Massachusetts 02454-9110
| | - Tracy Cui
- Cancer Program, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
- Departments of Cancer Biology and Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA
- Rosenstiel Center and Department of Biology, Brandeis University, Waltham, Massachusetts 02454-9110
| | - Victoria Liang
- Rosenstiel Center and Department of Biology, Brandeis University, Waltham, Massachusetts 02454-9110
| | - Rameen Beroukhim
- Cancer Program, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
- Departments of Cancer Biology and Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA
| | - James E. Haber
- Rosenstiel Center and Department of Biology, Brandeis University, Waltham, Massachusetts 02454-9110
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10
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Lynch M, Ali F, Lin T, Wang Y, Ni J, Long H. The divergence of mutation rates and spectra across the Tree of Life. EMBO Rep 2023; 24:e57561. [PMID: 37615267 PMCID: PMC10561183 DOI: 10.15252/embr.202357561] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/29/2023] [Revised: 08/01/2023] [Accepted: 08/02/2023] [Indexed: 08/25/2023] Open
Abstract
Owing to advances in genome sequencing, genome stability has become one of the most scrutinized cellular traits across the Tree of Life. Despite its centrality to all things biological, the mutation rate (per nucleotide site per generation) ranges over three orders of magnitude among species and several-fold within individual phylogenetic lineages. Within all major organismal groups, mutation rates scale negatively with the effective population size of a species and with the amount of functional DNA in the genome. This relationship is most parsimoniously explained by the drift-barrier hypothesis, which postulates that natural selection typically operates to reduce mutation rates until further improvement is thwarted by the power of random genetic drift. Despite this constraint, the molecular mechanisms underlying DNA replication fidelity and repair are free to wander, provided the performance of the entire system is maintained at the prevailing level. The evolutionary flexibility of the mutation rate bears on the resolution of several prior conundrums in phylogenetic and population-genetic analysis and raises challenges for future applications in these areas.
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Affiliation(s)
- Michael Lynch
- Biodesign Center for Mechanisms of EvolutionArizona State UniversityTempeAZUSA
| | - Farhan Ali
- Biodesign Center for Mechanisms of EvolutionArizona State UniversityTempeAZUSA
| | - Tongtong Lin
- Institute of Evolution and Marine Biodiversity, KLMMEOcean University of ChinaQingdaoChina
| | - Yaohai Wang
- Institute of Evolution and Marine Biodiversity, KLMMEOcean University of ChinaQingdaoChina
| | - Jiahao Ni
- Institute of Evolution and Marine Biodiversity, KLMMEOcean University of ChinaQingdaoChina
| | - Hongan Long
- Institute of Evolution and Marine Biodiversity, KLMMEOcean University of ChinaQingdaoChina
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11
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Polleys EJ, Del Priore I, Haber JE, Freudenreich CH. Structure-forming CAG/CTG repeats interfere with gap repair to cause repeat expansions and chromosome breaks. Nat Commun 2023; 14:2469. [PMID: 37120647 PMCID: PMC10148874 DOI: 10.1038/s41467-023-37901-2] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2022] [Accepted: 04/04/2023] [Indexed: 05/01/2023] Open
Abstract
Expanded CAG/CTG repeats are sites of DNA damage, leading to repeat length changes. Homologous recombination (HR) is one cause of repeat instability and we hypothesized that gap filling was a driver of repeat instability during HR. To test this, we developed an assay such that resection and ssDNA gap fill-in would occur across a (CAG)70 or (CTG)70 repeat tract. When the ssDNA template was a CTG sequence, there were increased repeat contractions and a fragile site was created leading to large-scale deletions. When the CTG sequence was on the resected strand, resection was inhibited, resulting in repeat expansions. Increased nucleolytic processing by deletion of Rad9, the ortholog of 53BP1, rescued repeat instability and chromosome breakage. Loss of Rad51 increased contractions implicating a protective role for Rad51 on ssDNA. Together, our work implicates structure-forming repeats as an impediment to resection and gap-filling which can lead to mutations and large-scale deletions.
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Affiliation(s)
- Erica J Polleys
- Department of Biology, Tufts University, Medford, MA, 02155, USA.
| | | | - James E Haber
- Department of Biology and Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA, 02454, USA
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12
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Shuffling the yeast genome using CRISPR/Cas9-generated DSBs that target the transposable Ty1 elements. PLoS Genet 2023; 19:e1010590. [PMID: 36701275 PMCID: PMC9879454 DOI: 10.1371/journal.pgen.1010590] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2022] [Accepted: 12/21/2022] [Indexed: 01/27/2023] Open
Abstract
Although homologous recombination between transposable elements can drive genomic evolution in yeast by facilitating chromosomal rearrangements, the details of the underlying mechanisms are not fully clarified. In the genome of the yeast Saccharomyces cerevisiae, the most common class of transposon is the retrotransposon Ty1. Here, we explored how Cas9-induced double-strand breaks (DSBs) directed to Ty1 elements produce genomic alterations in this yeast species. Following Cas9 induction, we observed a significant elevation of chromosome rearrangements such as deletions, duplications and translocations. In addition, we found elevated rates of mitotic recombination, resulting in loss of heterozygosity. Using Southern analysis coupled with short- and long-read DNA sequencing, we revealed important features of recombination induced in retrotransposons. Almost all of the chromosomal rearrangements reflect the repair of DSBs at Ty1 elements by non-allelic homologous recombination; clustered Ty elements were hotspots for chromosome rearrangements. In contrast, a large proportion (about three-fourths) of the allelic mitotic recombination events have breakpoints in unique sequences. Our analysis suggests that some of the latter events reflect extensive processing of the broken ends produced in the Ty element that extend into unique sequences resulting in break-induced replication. Finally, we found that haploid and diploid strain have different preferences for the pathways used to repair double-stranded DNA breaks. Our findings demonstrate the importance of DNA lesions in retrotransposons in driving genome evolution.
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13
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Al-Zain A, Nester MR, Symington LS. Double-strand breaks induce inverted duplication chromosome rearrangements by a DNA polymerase δ and Rad51-dependent mechanism. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.01.24.525421. [PMID: 36747747 PMCID: PMC9900772 DOI: 10.1101/2023.01.24.525421] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/27/2023]
Abstract
Inverted duplications, also known as foldback inversions, are commonly observed in cancers and are the major class of chromosome rearrangement recovered from yeast cells lacking Mre11 nuclease. Foldback priming at naturally occurring inverted repeats is one mechanism proposed for the generation of inverted duplications. However, the initiating lesion for these events and the mechanism by which they form has not been fully elucidated. Here, we show that a DNA double-strand break (DSB) induced near natural short, inverted repeats drives high frequency inverted duplication in Sae2 and Mre11-deficient cells. We find that DNA polymerase δ proof-reading activity acts non-redundantly with Rad1 nuclease to remove heterologous tails formed during foldback annealing. Additionally, Pol32 is required for the generation of inverted duplications, suggesting that Pol δ catalyzes fill-in synthesis primed from the foldback to create a hairpin-capped chromosome that is subsequently replicated to form a dicentric isochromosome. Stabilization of the dicentric chromosome after breakage involves telomere capture by non-reciprocal translocation mediated by repeat sequences and requires Rad51.
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14
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Geci R, Willis K, Burt A. Gene drive designs for efficient and localisable population suppression using Y-linked editors. PLoS Genet 2022; 18:e1010550. [PMID: 36574454 PMCID: PMC9829173 DOI: 10.1371/journal.pgen.1010550] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2022] [Revised: 01/09/2023] [Accepted: 11/29/2022] [Indexed: 12/28/2022] Open
Abstract
The sterile insect technique (SIT) has been successful in controlling some pest species but is not practicable for many others due to the large number of individuals that need to be reared and released. Previous computer modelling has demonstrated that the release of males carrying a Y-linked editor that kills or sterilises female descendants could be orders of magnitude more efficient than SIT while still remaining spatially restricted, particularly if combined with an autosomal sex distorter. In principle, further gains in efficiency could be achieved by using a self-propagating double drive design, in which each of the two components (the Y-linked editor and the sex ratio distorter) boosted the transmission of the other. To better understand the expected dynamics and impact of releasing constructs of this new design we have analysed a deterministic population genetic and population dynamic model. Our modelling demonstrates that this design can suppress a population from very low release rates, with no invasion threshold. Importantly, the design can work even if homing rates are low and sex chromosomes are silenced at meiosis, potentially expanding the range of species amenable to such control. Moreover, the predicted dynamics and impacts can be exquisitely sensitive to relatively small (e.g., 25%) changes in allele frequencies in the target population, which could be exploited for sequence-based population targeting. Analysis of published Anopheles gambiae genome sequences indicates that even for weakly differentiated populations with an FST of 0.02 there may be thousands of suitably differentiated genomic sites that could be used to restrict the spread and impact of a release. Our proposed design, which extends an already promising development pathway based on Y-linked editors, is therefore a potentially useful addition to the menu of options for genetic biocontrol.
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Affiliation(s)
- René Geci
- Dept. of Life Sciences, Imperial College London, Silwood Park, United Kingdom
| | - Katie Willis
- Dept. of Life Sciences, Imperial College London, Silwood Park, United Kingdom
| | - Austin Burt
- Dept. of Life Sciences, Imperial College London, Silwood Park, United Kingdom
- * E-mail:
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15
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Osia B, Twarowski J, Jackson T, Lobachev K, Liu L, Malkova A. Migrating bubble synthesis promotes mutagenesis through lesions in its template. Nucleic Acids Res 2022; 50:6870-6889. [PMID: 35748867 PMCID: PMC9262586 DOI: 10.1093/nar/gkac520] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2021] [Revised: 05/23/2022] [Accepted: 06/10/2022] [Indexed: 12/24/2022] Open
Abstract
Break-induced replication (BIR) proceeds via a migrating D-loop for hundreds of kilobases and is highly mutagenic. Previous studies identified long single-stranded (ss) nascent DNA that accumulates during leading strand synthesis to be a target for DNA damage and a primary source of BIR-induced mutagenesis. Here, we describe a new important source of mutagenic ssDNA formed during BIR: the ssDNA template for leading strand BIR synthesis formed during D-loop migration. Specifically, we demonstrate that this D-loop bottom template strand (D-BTS) is susceptible to APOBEC3A (A3A)-induced DNA lesions leading to mutations associated with BIR. Also, we demonstrate that BIR-associated ssDNA promotes an additional type of genetic instability: replication slippage between microhomologies stimulated by inverted DNA repeats. Based on our results we propose that these events are stimulated by both known sources of ssDNA formed during BIR, nascent DNA formed by leading strand synthesis, and the D-BTS that we describe here. Together we report a new source of mutagenesis during BIR that may also be shared by other homologous recombination pathways driven by D-loop repair synthesis.
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Affiliation(s)
| | | | - Tyler Jackson
- Department of Biology, University of Iowa, Iowa City, IA 52245, USA,Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA
| | - Kirill Lobachev
- School of Biological Sciences, Georgia Institute of Technology, Atlanta, GE 30332, USA
| | - Liping Liu
- Department of Biology, University of Iowa, Iowa City, IA 52245, USA
| | - Anna Malkova
- To whom correspondence should be addressed. Tel: +1 319 384 1285;
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16
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Mating-Type Switching in Budding Yeasts, from Flip/Flop Inversion to Cassette Mechanisms. Microbiol Mol Biol Rev 2022; 86:e0000721. [PMID: 35195440 PMCID: PMC8941940 DOI: 10.1128/mmbr.00007-21] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Mating-type switching is a natural but unusual genetic control process that regulates cell identity in ascomycete yeasts. It involves physically replacing one small piece of genomic DNA by another, resulting in replacement of the master regulatory genes in the mating pathway and hence a switch of cell type and mating behavior. In this review, we concentrate on recent progress that has been made on understanding the origins and evolution of mating-type switching systems in budding yeasts (subphylum Saccharomycotina). Because of the unusual nature and the complexity of the mechanism in Saccharomyces cerevisiae, mating-type switching was assumed until recently to have originated only once or twice during yeast evolution. However, comparative genomics analysis now shows that switching mechanisms arose many times independently-at least 11 times in budding yeasts and once in fission yeasts-a dramatic example of convergent evolution. Most of these lineages switch mating types by a flip/flop mechanism that inverts a section of a chromosome and is simpler than the well-characterized 3-locus cassette mechanism (MAT/HML/HMR) used by S. cerevisiae. Mating-type switching (secondary homothallism) is one of the two possible mechanisms by which a yeast species can become self-fertile. The other mechanism (primary homothallism) has also emerged independently in multiple evolutionary lineages of budding yeasts, indicating that homothallism has been favored strongly by natural selection. Recent work shows that HO endonuclease, which makes the double-strand DNA break that initiates switching at the S. cerevisiae MAT locus, evolved from an unusual mobile genetic element that originally targeted a glycolytic gene, FBA1.
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17
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Gene Overlapping as a Modulator of Begomovirus Evolution. Microorganisms 2022; 10:microorganisms10020366. [PMID: 35208820 PMCID: PMC8875319 DOI: 10.3390/microorganisms10020366] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2021] [Revised: 02/01/2022] [Accepted: 02/01/2022] [Indexed: 02/06/2023] Open
Abstract
In RNA viruses, which have high mutation—and fast evolutionary— rates, gene overlapping (i.e., genomic regions that encode more than one protein) is a major factor controlling mutational load and therefore the virus evolvability. Although DNA viruses use host high-fidelity polymerases for their replication, and therefore should have lower mutation rates, it has been shown that some of them have evolutionary rates comparable to those of RNA viruses. Notably, these viruses have large proportions of their genes with at least one overlapping instance. Hence, gene overlapping could be a modulator of virus evolution beyond the RNA world. To test this hypothesis, we use the genus Begomovirus of plant viruses as a model. Through comparative genomic approaches, we show that terminal gene overlapping decreases the rate of virus evolution, which is associated with lower frequency of both synonymous and nonsynonymous mutations. In contrast, terminal overlapping has little effect on the pace of virus evolution. Overall, our analyses support a role for gene overlapping in the evolution of begomoviruses and provide novel information on the factors that shape their genetic diversity.
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18
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Template switching in DNA replication can create and maintain RNA hairpins. Proc Natl Acad Sci U S A 2022; 119:2107005119. [PMID: 35046021 PMCID: PMC8794818 DOI: 10.1073/pnas.2107005119] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 12/14/2021] [Indexed: 11/18/2022] Open
Abstract
The evolutionary origin of RNA stem structures and the preservation of their base pairing under a spontaneous and random mutation process have puzzled theoretical evolutionary biologists. DNA replication-related template switching is a mutation mechanism that creates reverse-complement copies of sequence regions within a genome by replicating briefly along either the complementary or nascent DNA strand. Depending on the relative positions and context of the four switch points, this process may produce a reverse-complement repeat capable of forming the stem of a perfect DNA hairpin or fix the base pairing of an existing stem. Template switching is typically thought to trigger large structural changes, and its possible role in the origin and evolution of RNA genes has not been studied. Here, we show that the reconstructed ancestral histories of RNA genes contain mutation patterns consistent with the DNA replication-related template switching. In addition to multibase compensatory mutations, the mechanism can explain complex sequence changes, although mutations breaking the structure rarely get fixed in evolution. Our results suggest a solution for the long-standing dilemma of RNA gene evolution and demonstrate how template switching can both create perfect stems with a single mutation event and help maintaining the stem structure over time. Interestingly, template switching also provides an elegant explanation for the asymmetric base pair frequencies within RNA stems.
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19
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Structural switching/polymorphism by sequential base substitution at quasi-palindromic SNP site (G → A) in LCR of human β-globin gene cluster. Int J Biol Macromol 2021; 201:216-225. [PMID: 34973267 DOI: 10.1016/j.ijbiomac.2021.12.142] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2021] [Revised: 12/21/2021] [Accepted: 12/21/2021] [Indexed: 11/20/2022]
Abstract
The human β-globin gene Locus Control Region (LCR), a dominant regulator of globin gene expression contains five tissue-specific DNase I-hypersensitive sites (HSs). A single nucleotide polymorphism (SNP) (A → G) present in HS4 region of locus control region (LCR), have shown a notable association between the G allele and the occurrence of β-thalassemia. This SNP site exhibiting a hairpin - duplex equilibrium manifested in A → B like DNA transition has previously been reported from this laboratory. Since, DNA is a dynamic and adaptable molecule, so any change of a single base within a primary DNA sequence can produce major biological consequences commonly manifested in genetic disorders such as sickle cell anemia and β-thalassemia. Herein, the differential behavior of sequential single base substitutions G → A on the quasi-palindromic sequence (d-TGGGGGCCCCA; HPG11) has been explored. A combination of native gel electrophoresis, circular dichroism (CD), and UV-thermal denaturation (Tm) techniques have been used to investigate the structural polymorphism associated with various variants of HPG11 i.e. HPG11A2 to HPG11A5. The CD spectra confirmed that all the HPG11 variants exhibit a hairpin - duplex equilibrium. Oligomer concentration dependence on CD spectra has been correlated with A → B DNA conformational transition. However, as revealed in gel electrophoresis, HPG11A2 → A5 exhibit the formation of a tetramolecular structure (four-way junction) at higher oligomer concentration. UV-melting studies also supported the melting of hairpin, duplex and four-way junction structure. This polymorphism pattern may possibly be significant for DNA-protein recognition, in the process of regulation of LCR in the β-globin gene.
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20
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De Falco M, De Felice M. Take a Break to Repair: A Dip in the World of Double-Strand Break Repair Mechanisms Pointing the Gaze on Archaea. Int J Mol Sci 2021; 22:ijms222413296. [PMID: 34948099 PMCID: PMC8708640 DOI: 10.3390/ijms222413296] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2021] [Revised: 12/06/2021] [Accepted: 12/07/2021] [Indexed: 12/24/2022] Open
Abstract
All organisms have evolved many DNA repair pathways to counteract the different types of DNA damages. The detection of DNA damage leads to distinct cellular responses that bring about cell cycle arrest and the induction of DNA repair mechanisms. In particular, DNA double-strand breaks (DSBs) are extremely toxic for cell survival, that is why cells use specific mechanisms of DNA repair in order to maintain genome stability. The choice among the repair pathways is mainly linked to the cell cycle phases. Indeed, if it occurs in an inappropriate cellular context, it may cause genome rearrangements, giving rise to many types of human diseases, from developmental disorders to cancer. Here, we analyze the most recent remarks about the main pathways of DSB repair with the focus on homologous recombination. A thorough knowledge in DNA repair mechanisms is pivotal for identifying the most accurate treatments in human diseases.
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21
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Fijarczyk A, Hénault M, Marsit S, Charron G, Landry CR. Heterogeneous Mutation Rates and Spectra in Yeast Hybrids. Genome Biol Evol 2021; 13:6462191. [PMID: 34908117 PMCID: PMC8715523 DOI: 10.1093/gbe/evab282] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 12/10/2021] [Indexed: 12/11/2022] Open
Abstract
Mutation rates and spectra vary between species and among populations. Hybridization can contribute to this variation, but its role remains poorly understood. Estimating mutation rates requires controlled conditions where the effect of natural selection can be minimized. One way to achieve this is through mutation accumulation experiments coupled with genome sequencing. Here, we investigate 400 mutation accumulation lines initiated from 11 genotypes spanning intralineage, interlineage, and interspecific crosses of the yeasts Saccharomyces paradoxus and S. cerevisiae and propagated for 770 generations. We find significant differences in mutation rates and spectra among crosses, which are not related to the level of divergence of parental strains but are specific to some genotype combinations. Differences in number of generations and departures from neutrality play a minor role, whereas polyploidy and loss of heterozygosity impact mutation rates in some of the hybrid crosses in an opposite way.
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Affiliation(s)
- Anna Fijarczyk
- Département de Biologie, Université Laval, Québec, Québec, Canada.,Institut de Biologie Intégrative et des Systemes (IBIS), Université Laval, Québec, Québec, Canada.,Département de Biochimie, Microbiologie et Bioinformatique, Université Laval, Québec, Québec, Canada.,PROTEO, Le Réseau Québécois de Recherche sur la Fonction, La Structure et L'Ingénierie des Protéines, Université Laval, Québec, Québec, Canada.,Centre de Recherche en Données Massives (CRDM), Université Laval, Québec, Québec, Canada
| | - Mathieu Hénault
- Département de Biologie, Université Laval, Québec, Québec, Canada.,Institut de Biologie Intégrative et des Systemes (IBIS), Université Laval, Québec, Québec, Canada.,Département de Biochimie, Microbiologie et Bioinformatique, Université Laval, Québec, Québec, Canada.,PROTEO, Le Réseau Québécois de Recherche sur la Fonction, La Structure et L'Ingénierie des Protéines, Université Laval, Québec, Québec, Canada.,Centre de Recherche en Données Massives (CRDM), Université Laval, Québec, Québec, Canada
| | - Souhir Marsit
- Département de Biologie, Université Laval, Québec, Québec, Canada.,Institut de Biologie Intégrative et des Systemes (IBIS), Université Laval, Québec, Québec, Canada.,Département de Biochimie, Microbiologie et Bioinformatique, Université Laval, Québec, Québec, Canada.,PROTEO, Le Réseau Québécois de Recherche sur la Fonction, La Structure et L'Ingénierie des Protéines, Université Laval, Québec, Québec, Canada.,Centre de Recherche en Données Massives (CRDM), Université Laval, Québec, Québec, Canada
| | - Guillaume Charron
- Département de Biologie, Université Laval, Québec, Québec, Canada.,Institut de Biologie Intégrative et des Systemes (IBIS), Université Laval, Québec, Québec, Canada.,Département de Biochimie, Microbiologie et Bioinformatique, Université Laval, Québec, Québec, Canada.,PROTEO, Le Réseau Québécois de Recherche sur la Fonction, La Structure et L'Ingénierie des Protéines, Université Laval, Québec, Québec, Canada.,Centre de Recherche en Données Massives (CRDM), Université Laval, Québec, Québec, Canada
| | - Christian R Landry
- Département de Biologie, Université Laval, Québec, Québec, Canada.,Institut de Biologie Intégrative et des Systemes (IBIS), Université Laval, Québec, Québec, Canada.,Département de Biochimie, Microbiologie et Bioinformatique, Université Laval, Québec, Québec, Canada.,PROTEO, Le Réseau Québécois de Recherche sur la Fonction, La Structure et L'Ingénierie des Protéines, Université Laval, Québec, Québec, Canada.,Centre de Recherche en Données Massives (CRDM), Université Laval, Québec, Québec, Canada
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22
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Vu TV, Das S, Nguyen CC, Kim J, Kim JY. Single-strand annealing: Molecular mechanisms and potential applications in CRISPR-Cas-based precision genome editing. Biotechnol J 2021; 17:e2100413. [PMID: 34846104 DOI: 10.1002/biot.202100413] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/04/2021] [Revised: 11/22/2021] [Accepted: 11/29/2021] [Indexed: 12/24/2022]
Abstract
BACKGROUND Spontaneous double-stranded DNA breaks (DSBs) frequently occur within the genome of all living organisms and must be well repaired for survival. Recently, more important roles of the DSB repair pathways that were previously thought to be minor pathways, such as single-strand annealing (SSA), have been shown. Nevertheless, the biochemical mechanisms and applications of the SSA pathway in genome editing have not been updated. PURPOSE AND SCOPE Understanding the molecular mechanism of SSA is important to design potential applications in gene editing. This review provides insights into the recent progress of SSA studies and establishes a model for their potential applications in precision genome editing. SUMMARY AND CONCLUSION The SSA mechanism involved in DNA DSB repair appears to be activated by a complex signaling cascade starting with broken end sensing and 5'-3' resection to reveal homologous repeats on the 3' ssDNA overhangs that flank the DSB. Annealing the repeats would help to amend the discontinuous ends and restore the intact genome, resulting in the missing of one repeat and the intervening sequence between the repeats. We proposed a model for CRISPR-Cas-based precision insertion or replacement of DNA fragments to take advantage of the characteristics. The proposed model can add a tool to extend the choice for precision gene editing. Nevertheless, the model needs to be experimentally validated and optimized with SSA-favorable conditions for practical applications.
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Affiliation(s)
- Tien Van Vu
- Division of Applied Life Science (BK21 Four Program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju, Republic of Korea.,National Key Laboratory for Plant Cell Biotechnology, Agricultural Genetics Institute, Bac Tu Liem, Hanoi, Vietnam
| | - Swati Das
- Division of Applied Life Science (BK21 Four Program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju, Republic of Korea
| | - Cam Chau Nguyen
- Division of Applied Life Science (BK21 Four Program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju, Republic of Korea
| | - Jihae Kim
- Division of Applied Life Science (BK21 Four Program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju, Republic of Korea
| | - Jae-Yean Kim
- Division of Applied Life Science (BK21 Four Program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju, Republic of Korea.,Division of Life Science, Gyeongsang National University, Jinju, Republic of Korea
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23
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Zhu Q, Huang J, Huang H, Li H, Yi P, Kloeber JA, Yuan J, Chen Y, Deng M, Luo K, Gao M, Guo G, Tu X, Yin P, Zhang Y, Su J, Chen J, Lou Z. RNF19A-mediated ubiquitination of BARD1 prevents BRCA1/BARD1-dependent homologous recombination. Nat Commun 2021; 12:6653. [PMID: 34789768 PMCID: PMC8599684 DOI: 10.1038/s41467-021-27048-3] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2020] [Accepted: 11/01/2021] [Indexed: 12/19/2022] Open
Abstract
BRCA1-BARD1 heterodimers act in multiple steps during homologous recombination (HR) to ensure the prompt repair of DNA double strand breaks. Dysfunction of the BRCA1 pathway enhances the therapeutic efficiency of poly-(ADP-ribose) polymerase inhibitors (PARPi) in cancers, but the molecular mechanisms underlying this sensitization to PARPi are not fully understood. Here, we show that cancer cell sensitivity to PARPi is promoted by the ring between ring fingers (RBR) protein RNF19A. We demonstrate that RNF19A suppresses HR by ubiquitinating BARD1, which leads to dissociation of BRCA1-BARD1 complex and exposure of a nuclear export sequence in BARD1 that is otherwise masked by BRCA1, resulting in the export of BARD1 to the cytoplasm. We provide evidence that high RNF19A expression in breast cancer compromises HR and increases sensitivity to PARPi. We propose that RNF19A modulates the cancer cell response to PARPi by negatively regulating the BRCA1-BARD1 complex and inhibiting HR-mediated DNA repair.
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Affiliation(s)
- Qian Zhu
- Department of Radiation Oncology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, 200025, China
- Department of Oncology, Mayo Clinic, Rochester, MN, 55905, USA
| | - Jinzhou Huang
- Department of Oncology, Mayo Clinic, Rochester, MN, 55905, USA
| | - Hongyang Huang
- Department of Pathology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, 999077, China
| | - Huan Li
- Department of Radiation Oncology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, 200025, China
| | - Peiqiang Yi
- Department of Radiation Oncology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, 200025, China
| | - Jake A Kloeber
- Department of Oncology, Mayo Clinic, Rochester, MN, 55905, USA
- Mayo Clinic Medical Scientist Training Program, Mayo Clinic, Rochester, MN, 55905, USA
| | - Jian Yuan
- Research Center for Translational Medicine, East Hospital, Tongji University School of medicine, Shanghai, 200120, China
| | - Yuping Chen
- Research Center for Translational Medicine, East Hospital, Tongji University School of medicine, Shanghai, 200120, China
| | - Min Deng
- Department of Oncology, Mayo Clinic, Rochester, MN, 55905, USA
| | - Kuntian Luo
- Department of Oncology, Mayo Clinic, Rochester, MN, 55905, USA
| | - Ming Gao
- Department of Oncology, Mayo Clinic, Rochester, MN, 55905, USA
| | - Guijie Guo
- Department of Oncology, Mayo Clinic, Rochester, MN, 55905, USA
| | - Xinyi Tu
- Department of Oncology, Mayo Clinic, Rochester, MN, 55905, USA
| | - Ping Yin
- Department of Oncology, Mayo Clinic, Rochester, MN, 55905, USA
| | - Yong Zhang
- Department of Oncology, Mayo Clinic, Rochester, MN, 55905, USA
| | - Jun Su
- Department of Radiation Oncology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, 200025, China
| | - Jiayi Chen
- Department of Radiation Oncology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, 200025, China.
| | - Zhenkun Lou
- Department of Oncology, Mayo Clinic, Rochester, MN, 55905, USA.
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24
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Gallagher DN, Haber JE. Single-strand template repair: key insights to increase the efficiency of gene editing. Curr Genet 2021; 67:747-753. [PMID: 33881574 DOI: 10.1007/s00294-021-01186-z] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/04/2021] [Revised: 03/27/2021] [Accepted: 03/29/2021] [Indexed: 10/21/2022]
Abstract
DNA double-strand breaks (DSBs) pose a serious hazard for the stability of the genome. CRISPR-Cas9-mediated gene editing intentionally creates a site-specific DSB to modify the genomic sequence, typically from an introduced single-stranded DNA donor. However, unlike typical forms of homologous recombination, single-strand template repair (SSTR) is Rad51-independent. Moreover, this pathway is distinct from other previously characterized Rad51-independent processes. Here, we briefly review the work characterizing this pathway, and how these findings can be used to guide and improve current gene editing strategies.
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Affiliation(s)
- Danielle N Gallagher
- Department of Biology and Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA, 02154, USA
| | - James E Haber
- Department of Biology and Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA, 02154, USA.
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25
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Wu X, Malkova A. Break-induced replication mechanisms in yeast and mammals. Curr Opin Genet Dev 2021; 71:163-170. [PMID: 34481360 DOI: 10.1016/j.gde.2021.08.002] [Citation(s) in RCA: 23] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2021] [Revised: 07/17/2021] [Accepted: 08/05/2021] [Indexed: 11/26/2022]
Abstract
Break-induced replication (BIR) is a pathway specialized in repair of double-strand DNA breaks with only one end capable of invading homologous template that can arise following replication collapse, telomere erosion or DNA cutting by site-specific endonucleases. For a long time, yeast remained the only model system to study BIR. Studies in yeast demonstrated that BIR represents an unusual mode of DNA synthesis that is driven by a migrating bubble and leads to conservative inheritance of newly synthesized DNA. This unusual type of DNA synthesis leads to high levels of mutations and chromosome rearrangements. Recently, multiple examples of BIR were uncovered in mammalian cells that allowed the comparison of BIR between organisms. It appeared initially that BIR in mammalian cells is predominantly independent of RAD51, and therefore different from BIR that is predominantly Rad51-dependent in yeast. However, a series of systematic studies utilizing site-specific DNA breaks for BIR initiation in mammalian reporters led to the discovery of highly efficient RAD51-dependent BIR, allowing side-by side comparison with BIR in yeast which is the focus of this review.
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Affiliation(s)
- Xiaohua Wu
- Department of Molecular Medicine, The Scripps Research Institute, La Jolla, CA 92037, United States.
| | - Anna Malkova
- Department of Biology, University of Iowa, Iowa City, IA 52242, United States.
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26
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Stead ER, Bjedov I. Balancing DNA repair to prevent ageing and cancer. Exp Cell Res 2021; 405:112679. [PMID: 34102225 PMCID: PMC8361780 DOI: 10.1016/j.yexcr.2021.112679] [Citation(s) in RCA: 24] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/19/2020] [Revised: 04/25/2021] [Accepted: 04/29/2021] [Indexed: 02/06/2023]
Abstract
DNA damage is a constant stressor to the cell. Persistent damage to the DNA over time results in an increased risk of mutation and an accumulation of mutations with age. Loss of efficient DNA damage repair can lead to accelerated ageing phenotypes or an increased cancer risk, and the trade-off between cancer susceptibility and longevity is often driven by the cell's response to DNA damage. High levels of mutations in DNA repair mutants often leads to excessive cell death and stem cell exhaustion which may promote premature ageing. Stem cells themselves have distinct characteristics that enable them to retain low mutation rates. However, when mutations do arise, stem cell clonal expansion can also contribute to age-related tissue dysfunction as well as heightened cancer risk. In this review, we will highlight increasing DNA damage and mutation accumulation as hallmarks common to both ageing and cancer. We will propose that anti-ageing interventions might be cancer preventative and discuss the mechanisms through which they may act.
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Affiliation(s)
- Eleanor Rachel Stead
- UCL Cancer Institute, Paul O'Gorman Building, University College London, 72 Huntley Street London, London WC1E 6DD, UK
| | - Ivana Bjedov
- UCL Cancer Institute, Paul O'Gorman Building, University College London, 72 Huntley Street London, London WC1E 6DD, UK; University College London, Department of Medical Physics and Biomedical Engineering, Malet Place Engineering Building, Gower Street, London WC1E 6BT, UK.
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27
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Nickoloff JA, Sharma N, Allen CP, Taylor L, Allen SJ, Jaiswal AS, Hromas R. Roles of homologous recombination in response to ionizing radiation-induced DNA damage. Int J Radiat Biol 2021; 99:903-914. [PMID: 34283012 PMCID: PMC9629169 DOI: 10.1080/09553002.2021.1956001] [Citation(s) in RCA: 25] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/29/2021] [Revised: 03/04/2021] [Accepted: 07/05/2021] [Indexed: 02/06/2023]
Abstract
PURPOSE Ionizing radiation induces a vast array of DNA lesions including base damage, and single- and double-strand breaks (SSB, DSB). DSBs are among the most cytotoxic lesions, and mis-repair causes small- and large-scale genome alterations that can contribute to carcinogenesis. Indeed, ionizing radiation is a 'complete' carcinogen. DSBs arise immediately after irradiation, termed 'frank DSBs,' as well as several hours later in a replication-dependent manner, termed 'secondary' or 'replication-dependent DSBs. DSBs resulting from replication fork collapse are single-ended and thus pose a distinct problem from two-ended, frank DSBs. DSBs are repaired by error-prone nonhomologous end-joining (NHEJ), or generally error-free homologous recombination (HR), each with sub-pathways. Clarifying how these pathways operate in normal and tumor cells is critical to increasing tumor control and minimizing side effects during radiotherapy. CONCLUSIONS The choice between NHEJ and HR is regulated during the cell cycle and by other factors. DSB repair pathways are major contributors to cell survival after ionizing radiation, including tumor-resistance to radiotherapy. Several nucleases are important for HR-mediated repair of replication-dependent DSBs and thus replication fork restart. These include three structure-specific nucleases, the 3' MUS81 nuclease, and two 5' nucleases, EEPD1 and Metnase, as well as three end-resection nucleases, MRE11, EXO1, and DNA2. The three structure-specific nucleases evolved at very different times, suggesting incremental acceleration of replication fork restart to limit toxic HR intermediates and genome instability as genomes increased in size during evolution, including the gain of large numbers of HR-prone repetitive elements. Ionizing radiation also induces delayed effects, observed days to weeks after exposure, including delayed cell death and delayed HR. In this review we highlight the roles of HR in cellular responses to ionizing radiation, and discuss the importance of HR as an exploitable target for cancer radiotherapy.
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Affiliation(s)
- Jac A. Nickoloff
- Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, CO, USA
| | - Neelam Sharma
- Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, CO, USA
| | - Christopher P. Allen
- Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, CO, USA
- Department of Microbiology, Immunology and Pathology, Flow Cytometry and Cell Sorting Facility, Colorado State University, Fort Collins, CO, USA
| | - Lynn Taylor
- Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, CO, USA
| | - Sage J. Allen
- Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, CO, USA
| | - Aruna S. Jaiswal
- Division of Hematology and Medical Oncology, Department of Medicine and the Mays Cancer Center, University of Texas Health Science Center, San Antonio, TX, USA
| | - Robert Hromas
- Division of Hematology and Medical Oncology, Department of Medicine and the Mays Cancer Center, University of Texas Health Science Center, San Antonio, TX, USA
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28
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Sanchez A, Reginato G, Cejka P. Crossover or non-crossover outcomes: tailored processing of homologous recombination intermediates. Curr Opin Genet Dev 2021; 71:39-47. [PMID: 34293660 DOI: 10.1016/j.gde.2021.06.012] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2021] [Revised: 06/15/2021] [Accepted: 06/23/2021] [Indexed: 12/14/2022]
Abstract
DNA breaks may arise accidentally in vegetative cells or in a programmed manner in meiosis. The usage of a DNA template makes homologous recombination potentially error-free, however, recombination is not always accurate. Cells possess a remarkable capacity to tailor processing of recombination intermediates to fulfill a particular need. Vegetatively growing cells aim to maintain genome stability and therefore repair accidental breaks largely accurately, using sister chromatids as templates, into mostly non-crossovers products. Recombination in meiotic cells is instead more likely to employ homologous chromosomes as templates and result in crossovers to allow proper chromosome segregation and promote genetic diversity. Here we review models explaining the processing of recombination intermediates in vegetative and meiotic cells and its regulation, with a focus on MLH1-MLH3-dependent crossing-over during meiotic recombination.
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Affiliation(s)
- Aurore Sanchez
- Institute for Research in Biomedicine, Università della Svizzera italiana (USI), Faculty of Biomedical Sciences, Bellinzona, Switzerland
| | - Giordano Reginato
- Institute for Research in Biomedicine, Università della Svizzera italiana (USI), Faculty of Biomedical Sciences, Bellinzona, Switzerland; Department of Biology, Institute of Biochemistry, Eidgenössische Technische Hochschule (ETH), Zürich, Switzerland
| | - Petr Cejka
- Institute for Research in Biomedicine, Università della Svizzera italiana (USI), Faculty of Biomedical Sciences, Bellinzona, Switzerland; Department of Biology, Institute of Biochemistry, Eidgenössische Technische Hochschule (ETH), Zürich, Switzerland.
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29
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Wu M, Wei H, Tan H, Pan S, Liu Q, Bejarano ER, Lozano-Durán R. Plant DNA polymerases α and δ mediate replication of geminiviruses. Nat Commun 2021; 12:2780. [PMID: 33986276 PMCID: PMC8119979 DOI: 10.1038/s41467-021-23013-2] [Citation(s) in RCA: 27] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2020] [Accepted: 04/06/2021] [Indexed: 12/19/2022] Open
Abstract
Geminiviruses are causal agents of devastating diseases in crops. Geminiviruses have circular single-stranded (ss) DNA genomes that are replicated in the nucleus of the infected plant cell through double-stranded (ds) DNA intermediates by the plant DNA replication machinery. Which host DNA polymerase mediates geminiviral multiplication, however, has so far remained elusive. Here, we show that subunits of the nuclear replicative DNA polymerases α and δ physically interact with the geminivirus-encoded replication enhancer protein, C3, and that these polymerases are required for viral replication. Our results suggest that, while DNA polymerase α is essential to generate the viral dsDNA intermediate, DNA polymerase δ mediates the synthesis of new copies of the geminiviral ssDNA genome, and that the virus-encoded C3 may act selectively, recruiting DNA polymerase δ over ε to favour productive replication.
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Affiliation(s)
- Mengshi Wu
- Shanghai Center for Plant Stress Biology, CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China
- Bioinformatics Department, School of Life Sciences and Technology, Tongji University, Shanghai, China
- University of the Chinese Academy of Sciences, Beijing, China
| | - Hua Wei
- Shanghai Center for Plant Stress Biology, CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China
- University of the Chinese Academy of Sciences, Beijing, China
| | - Huang Tan
- Shanghai Center for Plant Stress Biology, CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China
- University of the Chinese Academy of Sciences, Beijing, China
| | - Shaojun Pan
- Shanghai Center for Plant Stress Biology, CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China
- University of the Chinese Academy of Sciences, Beijing, China
| | - Qi Liu
- Bioinformatics Department, School of Life Sciences and Technology, Tongji University, Shanghai, China
| | - Eduardo R Bejarano
- Instituto de Hortofruticultura Subtropical y Mediterránea "La Mayora" (IHSM-UMA-CSIC), Area de Genética, Facultad de Ciencias, Universidad de Málaga, Campus de Teatinos s/n, Málaga, Spain
| | - Rosa Lozano-Durán
- Shanghai Center for Plant Stress Biology, CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China.
- Department of Plant Biochemistry, Centre for Plant Molecular Biology (ZMBP), Eberhard Karls University, Tübingen, Germany.
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30
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Learning Yeast Genetics from Miro Radman. Cells 2021; 10:cells10040945. [PMID: 33923882 PMCID: PMC8072546 DOI: 10.3390/cells10040945] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/29/2021] [Revised: 04/13/2021] [Accepted: 04/13/2021] [Indexed: 11/17/2022] Open
Abstract
Miroslav Radman's far-sighted ideas have penetrated many aspects of our study of the repair of broken eukaryotic chromosomes. For over 35 years my lab has studied different aspects of the repair of chromosomal breaks in the budding yeast, Saccharomyces cerevisiae. From the start, we have made what we thought were novel observations that turned out to have been predicted by Miro's extraordinary work in the bacterium Escherichia coli and then later in the radiation-resistant Dienococcus radiodurans. In some cases, we have been able to extend some of his ideas a bit further.
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31
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Pham N, Yan Z, Yu Y, Faria Afreen M, Malkova A, Haber JE, Ira G. Mechanisms restraining break-induced replication at two-ended DNA double-strand breaks. EMBO J 2021; 40:e104847. [PMID: 33844333 DOI: 10.15252/embj.2020104847] [Citation(s) in RCA: 38] [Impact Index Per Article: 9.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2020] [Revised: 03/04/2021] [Accepted: 03/09/2021] [Indexed: 11/09/2022] Open
Abstract
DNA synthesis during homologous recombination is highly mutagenic and prone to template switches. Two-ended DNA double-strand breaks (DSBs) are usually repaired by gene conversion with a short patch of DNA synthesis, thus limiting the mutation load to the vicinity of the DSB. Single-ended DSBs are repaired by break-induced replication (BIR), which involves extensive and mutagenic DNA synthesis spanning up to hundreds of kilobases. It remains unknown how mutagenic BIR is suppressed at two-ended DSBs. Here, we demonstrate that BIR is suppressed at two-ended DSBs by proteins coordinating the usage of two ends of a DSB: (i) ssDNA annealing proteins Rad52 and Rad59 that promote second end capture, (ii) D-loop unwinding helicase Mph1, and (iii) Mre11-Rad50-Xrs2 complex that promotes synchronous resection of two ends of a DSB. Finally, BIR is also suppressed when Sir2 silences a normally heterochromatic repair template. All of these proteins are particularly important for limiting BIR when recombination occurs between short repetitive sequences, emphasizing the significance of these mechanisms for species carrying many repetitive elements such as humans.
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Affiliation(s)
- Nhung Pham
- Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA
| | - Zhenxin Yan
- Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA
| | - Yang Yu
- Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA
| | - Mosammat Faria Afreen
- Department of Biology, Rosenstiel Basic Medical Sciences Research Center, Waltham, MA, USA
| | - Anna Malkova
- Department of Biology, University of Iowa, Iowa City, IA, USA
| | - James E Haber
- Department of Biology, Rosenstiel Basic Medical Sciences Research Center, Waltham, MA, USA
| | - Grzegorz Ira
- Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA
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32
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Toraason E, Horacek A, Clark C, Glover ML, Adler VL, Premkumar T, Salagean A, Cole F, Libuda DE. Meiotic DNA break repair can utilize homolog-independent chromatid templates in C. elegans. Curr Biol 2021; 31:1508-1514.e5. [PMID: 33740427 DOI: 10.1016/j.cub.2021.03.008] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/23/2020] [Revised: 02/09/2021] [Accepted: 03/02/2021] [Indexed: 02/06/2023]
Abstract
During meiosis, the maintenance of genome integrity is critical for generating viable haploid gametes.1 In meiotic prophase I, double-strand DNA breaks (DSBs) are induced and a subset of these DSBs are repaired as interhomolog crossovers to ensure proper chromosome segregation. DSBs not resolved as crossovers with the homolog must be repaired by other pathways to ensure genome integrity.2 To determine if alternative repair templates can be engaged for meiotic DSB repair during oogenesis, we developed an assay to detect sister and/or intra-chromatid repair events at a defined DSB site during Caenorhabditis elegans meiosis. Using this assay, we directly demonstrate that the sister chromatid or the same DNA molecule can be engaged as a meiotic repair template for both crossover and noncrossover recombination, with noncrossover events being the predominant recombination outcome. We additionally find that the sister or intra-chromatid substrate is available as a recombination partner for DSBs induced throughout meiotic prophase I, including late prophase when the homolog is unavailable. Analysis of noncrossover conversion tract sequences reveals that DSBs are processed similarly throughout prophase I. We further present data indicating that the XPF-1 nuclease functions in late prophase to promote sister or intra-chromatid repair at steps of recombination following joint molecule processing. Despite its function in sister or intra-chromatid repair, we find that xpf-1 mutants do not exhibit severe defects in progeny viability following exposure to ionizing radiation. Overall, we propose that C. elegans XPF-1 may assist as an intersister or intrachromatid resolvase only in late prophase I.
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Affiliation(s)
- Erik Toraason
- Institute of Molecular Biology, Department of Biology, University of Oregon, 1229 Franklin Boulevard, Eugene, OR 97403, USA
| | - Anna Horacek
- Institute of Molecular Biology, Department of Biology, University of Oregon, 1229 Franklin Boulevard, Eugene, OR 97403, USA
| | - Cordell Clark
- Institute of Molecular Biology, Department of Biology, University of Oregon, 1229 Franklin Boulevard, Eugene, OR 97403, USA
| | - Marissa L Glover
- Institute of Molecular Biology, Department of Biology, University of Oregon, 1229 Franklin Boulevard, Eugene, OR 97403, USA
| | - Victoria L Adler
- Institute of Molecular Biology, Department of Biology, University of Oregon, 1229 Franklin Boulevard, Eugene, OR 97403, USA
| | - Tolkappiyan Premkumar
- Department of Epigenetics and Molecular Carcinogenesis, MD Anderson Cancer Center, 1808 Park Road 1C, Smithville, TX 78957, USA
| | - Alina Salagean
- Institute of Molecular Biology, Department of Biology, University of Oregon, 1229 Franklin Boulevard, Eugene, OR 97403, USA
| | - Francesca Cole
- Department of Epigenetics and Molecular Carcinogenesis, MD Anderson Cancer Center, 1808 Park Road 1C, Smithville, TX 78957, USA
| | - Diana E Libuda
- Institute of Molecular Biology, Department of Biology, University of Oregon, 1229 Franklin Boulevard, Eugene, OR 97403, USA.
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33
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Willis K, Burt A. Double drives and private alleles for localised population genetic control. PLoS Genet 2021; 17:e1009333. [PMID: 33755671 PMCID: PMC8018619 DOI: 10.1371/journal.pgen.1009333] [Citation(s) in RCA: 17] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/18/2020] [Revised: 04/02/2021] [Accepted: 03/07/2021] [Indexed: 12/12/2022] Open
Abstract
Synthetic gene drive constructs could, in principle, provide the basis for highly efficient interventions to control disease vectors and other pest species. This efficiency derives in part from leveraging natural processes of dispersal and gene flow to spread the construct and its impacts from one population to another. However, sometimes (for example, with invasive species) only specific populations are in need of control, and impacts on non-target populations would be undesirable. Many gene drive designs use nucleases that recognise and cleave specific genomic sequences, and one way to restrict their spread would be to exploit sequence differences between target and non-target populations. In this paper we propose and model a series of low threshold double drive designs for population suppression, each consisting of two constructs, one imposing a reproductive load on the population and the other inserted into a differentiated locus and controlling the drive of the first. Simple deterministic, discrete-generation computer simulations are used to assess the alternative designs. We find that the simplest double drive designs are significantly more robust to pre-existing cleavage resistance at the differentiated locus than single drive designs, and that more complex designs incorporating sex ratio distortion can be more efficient still, even allowing for successful control when the differentiated locus is neutral and there is up to 50% pre-existing resistance in the target population. Similar designs can also be used for population replacement, with similar benefits. A population genomic analysis of CRISPR PAM sites in island and mainland populations of the malaria mosquito Anopheles gambiae indicates that the differentiation needed for our methods to work can exist in nature. Double drives should be considered when efficient but localised population genetic control is needed and there is some genetic differentiation between target and non-target populations.
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Affiliation(s)
- Katie Willis
- Department of Life Sciences, Imperial College London, Silwood Park, Ascot, United Kingdom
| | - Austin Burt
- Department of Life Sciences, Imperial College London, Silwood Park, Ascot, United Kingdom
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34
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Stivison EA, Young KJ, Symington LS. Interstitial telomere sequences disrupt break-induced replication and drive formation of ectopic telomeres. Nucleic Acids Res 2021; 48:12697-12710. [PMID: 33264397 PMCID: PMC7736798 DOI: 10.1093/nar/gkaa1081] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2019] [Revised: 10/20/2020] [Accepted: 10/22/2020] [Indexed: 01/18/2023] Open
Abstract
Break-induced replication (BIR) is a mechanism used to heal one-ended DNA double-strand breaks, such as those formed at collapsed replication forks or eroded telomeres. Instead of utilizing a canonical replication fork, BIR is driven by a migrating D-loop and is associated with a high frequency of mutagenesis. Here we show that when BIR encounters an interstitial telomere sequence (ITS), the machinery frequently terminates, resulting in the formation of an ectopic telomere. The primary mechanism to convert the ITS to a functional telomere is by telomerase-catalyzed addition of telomeric repeats with homology-directed repair serving as a back-up mechanism. Termination of BIR and creation of an ectopic telomere is promoted by Mph1/FANCM helicase, which has the capacity to disassemble D-loops. Other sequences that have the potential to seed new telomeres but lack the unique features of a natural telomere sequence, do not terminate BIR at a significant frequency in wild-type cells. However, these sequences can form ectopic telomeres if BIR is made less processive. Our results support a model in which features of the ITS itself, such as the propensity to form secondary structures and telomeric protein binding, pose a challenge to BIR and increase the vulnerability of the D-loop to dissociation by helicases, thereby promoting ectopic telomere formation.
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Affiliation(s)
- Elizabeth A Stivison
- Program in Nutritional and Metabolic Biology, Columbia University Irving Medical Center, New York, NY 10032, USA.,Department of Microbiology and Immunology, Columbia University Irving Medical Center, New York, NY 10032, USA
| | - Kati J Young
- Department of Microbiology and Immunology, Columbia University Irving Medical Center, New York, NY 10032, USA
| | - Lorraine S Symington
- Department of Microbiology and Immunology, Columbia University Irving Medical Center, New York, NY 10032, USA.,Department of Genetics and Development, Columbia University Irving Medical Center, New York, NY 10032, USA
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35
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Hay BA, Oberhofer G, Guo M. Engineering the Composition and Fate of Wild Populations with Gene Drive. ANNUAL REVIEW OF ENTOMOLOGY 2021; 66:407-434. [PMID: 33035437 DOI: 10.1146/annurev-ento-020117-043154] [Citation(s) in RCA: 41] [Impact Index Per Article: 10.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/11/2023]
Abstract
Insects play important roles as predators, prey, pollinators, recyclers, hosts, parasitoids, and sources of economically important products. They can also destroy crops; wound animals; and serve as vectors for plant, animal, and human diseases. Gene drive-a process by which genes, gene complexes, or chromosomes encoding specific traits are made to spread through wild populations, even if these traits result in a fitness cost to carriers-provides new opportunities for altering populations to benefit humanity and the environment in ways that are species specific and sustainable. Gene drive can be used to alter the genetic composition of an existing population, referred to as population modification or replacement, or to bring about population suppression or elimination. We describe technologies under consideration, progress that has been made, and remaining technological hurdles, particularly with respect to evolutionary stability and our ability to control the spread and ultimate fate of genes introduced into populations.
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Affiliation(s)
- Bruce A Hay
- Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California 91125, USA; ,
- St. John's College, University of Cambridge, Cambridge CB2 1TP, United Kingdom
| | - Georg Oberhofer
- Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California 91125, USA; ,
| | - Ming Guo
- Departments of Neurology and Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, California 90095, USA;
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36
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Cano‐Linares MI, Yáñez‐Vilches A, García‐Rodríguez N, Barrientos‐Moreno M, González‐Prieto R, San‐Segundo P, Ulrich HD, Prado F. Non-recombinogenic roles for Rad52 in translesion synthesis during DNA damage tolerance. EMBO Rep 2021; 22:e50410. [PMID: 33289333 PMCID: PMC7788459 DOI: 10.15252/embr.202050410] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/11/2020] [Revised: 10/09/2020] [Accepted: 10/15/2020] [Indexed: 01/09/2023] Open
Abstract
DNA damage tolerance relies on homologous recombination (HR) and translesion synthesis (TLS) mechanisms to fill in the ssDNA gaps generated during passing of the replication fork over DNA lesions in the template. Whereas TLS requires specialized polymerases able to incorporate a dNTP opposite the lesion and is error-prone, HR uses the sister chromatid and is mostly error-free. We report that the HR protein Rad52-but not Rad51 and Rad57-acts in concert with the TLS machinery (Rad6/Rad18-mediated PCNA ubiquitylation and polymerases Rev1/Pol ζ) to repair MMS and UV light-induced ssDNA gaps through a non-recombinogenic mechanism, as inferred from the different phenotypes displayed in the absence of Rad52 and Rad54 (essential for MMS- and UV-induced HR); accordingly, Rad52 is required for efficient DNA damage-induced mutagenesis. In addition, Rad52, Rad51, and Rad57, but not Rad54, facilitate Rad6/Rad18 binding to chromatin and subsequent DNA damage-induced PCNA ubiquitylation. Therefore, Rad52 facilitates the tolerance process not only by HR but also by TLS through Rad51/Rad57-dependent and -independent processes, providing a novel role for the recombination proteins in maintaining genome integrity.
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Affiliation(s)
- María I Cano‐Linares
- Department of Genome BiologyAndalusian Molecular Biology and Regenerative Medicine Center (CABIMER)CSIC‐University of Seville‐University Pablo de OlavideSevilleSpain
| | - Aurora Yáñez‐Vilches
- Department of Genome BiologyAndalusian Molecular Biology and Regenerative Medicine Center (CABIMER)CSIC‐University of Seville‐University Pablo de OlavideSevilleSpain
| | - Néstor García‐Rodríguez
- Institute of Molecular Biology (IMB)MainzGermany
- Present address:
Department of Genome BiologyAndalusian Molecular Biology and Regenerative Medicine Center (CABIMER)CSIC‐University of Seville‐University Pablo de OlavideSevilleSpain
| | - Marta Barrientos‐Moreno
- Department of Genome BiologyAndalusian Molecular Biology and Regenerative Medicine Center (CABIMER)CSIC‐University of Seville‐University Pablo de OlavideSevilleSpain
| | - Román González‐Prieto
- Department of Genome BiologyAndalusian Molecular Biology and Regenerative Medicine Center (CABIMER)CSIC‐University of Seville‐University Pablo de OlavideSevilleSpain
- Present address:
Department of Cell and Chemical BiologyLeiden University Medical CenterLeidenThe Netherlands
| | - Pedro San‐Segundo
- Institute of Functional Biology and Genomics (IBFG)CSIC‐University of SalamancaSalamancaSpain
| | | | - Félix Prado
- Department of Genome BiologyAndalusian Molecular Biology and Regenerative Medicine Center (CABIMER)CSIC‐University of Seville‐University Pablo de OlavideSevilleSpain
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37
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Gallagher DN, Pham N, Tsai AM, Janto NV, Choi J, Ira G, Haber JE. A Rad51-independent pathway promotes single-strand template repair in gene editing. PLoS Genet 2020; 16:e1008689. [PMID: 33057349 PMCID: PMC7591047 DOI: 10.1371/journal.pgen.1008689] [Citation(s) in RCA: 25] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/18/2020] [Revised: 10/27/2020] [Accepted: 08/03/2020] [Indexed: 01/26/2023] Open
Abstract
The Rad51/RecA family of recombinases perform a critical function in typical repair of double-strand breaks (DSBs): strand invasion of a resected DSB end into a homologous double-stranded DNA (dsDNA) template sequence to initiate repair. However, repair of a DSB using single stranded DNA (ssDNA) as a template, a common method of CRISPR/Cas9-mediated gene editing, is Rad51-independent. We have analyzed the genetic requirements for these Rad51-independent events in Saccharomyces cerevisiae by creating a DSB with the site-specific HO endonuclease and repairing the DSB with 80-nt single-stranded oligonucleotides (ssODNs), and confirmed these results by Cas9-mediated DSBs in combination with a bacterial retron system that produces ssDNA templates in vivo. We show that single strand template repair (SSTR), is dependent on Rad52, Rad59, Srs2 and the Mre11-Rad50-Xrs2 (MRX) complex, but unlike other Rad51-independent recombination events, independent of Rdh54. We show that Rad59 acts to alleviate the inhibition of Rad51 on Rad52's strand annealing activity both in SSTR and in single strand annealing (SSA). Gene editing is Rad51-dependent when double-stranded oligonucleotides of the same size and sequence are introduced as templates. The assimilation of mismatches during gene editing is dependent on the activity of Msh2, which acts very differently on the 3' side of the ssODN which can anneal directly to the resected DSB end compared to the 5' end. In addition DNA polymerase Polδ's 3' to 5' proofreading activity frequently excises a mismatch very close to the 3' end of the template. We further report that SSTR is accompanied by as much as a 600-fold increase in mutations in regions adjacent to the sequences directly undergoing repair. These DNA polymerase ζ-dependent mutations may compromise the accuracy of gene editing.
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Affiliation(s)
- Danielle N. Gallagher
- Department of Biology and Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA, United States of America
| | - Nhung Pham
- Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, United States of America
| | - Annie M. Tsai
- Department of Biology and Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA, United States of America
| | - Nicolas V. Janto
- Department of Biology and Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA, United States of America
| | - Jihyun Choi
- Department of Biology and Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA, United States of America
| | - Grzegorz Ira
- Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, United States of America
| | - James E. Haber
- Department of Biology and Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA, United States of America
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38
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Mechanism and significance of chromosome damage repair by homologous recombination. Essays Biochem 2020; 64:779-790. [DOI: 10.1042/ebc20190093] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2020] [Revised: 06/22/2020] [Accepted: 06/29/2020] [Indexed: 12/29/2022]
Abstract
Abstract
Homologous recombination (HR) is a major, conserved pathway of chromosome damage repair. It not only fulfills key functions in the removal of deleterious lesions such as DNA double-strand breaks (DSBs) and interstrand cross-links (ICLs), but also in replication fork repair and protection. Several familial and acquired cancer predisposition syndromes stem from defects in HR. In particular, individuals with mutations in HR genes exhibit predisposition to breast, ovarian, pancreatic, and prostate cancers, and they also show signs of accelerated aging. However, aberrant and untimely HR events can lead to the loss of heterozygosity, genomic rearrangements, and cytotoxic nucleoprotein intermediates. Thus, it is critically important that HR be tightly regulated. In addition to DNA repair, HR is also involved in meiotic chromosome segregation and telomere maintenance in cells that lack telomerase. In this review, we focus on the role of HR in DSB repair (DSBR) and summarize the current state of the field.
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Regulation of DNA Damage Response and Homologous Recombination Repair by microRNA in Human Cells Exposed to Ionizing Radiation. Cancers (Basel) 2020; 12:cancers12071838. [PMID: 32650508 PMCID: PMC7408912 DOI: 10.3390/cancers12071838] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/07/2020] [Revised: 06/26/2020] [Accepted: 06/29/2020] [Indexed: 12/12/2022] Open
Abstract
Ionizing radiation may be of both artificial and natural origin and causes cellular damage in living organisms. Radioactive isotopes have been used significantly in cancer therapy for many years. The formation of DNA double-strand breaks (DSBs) is the most dangerous effect of ionizing radiation on the cellular level. After irradiation, cells activate a DNA damage response, the molecular path that determines the fate of the cell. As an important element of this, homologous recombination repair is a crucial pathway for the error-free repair of DNA lesions. All components of DNA damage response are regulated by specific microRNAs. MicroRNAs are single-stranded short noncoding RNAs of 20–25 nt in length. They are directly involved in the regulation of gene expression by repressing translation or by cleaving target mRNA. In the present review, we analyze the biological mechanisms by which miRNAs regulate cell response to ionizing radiation-induced double-stranded breaks with an emphasis on DNA repair by homologous recombination, and its main component, the RAD51 recombinase. On the other hand, we discuss the ability of DNA damage response proteins to launch particular miRNA expression and modulate the course of this process. A full understanding of cell response processes to radiation-induced DNA damage will allow us to develop new and more effective methods of ionizing radiation therapy for cancers, and may help to develop methods for preventing the harmful effects of ionizing radiation on healthy organisms.
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40
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Saini N, Gordenin DA. Hypermutation in single-stranded DNA. DNA Repair (Amst) 2020; 91-92:102868. [PMID: 32438271 PMCID: PMC7234795 DOI: 10.1016/j.dnarep.2020.102868] [Citation(s) in RCA: 27] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2020] [Revised: 05/02/2020] [Accepted: 05/04/2020] [Indexed: 12/15/2022]
Abstract
Regions of genomic DNA can become single-stranded in the course of normal replication and transcription as well as during DNA repair. Abnormal repair and replication intermediates can contain large stretches of persistent single-stranded DNA, which is extremely vulnerable to DNA damaging agents and hypermutation. Since such single-stranded DNA spans only a fraction of the genome at a given instance, hypermutation in these regions leads to tightly-spaced mutation clusters. This phenomenon of hypermutation in single-stranded DNA has been documented in several experimental models as well as in cancer genomes. Recently, hypermutated single-stranded RNA viral genomes also have been documented. Moreover, indications of hypermutation in single-stranded DNA may also be found in the human germline. This review will summarize key current knowledge and the recent developments in understanding the diverse mechanisms and sources of ssDNA hypermutation.
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Affiliation(s)
- Natalie Saini
- Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, US National Institutes of Health, Research Triangle Park, NC, USA
| | - Dmitry A Gordenin
- Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, US National Institutes of Health, Research Triangle Park, NC, USA.
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41
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Identifying Small Molecules That Promote Quasipalindrome-Associated Template-Switch Mutations in Escherichia coli. G3-GENES GENOMES GENETICS 2020; 10:1809-1815. [PMID: 32220953 PMCID: PMC7202029 DOI: 10.1534/g3.120.401106] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 11/18/2022]
Abstract
DNA can assemble into non-B form structures that stall replication and cause genomic instability. One such secondary structure results from an inverted DNA repeat that can assemble into hairpin and cruciform structures during DNA replication. Quasipalindromes (QP), imperfect inverted repeats, are sites of mutational hotspots. Quasipalindrome-associated mutations (QPMs) occur through a template-switch mechanism in which the replicative polymerase stalls at a QP site and uses the nascent strand as a template instead of the correct template strand. This mutational event causes the QP to become a perfect or more perfect inverted repeat. Since it is not fully understood how template-switch events are stimulated or repressed, we designed a high-throughput screen to discover drugs that affect these events. QP reporters were engineered in the Escherichia coli lacZ gene to allow us to study template-switch events specifically. We tested 700 compounds from the NIH Clinical Collection through a disk diffusion assay and identified 11 positive hits. One of the hits was azidothymidine (zidovudine, AZT), a thymidine analog and DNA chain terminator. The other ten were found to be fluoroquinolone antibiotics, which induce DNA-protein crosslinks. This work shows that our screen is useful in identifying small molecules that affect quasipalindrome-associated template-switch mutations. We are currently assessing more small molecule libraries and applying this method to study other types of mutations.
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42
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Timing of appearance of new mutations during yeast meiosis and their association with recombination. Curr Genet 2020; 66:577-592. [DOI: 10.1007/s00294-019-01051-0] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2019] [Revised: 12/26/2019] [Accepted: 12/29/2019] [Indexed: 01/18/2023]
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43
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Scully R, Panday A, Elango R, Willis NA. DNA double-strand break repair-pathway choice in somatic mammalian cells. Nat Rev Mol Cell Biol 2019; 20:698-714. [PMID: 31263220 PMCID: PMC7315405 DOI: 10.1038/s41580-019-0152-0] [Citation(s) in RCA: 935] [Impact Index Per Article: 155.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 05/23/2019] [Indexed: 11/09/2022]
Abstract
The major pathways of DNA double-strand break (DSB) repair are crucial for maintaining genomic stability. However, if deployed in an inappropriate cellular context, these same repair functions can mediate chromosome rearrangements that underlie various human diseases, ranging from developmental disorders to cancer. The two major mechanisms of DSB repair in mammalian cells are non-homologous end joining (NHEJ) and homologous recombination. In this Review, we consider DSB repair-pathway choice in somatic mammalian cells as a series of 'decision trees', and explore how defective pathway choice can lead to genomic instability. Stalled, collapsed or broken DNA replication forks present a distinctive challenge to the DSB repair system. Emerging evidence suggests that the 'rules' governing repair-pathway choice at stalled replication forks differ from those at replication-independent DSBs.
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Affiliation(s)
- Ralph Scully
- Department of Medicine, Division of Hematology-Oncology and Cancer Research Institute, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, USA.
| | - Arvind Panday
- Department of Medicine, Division of Hematology-Oncology and Cancer Research Institute, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, USA
| | - Rajula Elango
- Department of Medicine, Division of Hematology-Oncology and Cancer Research Institute, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, USA
| | - Nicholas A Willis
- Department of Medicine, Division of Hematology-Oncology and Cancer Research Institute, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, USA.
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44
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James TY, Michelotti LA, Glasco AD, Clemons RA, Powers RA, James ES, Simmons DR, Bai F, Ge S. Adaptation by Loss of Heterozygosity in Saccharomyces cerevisiae Clones Under Divergent Selection. Genetics 2019; 213:665-683. [PMID: 31371407 PMCID: PMC6781901 DOI: 10.1534/genetics.119.302411] [Citation(s) in RCA: 24] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/07/2019] [Accepted: 07/29/2019] [Indexed: 01/14/2023] Open
Abstract
Loss of heterozygosity (LOH) is observed during vegetative growth and reproduction of diploid genotypes through mitotic crossovers, aneuploidy caused by nondisjunction, and gene conversion. We aimed to test the role that LOH plays during adaptation of two highly heterozygous Saccharomyces cerevisiae genotypes to multiple environments over a short time span in the laboratory. We hypothesized that adaptation would be observed through parallel LOH events across replicate populations. Using genome resequencing of 70 clones, we found that LOH was widespread with 5.2 LOH events per clone after ∼500 generations. The most common mode of LOH was gene conversion (51%) followed by crossing over consistent with either break-induced replication or double Holliday junction resolution. There was no evidence that LOH involved nondisjunction of whole chromosomes. We observed parallel LOH in both an environment-specific and environment-independent manner. LOH largely involved recombining existing variation between the parental genotypes, but also was observed after de novo, presumably beneficial, mutations occurred in the presence of canavanine, a toxic analog of arginine. One highly parallel LOH event involved the ENA salt efflux pump locus on chromosome IV, which showed repeated LOH to the allele from the European parent, an allele originally derived by introgression from S. paradoxus Using CRISPR-engineered LOH we showed that the fitness advantage provided by this single LOH event was 27%. Overall, we found extensive evidence that LOH could be adaptive and is likely to be a greater source of initial variation than de novo mutation for rapid evolution of diploid genotypes.
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Affiliation(s)
- Timothy Y James
- Department of Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, Michigan 48109
| | - Lucas A Michelotti
- Department of Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, Michigan 48109
| | - Alexander D Glasco
- Department of Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, Michigan 48109
| | - Rebecca A Clemons
- Department of Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, Michigan 48109
| | - Robert A Powers
- Department of Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, Michigan 48109
| | - Ellen S James
- Department of Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, Michigan 48109
| | - D Rabern Simmons
- Department of Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, Michigan 48109
| | - Fengyan Bai
- Institute of Microbiology, Chinese Academy of Sciences, State Key Laboratory of Mycology, Chaoyang District, Beijing 100101, China
| | - Shuhua Ge
- Technology Development and Transfer Center, Institute of Microbiology, Chinese Academy of Sciences, Chaoyang District, Beijing 100029, China
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45
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DNA Rereplication Is Susceptible to Nucleotide-Level Mutagenesis. Genetics 2019; 212:445-460. [PMID: 31028114 PMCID: PMC6553831 DOI: 10.1534/genetics.119.302194] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2018] [Accepted: 04/15/2019] [Indexed: 12/12/2022] Open
Abstract
The initiation of eukaryotic DNA replication at replication origins is tightly regulated to prevent re-initiation and re-replication within each cell cycle. This regulation is critical for genome stability as re-replication is an extremely potent inducer... The sources of genome instability, a hallmark of cancer, remain incompletely understood. One potential source is DNA rereplication, which arises when the mechanisms that prevent the reinitiation of replication origins within a single cell cycle are compromised. Using the budding yeast Saccharomyces cerevisiae, we previously showed that DNA rereplication is extremely potent at inducing gross chromosomal alterations and that this arises in part because of the susceptibility of rereplication forks to break. Here, we examine the ability of DNA rereplication to induce nucleotide-level mutations. During normal replication these mutations are restricted by three overlapping error-avoidance mechanisms: the nucleotide selectivity of replicative polymerases, their proofreading activity, and mismatch repair. Using lys2InsEA14, a frameshift reporter that is poorly proofread, we show that rereplication induces up to a 30× higher rate of frameshift mutations and that this mutagenesis is due to passage of the rereplication fork, not secondary to rereplication fork breakage. Rereplication can also induce comparable rates of frameshift and base-substitution mutations in a more general mutagenesis reporter CAN1, when the proofreading activity of DNA polymerase ε is inactivated. Finally, we show that the rereplication-induced mutagenesis of both lys2InsEA14 and CAN1 disappears in the absence of mismatch repair. These results suggest that mismatch repair is attenuated during rereplication, although at most sequences DNA polymerase proofreading provides enough error correction to mitigate the mutagenic consequences. Thus, rereplication can facilitate nucleotide-level mutagenesis in addition to inducing gross chromosomal alterations, broadening its potential role in genome instability.
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46
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Arbel‐Eden A, Simchen G. Elevated Mutagenicity in Meiosis and Its Mechanism. Bioessays 2019; 41:e1800235. [DOI: 10.1002/bies.201800235] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/23/2018] [Revised: 01/31/2019] [Indexed: 12/25/2022]
Affiliation(s)
| | - Giora Simchen
- Department of GeneticsThe Hebrew University of JerusalemJerusalem 91904 Israel
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47
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Piazza A, Heyer WD. Homologous Recombination and the Formation of Complex Genomic Rearrangements. Trends Cell Biol 2019; 29:135-149. [PMID: 30497856 PMCID: PMC6402879 DOI: 10.1016/j.tcb.2018.10.006] [Citation(s) in RCA: 65] [Impact Index Per Article: 10.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2018] [Revised: 10/28/2018] [Accepted: 10/29/2018] [Indexed: 12/13/2022]
Abstract
The maintenance of genome integrity involves multiple independent DNA damage avoidance and repair mechanisms. However, the origin and pathways of the focal chromosomal reshuffling phenomena collectively referred to as chromothripsis remain mechanistically obscure. We discuss here the role, mechanisms, and regulation of homologous recombination (HR) in the formation of simple and complex chromosomal rearrangements. We emphasize features of the recently characterized multi-invasion (MI)-induced rearrangement (MIR) pathway which uniquely amplifies the initial DNA damage. HR intermediates and cellular contexts that endanger genomic stability are discussed as well as the emerging roles of various classes of nucleases in the formation of genome rearrangements. Long-read sequencing and improved mapping of repeats should enable better appreciation of the significance of recombination in generating genomic rearrangements.
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Affiliation(s)
- Aurèle Piazza
- Department of Microbiology and Molecular Genetics, University of California, Davis, CA 95616, USA; Spatial Regulation of Genomes, Department of Genomes and Genetics, Centre National de la Recherche Scientifique (CNRS) Unité Mixte de Recherche 3525, Institut Pasteur, 75015 Paris, France
| | - Wolf-Dietrich Heyer
- Department of Microbiology and Molecular Genetics, University of California, Davis, CA 95616, USA; Department of Molecular and Cellular Biology, University of California, Davis, CA 95616, USA.
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48
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Klein HL, Bačinskaja G, Che J, Cheblal A, Elango R, Epshtein A, Fitzgerald DM, Gómez-González B, Khan SR, Kumar S, Leland BA, Marie L, Mei Q, Miné-Hattab J, Piotrowska A, Polleys EJ, Putnam CD, Radchenko EA, Saada AA, Sakofsky CJ, Shim EY, Stracy M, Xia J, Yan Z, Yin Y, Aguilera A, Argueso JL, Freudenreich CH, Gasser SM, Gordenin DA, Haber JE, Ira G, Jinks-Robertson S, King MC, Kolodner RD, Kuzminov A, Lambert SAE, Lee SE, Miller KM, Mirkin SM, Petes TD, Rosenberg SM, Rothstein R, Symington LS, Zawadzki P, Kim N, Lisby M, Malkova A. Guidelines for DNA recombination and repair studies: Cellular assays of DNA repair pathways. MICROBIAL CELL (GRAZ, AUSTRIA) 2019; 6:1-64. [PMID: 30652105 PMCID: PMC6334234 DOI: 10.15698/mic2019.01.664] [Citation(s) in RCA: 37] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/24/2018] [Revised: 08/29/2018] [Accepted: 09/14/2018] [Indexed: 12/29/2022]
Abstract
Understanding the plasticity of genomes has been greatly aided by assays for recombination, repair and mutagenesis. These assays have been developed in microbial systems that provide the advantages of genetic and molecular reporters that can readily be manipulated. Cellular assays comprise genetic, molecular, and cytological reporters. The assays are powerful tools but each comes with its particular advantages and limitations. Here the most commonly used assays are reviewed, discussed, and presented as the guidelines for future studies.
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Affiliation(s)
- Hannah L. Klein
- Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY, USA
| | - Giedrė Bačinskaja
- Department of Biology, University of Copenhagen, DK-2200 Copenhagen N, Denmark
| | - Jun Che
- Department of Radiation Oncology, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX, USA
| | - Anais Cheblal
- Friedrich Miescher Institute for Biomedical Research (FMI), 4058 Basel, Switzerland
| | - Rajula Elango
- Department of Biology, University of Iowa, Iowa City, IA, USA
| | - Anastasiya Epshtein
- Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY, USA
| | - Devon M. Fitzgerald
- Dan L Duncan Comprehensive Cancer Center, Baylor College of Medicine, Houston, TX, USA
- Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA
- Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX, USA
- Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX, USA
| | - Belén Gómez-González
- Centro Andaluz de BIología Molecular y Medicina Regenerativa-CABIMER, Universidad de Sevilla, Seville, Spain
| | - Sharik R. Khan
- Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, IL, USA
| | - Sandeep Kumar
- Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA
| | | | - Léa Marie
- Department of Microbiology and Immunology, Columbia University Medical Center, New York, NY, USA
| | - Qian Mei
- Systems, Synthetic and Physical Biology Graduate Program, Rice University, Houston, TX, USA
| | - Judith Miné-Hattab
- Institut Curie, PSL Research University, CNRS, UMR3664, F-75005 Paris, France
- Sorbonne Université, Institut Curie, CNRS, UMR3664, F-75005 Paris, France
| | - Alicja Piotrowska
- NanoBioMedical Centre, Faculty of Physics, Adam Mickiewicz University, Umultowska 85, 61-614 Poznan, Poland
| | | | - Christopher D. Putnam
- Ludwig Institute for Cancer Research, University of California School of Medicine, San Diego, La Jolla, CA, USA
- Department of Medicine, University of California School of Medicine, San Diego, La Jolla, CA, USA
| | | | - Anissia Ait Saada
- Institut Curie, PSL Research University, CNRS, UMR3348 F-91405, Orsay, France
- University Paris Sud, Paris-Saclay University, CNRS, UMR3348, F-91405, Orsay, France
| | - Cynthia J. Sakofsky
- Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, Durham, NC, USA
| | - Eun Yong Shim
- Department of Radiation Oncology, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX, USA
| | - Mathew Stracy
- Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK
| | - Jun Xia
- Dan L Duncan Comprehensive Cancer Center, Baylor College of Medicine, Houston, TX, USA
- Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA
- Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX, USA
- Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX, USA
| | - Zhenxin Yan
- Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA
| | - Yi Yin
- Department of Molecular Genetics and Microbiology and University Program in Genetics and Genomics, Duke University Medical Center, Durham, NC USA
| | - Andrés Aguilera
- Centro Andaluz de BIología Molecular y Medicina Regenerativa-CABIMER, Universidad de Sevilla, Seville, Spain
| | - Juan Lucas Argueso
- Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, CO, USA
| | - Catherine H. Freudenreich
- Department of Biology, Tufts University, Medford, MA USA
- Program in Genetics, Tufts University, Boston, MA, USA
| | - Susan M. Gasser
- Friedrich Miescher Institute for Biomedical Research (FMI), 4058 Basel, Switzerland
| | - Dmitry A. Gordenin
- Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, Durham, NC, USA
| | - James E. Haber
- Department of Biology and Rosenstiel Basic Medical Sciences Research Center Brandeis University, Waltham, MA, USA
| | - Grzegorz Ira
- Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA
| | - Sue Jinks-Robertson
- Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC USA
| | | | - Richard D. Kolodner
- Ludwig Institute for Cancer Research, University of California School of Medicine, San Diego, La Jolla, CA, USA
- Department of Cellular and Molecular Medicine, University of California School of Medicine, San Diego, La Jolla, CA, USA
- Moores-UCSD Cancer Center, University of California School of Medicine, San Diego, La Jolla, CA, USA
- Institute of Genomic Medicine, University of California School of Medicine, San Diego, La Jolla, CA, USA
| | - Andrei Kuzminov
- Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, IL, USA
| | - Sarah AE Lambert
- Institut Curie, PSL Research University, CNRS, UMR3348 F-91405, Orsay, France
- University Paris Sud, Paris-Saclay University, CNRS, UMR3348, F-91405, Orsay, France
| | - Sang Eun Lee
- Department of Radiation Oncology, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX, USA
| | - Kyle M. Miller
- Dan L Duncan Comprehensive Cancer Center, Baylor College of Medicine, Houston, TX, USA
- Department of Molecular Biosciences, University of Texas at Austin, Austin, TX, USA
| | | | - Thomas D. Petes
- Department of Molecular Genetics and Microbiology and University Program in Genetics and Genomics, Duke University Medical Center, Durham, NC USA
| | - Susan M. Rosenberg
- Dan L Duncan Comprehensive Cancer Center, Baylor College of Medicine, Houston, TX, USA
- Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA
- Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX, USA
- Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX, USA
- Systems, Synthetic and Physical Biology Graduate Program, Rice University, Houston, TX, USA
| | - Rodney Rothstein
- Department of Genetics & Development, Columbia University Irving Medical Center, New York, NY, USA
| | - Lorraine S. Symington
- Department of Microbiology and Immunology, Columbia University Medical Center, New York, NY, USA
| | - Pawel Zawadzki
- NanoBioMedical Centre, Faculty of Physics, Adam Mickiewicz University, Umultowska 85, 61-614 Poznan, Poland
| | - Nayun Kim
- Department of Microbiology and Molecular Genetics, The University of Texas Health Science Center at Houston, Houston, TX, USA
| | - Michael Lisby
- Department of Biology, University of Copenhagen, DK-2200 Copenhagen N, Denmark
| | - Anna Malkova
- Department of Biology, University of Iowa, Iowa City, IA, USA
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49
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Kaiser VB, Semple CA. Chromatin loop anchors are associated with genome instability in cancer and recombination hotspots in the germline. Genome Biol 2018; 19:101. [PMID: 30060743 PMCID: PMC6066925 DOI: 10.1186/s13059-018-1483-4] [Citation(s) in RCA: 44] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/14/2017] [Accepted: 07/13/2018] [Indexed: 01/07/2023] Open
Abstract
Background Chromatin loops form a basic unit of interphase nuclear organization, with chromatin loop anchor points providing contacts between regulatory regions and promoters. However, the mutational landscape at these anchor points remains under-studied. Here, we describe the unusual patterns of somatic mutations and germline variation associated with loop anchor points and explore the underlying features influencing these patterns. Results Analyses of whole genome sequencing datasets reveal that anchor points are strongly depleted for single nucleotide variants (SNVs) in tumours. Despite low SNV rates in their genomic neighbourhood, anchor points emerge as sites of evolutionary innovation, showing enrichment for structural variant (SV) breakpoints and a peak of SNVs at focal CTCF sites within the anchor points. Both CTCF-bound and non-CTCF anchor points harbour an excess of SV breakpoints in multiple tumour types and are prone to double-strand breaks in cell lines. Common fragile sites, which are hotspots for genome instability, also show elevated numbers of intersecting loop anchor points. Recurrently disrupted anchor points are enriched for genes with functions in cell cycle transitions and regions associated with predisposition to cancer. We also discover a novel class of CTCF-bound anchor points which overlap meiotic recombination hotspots and are enriched for the core PRDM9 binding motif, suggesting that the anchor points have been foci for diversity generated during recent human evolution. Conclusions We suggest that the unusual chromatin environment at loop anchor points underlies the elevated rates of variation observed, marking them as sites of regulatory importance but also genomic fragility. Electronic supplementary material The online version of this article (10.1186/s13059-018-1483-4) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Vera B Kaiser
- MRC Human Genetics Unit, MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Western General Hospital, Crewe Road, Edinburgh, EH4 2XU, UK.
| | - Colin A Semple
- MRC Human Genetics Unit, MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Western General Hospital, Crewe Road, Edinburgh, EH4 2XU, UK
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Brinkman EK, Chen T, de Haas M, Holland HA, Akhtar W, van Steensel B. Kinetics and Fidelity of the Repair of Cas9-Induced Double-Strand DNA Breaks. Mol Cell 2018; 70:801-813.e6. [PMID: 29804829 PMCID: PMC5993873 DOI: 10.1016/j.molcel.2018.04.016] [Citation(s) in RCA: 175] [Impact Index Per Article: 25.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/26/2017] [Revised: 01/29/2018] [Accepted: 04/18/2018] [Indexed: 12/26/2022]
Abstract
The RNA-guided DNA endonuclease Cas9 is a powerful tool for genome editing. Little is known about the kinetics and fidelity of the double-strand break (DSB) repair process that follows a Cas9 cutting event in living cells. Here, we developed a strategy to measure the kinetics of DSB repair for single loci in human cells. Quantitative modeling of repaired DNA in time series after Cas9 activation reveals variable and often slow repair rates, with half-life times up to ∼10 hr. Furthermore, repair of the DSBs tends to be error prone. Both classical and microhomology-mediated end joining pathways contribute to the erroneous repair. Estimation of their individual rate constants indicates that the balance between these two pathways changes over time and can be altered by additional ionizing radiation. Our approach provides quantitative insights into DSB repair kinetics and fidelity in single loci and indicates that Cas9-induced DSBs are repaired in an unusual manner.
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Affiliation(s)
- Eva K Brinkman
- Oncode Institute; Division of Gene Regulation, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, the Netherlands
| | - Tao Chen
- Division of Gene Regulation, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, the Netherlands
| | - Marcel de Haas
- Oncode Institute; Division of Gene Regulation, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, the Netherlands
| | - Hanna A Holland
- Division of Gene Regulation, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, the Netherlands
| | - Waseem Akhtar
- Division of Molecular Genetics, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, the Netherlands
| | - Bas van Steensel
- Oncode Institute; Division of Gene Regulation, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, the Netherlands.
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