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1
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Brown NK, Merkens H, Rozemuller EH, Bell D, Bui TM, Kearns J. Reduced PCR-generated errors from a hybrid capture-based NGS assay for HLA typing. Hum Immunol 2021; 82:296-301. [PMID: 33676750 DOI: 10.1016/j.humimm.2021.02.010] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2020] [Revised: 02/17/2021] [Accepted: 02/18/2021] [Indexed: 11/28/2022]
Abstract
Next generation sequencing (NGS) assays are state of the art for HLA genotyping. To sequence on an Illumina sequencer, the DNA of interest must be enriched, fragmented, and bookended with known oligonucleotide sequences, a process known as library construction. Many HLA genotyping assays enrich the target loci by long-range PCR (LR-PCR), prior to fragmentation. This PCR step has been reported to introduce errors in the DNA to be sequenced, including inaccurate replication of repeated sequences, and the in vitro recombination of alleles encoded on separate chromosomes. An alternative library construction method involves fragmentation of genomic DNA, followed by hybrid-capture (HC) enrichment of target HLA loci. This HC-based method involves PCR, but with far fewer cycles. Consequently, the HC method had significantly fewer PCR-induced errors, including more faithful replication of repeated sequences, and the near elimination of recombinant sequences. These improvements likely produce more accurate NGS sequencing data of HLA loci.
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Affiliation(s)
- Nicholas K Brown
- Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, United States.
| | | | | | - Derrick Bell
- Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, United States
| | - Thanh-Mai Bui
- Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, United States
| | - Jane Kearns
- Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, United States
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2
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Khristich AN, Mirkin SM. On the wrong DNA track: Molecular mechanisms of repeat-mediated genome instability. J Biol Chem 2020; 295:4134-4170. [PMID: 32060097 PMCID: PMC7105313 DOI: 10.1074/jbc.rev119.007678] [Citation(s) in RCA: 188] [Impact Index Per Article: 37.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
Expansions of simple tandem repeats are responsible for almost 50 human diseases, the majority of which are severe, degenerative, and not currently treatable or preventable. In this review, we first describe the molecular mechanisms of repeat-induced toxicity, which is the connecting link between repeat expansions and pathology. We then survey alternative DNA structures that are formed by expandable repeats and review the evidence that formation of these structures is at the core of repeat instability. Next, we describe the consequences of the presence of long structure-forming repeats at the molecular level: somatic and intergenerational instability, fragility, and repeat-induced mutagenesis. We discuss the reasons for gender bias in intergenerational repeat instability and the tissue specificity of somatic repeat instability. We also review the known pathways in which DNA replication, transcription, DNA repair, and chromatin state interact and thereby promote repeat instability. We then discuss possible reasons for the persistence of disease-causing DNA repeats in the genome. We describe evidence suggesting that these repeats are a payoff for the advantages of having abundant simple-sequence repeats for eukaryotic genome function and evolvability. Finally, we discuss two unresolved fundamental questions: (i) why does repeat behavior differ between model systems and human pedigrees, and (ii) can we use current knowledge on repeat instability mechanisms to cure repeat expansion diseases?
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Affiliation(s)
| | - Sergei M Mirkin
- Department of Biology, Tufts University, Medford, Massachusetts 02155.
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3
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van Deutekom HW, Mulder W, Rozemuller EH. Accuracy of NGS HLA typing data influenced by STR. Hum Immunol 2019; 80:461-464. [DOI: 10.1016/j.humimm.2019.03.007] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2018] [Revised: 03/06/2019] [Accepted: 03/06/2019] [Indexed: 11/30/2022]
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4
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Castillo-Lizardo M, Henneke G, Viguera E. Replication slippage of the thermophilic DNA polymerases B and D from the Euryarchaeota Pyrococcus abyssi. Front Microbiol 2014; 5:403. [PMID: 25177316 PMCID: PMC4134008 DOI: 10.3389/fmicb.2014.00403] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2014] [Accepted: 07/17/2014] [Indexed: 11/30/2022] Open
Abstract
Replication slippage or slipped-strand mispairing involves the misalignment of DNA strands during the replication of repeated DNA sequences, and can lead to genetic rearrangements such as microsatellite instability. Here, we show that PolB and PolD replicative DNA polymerases from the archaeal model Pyrococcus abyssi (Pab) slip in vitro during replication of a single-stranded DNA template carrying a hairpin structure and short direct repeats. We find that this occurs in both their wild-type (exo+) and exonuclease deficient (exo-) forms. The slippage behavior of PabPolB and PabPolD, probably due to limited strand displacement activity, resembles that observed for the high fidelity P. furiosus (Pfu) DNA polymerase. The presence of PabPCNA inhibited PabPolB and PabPolD slippage. We propose a model whereby PabPCNA stimulates strand displacement activity and polymerase progression through the hairpin, thus permitting the error-free replication of repetitive sequences.
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Affiliation(s)
- Melissa Castillo-Lizardo
- Departamento de Biología Celular, Genética y Fisiología, Facultad de Ciencias, Universidad de Malaga Málaga, Spain
| | - Ghislaine Henneke
- Laboratoire de Microbiologie des Environnements Extrêmes, UMR 6197, Institut Français de Recherche pour l'Exploitation de la Mer, Université de Bretagne Occidentale Plouzané, France ; CNRS, UMR 6197, Laboratoire de Microbiologie des Environnements Extrêmes Plouzané, France
| | - Enrique Viguera
- Departamento de Biología Celular, Genética y Fisiología, Facultad de Ciencias, Universidad de Malaga Málaga, Spain
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Aza A, Martin MJ, Juarez R, Blanco L, Terrados G. DNA expansions generated by human Polμ on iterative sequences. Nucleic Acids Res 2012; 41:253-63. [PMID: 23143108 PMCID: PMC3592450 DOI: 10.1093/nar/gks1054] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022] Open
Abstract
Polµ is the only DNA polymerase equipped with template-directed and terminal transferase activities. Polµ is also able to accept distortions in both primer and template strands, resulting in misinsertions and extension of realigned mismatched primer terminus. In this study, we propose a model for human Polµ-mediated dinucleotide expansion as a function of the sequence context. In this model, Polµ requires an initial dislocation, that must be subsequently stabilized, to generate large sequence expansions at different 5′-P-containing DNA substrates, including those that mimic non-homologous end-joining (NHEJ) intermediates. Our mechanistic studies point at human Polµ residues His329 and Arg387 as responsible for regulating nucleotide expansions occurring during DNA repair transactions, either promoting or blocking, respectively, iterative polymerization. This is reminiscent of the role of both residues in the mechanism of terminal transferase activity. The iterative synthesis performed by Polµ at various contexts may lead to frameshift mutations producing DNA damage and instability, which may end in different human disorders, including cancer or congenital abnormalities.
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Affiliation(s)
- Ana Aza
- Centro de Biologia Molecular Severo Ochoa, CSIC-UAM, 28049 Madrid, Spain
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6
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Kato T, Liang X, Asanuma H. Model of elongation of short DNA sequence by thermophilic DNA polymerase under isothermal conditions. Biochemistry 2012; 51:7846-53. [PMID: 22992125 DOI: 10.1021/bi3010413] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Abstract
Short DNA sequences, especially those that are repetitive or palindromic, can be used as the seeds for synthesis of long DNA by some DNA polymerases in an unusual manner. Although several elongation mechanisms have been proposed, there is no well-established model that explains highly efficient elongation under isothermal conditions. In the present study, we analyzed the elongation of nonrepetitive sequences with distinct hairpins at each end. These DNAs were elongated efficiently under isothermal conditions by thermophilic Vent (exo(-)) DNA polymerase, and the products were longer than 10 kb within 10 min of the reaction. A 20-nucleotide DNA with only one hairpin was also elongated. Sequence analysis revealed that the long products are mainly tandem repeats of the short seed sequences. The thermal melting temperatures of the products were much higher than the reaction temperature, indicating that most DNAs form duplexes during the reaction. Accordingly, a terminal hairpin formation and self-priming extension model was proposed in detail, and the efficient elongation was explained. Formation of the hairpin at the 5' end plays an important role during the elongation.
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Affiliation(s)
- Tomohiro Kato
- Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa, Nagoya 464-8603, Japan
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7
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Pan X, Liao Y, Liu Y, Chang P, Liao L, Yang L, Li H. Transcription of AAT•ATT triplet repeats in Escherichia coli is silenced by H-NS and IS1E transposition. PLoS One 2010; 5:e14271. [PMID: 21151567 PMCID: PMC3000339 DOI: 10.1371/journal.pone.0014271] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/23/2010] [Accepted: 11/15/2010] [Indexed: 11/18/2022] Open
Abstract
Background The trinucleotide repeats AAT•ATT are simple DNA sequences that potentially form different types of non-B DNA secondary structures and cause genomic instabilities in vivo. Methodology and Principal Findings The molecular mechanism underlying the maintenance of a 24-triplet AAT•ATT repeat was examined in E.coli by cloning the repeats into the EcoRI site in plasmid pUC18 and into the attB site on the E.coli genome. Either the AAT or the ATT strand acted as lagging strand template in a replication fork. Propagations of the repeats in either orientation on plasmids did not affect colony morphology when triplet repeat transcription using the lacZ promoter was repressed either by supplementing LacIQin trans or by adding glucose into the medium. In contrast, transparent colonies were formed by inducing transcription of the repeats, suggesting that transcription of AAT•ATT repeats was toxic to cell growth. Meanwhile, significant IS1E transposition events were observed both into the triplet repeats region proximal to the promoter side, the promoter region of the lacZ gene, and into the AAT•ATT region itself. Transposition reversed the transparent colony phenotype back into healthy, convex colonies. In contrast, transcription of an 8-triplet AAT•ATT repeat in either orientation on plasmids did not produce significant changes in cell morphology and did not promote IS1E transposition events. We further found that a role of IS1E transposition into plasmids was to inhibit transcription through the repeats, which was influenced by the presence of the H-NS protein, but not of its paralogue StpA. Conclusions and Significance Our findings thus suggest that the longer AAT•ATT triplet repeats in E.coli become vulnerable after transcription. H-NS and its facilitated IS1E transposition can silence long triplet repeats transcription and preserve cell growth and survival.
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Affiliation(s)
- Xuefeng Pan
- School of Life Science, Beijing Institute of Technology, Beijing, China.
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8
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A bulge binding agent with novel wedge-shape topology for stimulation of DNA triplet repeat strand slippage synthesis. Bioorg Med Chem Lett 2008; 18:6184-8. [DOI: 10.1016/j.bmcl.2008.10.038] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/29/2008] [Revised: 09/25/2008] [Accepted: 10/01/2008] [Indexed: 11/15/2022]
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9
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Ouyang D, Yi L, Liu L, Mu HT, Xi Z. In vitro expansion of DNA triplet repeats with bulge binders and different DNA polymerases. FEBS J 2008; 275:4510-21. [DOI: 10.1111/j.1742-4658.2008.06593.x] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
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10
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Liu L, Yi L, Yang X, Yu Z, Wen X, Xi Z. Synthesis and spectroscopic characterization of binaphthol aminosugars for stimulation of DNA strand slippage synthesis. Tetrahedron 2008. [DOI: 10.1016/j.tet.2008.04.047] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/22/2022]
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11
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Vondrusková J, Parízková N, Kypr J. Factors influencing DNA expansion in the course of polymerase chain reaction. NUCLEOSIDES NUCLEOTIDES & NUCLEIC ACIDS 2007; 26:65-82. [PMID: 17162588 DOI: 10.1080/15257770601052299] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 10/23/2022]
Abstract
We performed more than 3,500 polymerase chain reactions (PCRs) under various conditions with more than 400 DNA fragments of 4-150 nucleotides in length. Some of the PCRs provided expanded DNA molecules of kilobase lengths whereas others led to no expansion. Repetitiveness of the primary structure was mostly found to be necessary but not sufficient for the expansion. (A+T)-rich fragments expand better than (G+C)-rich ones and pyrimidine-rich fragments expand better than purine-rich fragments. Terminal nucleotides and the fragment length also are important for the expansion. Examples are presented when relatively small alterations of the DNA primary structure caused a dramatic change in the expansion. For example, A8T8 expanded a lot whereas T8A8 did not expand at all. The present work has implications for pathological expansions of microsatellites in the human genome as well as regarding the genome evolution in general.
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Affiliation(s)
- Jitka Vondrusková
- Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic
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12
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Brylawski BP, Chastain PD, Cohen SM, Cordeiro-Stone M, Kaufman DG. Mapping of an origin of DNA replication in the promoter of fragile X gene FMR1. Exp Mol Pathol 2006; 82:190-6. [PMID: 17196195 PMCID: PMC1934615 DOI: 10.1016/j.yexmp.2006.10.004] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2006] [Revised: 10/20/2006] [Accepted: 10/20/2006] [Indexed: 09/30/2022]
Abstract
An origin of bidirectional DNA replication was mapped to the promoter of the FMR1 gene in human chromosome Xq27.3, which has been linked to the fragile X syndrome. This origin is adjacent to a CpG island and overlaps the site of expansion of the triplet repeat (CGG) at the fragile X instability site, FRAXA. The promoter region of FMR2 in the FRAXE site (approximately 600 kb away, in chromosome band Xq28) also includes an origin of replication, as previously described [Chastain II, P.D., Cohen, S.M., Brylawski, B.P., Cordeiro-Stone, M., Kaufman, D.G., 2006. A late origin of DNA replication in the trinucleotide repeat region of the human FMR2 gene. Cell Cycle 5, 869-872]. FMR1 transcripts were detected in foreskin and male fetal lung fibroblasts, while FMR2 transcripts were not. However, both FMR1 and FMR2 were found to replicate late in S phase (approximately 6 h into the S phase of normal human fibroblasts). The position of the origin of replication relative to the CGG repeat, and perhaps the late replication of these genes, might be important factors in the susceptibility to triplet repeat amplification at the FRAXA and FRAXE sites.
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Affiliation(s)
- Bruna P Brylawski
- Department of Pathology and Laboratory Medicine, C.B. #7525, University of North Carolina, Chapel Hill, NC 27599-7525, USA.
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13
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Trotta E, Del Grosso N, Erba M, Melino S, Cicero D, Paci M. Interaction of DAPI with individual strands of trinucleotide repeats. Effects of replication in vitro of the AAT x ATT triplet. ACTA ACUST UNITED AC 2004; 270:4755-61. [PMID: 14622264 DOI: 10.1046/j.1432-1033.2003.03877.x] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
The structural changes produced by the minor-groove binding ligand DAPI (4',6-diamidine-2-phenylindole) on individual strands of trinucleotide repeat sequences were detected by electrophoretic band-shift analysis and related to their effects on DNA replication in vitro. Among the 20 possible single-stranded trinucleotide repeats, only the T-rich strand of the AAT.ATT triplet exhibits an observable fluorescence band and a change in electrophoretic mobility due to the drug binding. This is attributable to the property of DAPI that favours folding of the random coil ATT strand into a fast-migrating hairpin structure by a minor-groove binding mechanism. Electrophoretic characteristics of AAT, ACT, AGT, ATG and ATC are unchanged by DAPI, suggesting the crucial role of T.T with respect to A.A, C.C and G.G mismatch, in favouring the binding properties and the structural features of the ATT-DAPI complexes. Primer extension experiments, using the Klenow fragment of DNA polymerase I, demonstrate that such a selective structural change at ATT targets presents a marked property to stall DNA replication in vitro in comparison with the complementary AAT and a random GC-rich sequence. The results suggest a novel molecular mechanism of action of the DNA minor-groove binding ligand DAPI.
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Affiliation(s)
- Edoardo Trotta
- Istituto di Neurobiologia e Medicina Molecolare, Consiglio Nazionale delle Ricerche, Roma, Italy.
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14
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Heidenfelder BL, Makhov AM, Topal MD. Hairpin formation in Friedreich's ataxia triplet repeat expansion. J Biol Chem 2003; 278:2425-31. [PMID: 12441336 DOI: 10.1074/jbc.m210643200] [Citation(s) in RCA: 54] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Triplet repeat tracts occur throughout the human genome. Expansions of a (GAA)(n)/(TTC)(n) repeat tract during its transmission from parent to child are tightly associated with the occurrence of Friedreich's ataxia. Evidence supports DNA slippage during DNA replication as the cause of the expansions. DNA slippage results in single-stranded expansion intermediates. Evidence has accumulated that predicts that hairpin structures protect from DNA repair the expansion intermediates of all of the disease-associated repeats except for those of Friedreich's ataxia. How the latter repeat expansions avoid repair remains a mystery because (GAA)(n) and (TTC)(n) repeats are reported not to self-anneal. To characterize the Friedreich's ataxia intermediates, we generated massive expansions of (GAA)(n) and (TTC)(n) during DNA replication in vitro using human polymerase beta and the Klenow fragment of Escherichia coli polymerase I. Electron microscopy, endonuclease cleavage, and DNA sequencing of the expansion products demonstrate, for the first time, the occurrence of large and growing (GAA)(n) and (TTC)(n) hairpins during DNA synthesis. The results provide unifying evidence that predicts that hairpin formation during DNA synthesis mediates all of the disease-associated, triplet repeat expansions.
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Affiliation(s)
- Brooke L Heidenfelder
- Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, 27599-7295, USA
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15
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Clarke LA, Rebelo CS, Gonçalves J, Boavida MG, Jordan P. PCR amplification introduces errors into mononucleotide and dinucleotide repeat sequences. Mol Pathol 2001; 54:351-3. [PMID: 11577179 PMCID: PMC1187094 DOI: 10.1136/mp.54.5.351] [Citation(s) in RCA: 93] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/03/2022]
Abstract
The polymerase chain reaction (PCR) is used universally for accurate exponential amplification of DNA. We describe a high error rate at mononucleotide and dinucleotide repeat sequence motifs. Subcloning of PCR products allowed sequence analysis of individual DNA molecules from the product pool and revealed that: (1) monothymidine repeats longer than 11 bp are amplified with decreasing accuracy, (2) repeats generally contract during PCR because of the loss of repeat units, (3) Taq and proofreading polymerase Pfu generate similar errors at mononucleotide and dinucleotide repeats, and (4) unlike the parent PCR product pool, individual clones containing a single repeat length produce no "shadow bands". These data demonstrate that routine PCR amplification alters mononucleotide and dinucleotide repeat lengths. Such sequences are common components of genetic markers, disease genes, and intronic splicing motifs, and the amplification errors described here can be mistaken for polymorphisms or mutations.
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Affiliation(s)
- L A Clarke
- Centro de Genética Humana, Instituto Nacional de Saúde Dr. Ricardo Jorge, Avenida Padre Cruz, 1649-016 Lisboa, Portugal
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16
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Bowater RP, Wells RD. The intrinsically unstable life of DNA triplet repeats associated with human hereditary disorders. PROGRESS IN NUCLEIC ACID RESEARCH AND MOLECULAR BIOLOGY 2001; 66:159-202. [PMID: 11051764 DOI: 10.1016/s0079-6603(00)66029-4] [Citation(s) in RCA: 84] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
Expansions of specific DNA triplet repeats are the cause of an increasing number of hereditary neurological disorders in humans. In some diseases, such as Huntington's and several spinocerebellar ataxias, the repetitive DNA sequences are translated into long tracts of the same amino acid (usually glutamine), which alters interactions with cellular constituents and leads to the development of disease. For other disorders, including common genetic disorders such as myotonic dystrophy and fragile X syndrome, the DNA repeat is located in noncoding regions of transcribed sequences and disease is probably caused by altered gene expression. In studies in lower organisms, mammalian cells, and transgenic mice, high frequencies of length changes (increases and decreases) occur in long DNA triplet repeats. These observations are similar to other types of repetitive DNA sequences, which also undergo frequent length changes at genomic loci. A variety of processes acting on DNA influence the genetic stability of DNA triplet repeats, including replication, recombination, repair, and transcription. It is not yet known how these different multienzyme systems interact to produce the genetic mutation of expanded repeats. In vitro studies have identified that DNA triplet repeats can adopt several unusual DNA structures, including hairpins, triplexes, quadruplexes, slipped structures, and highly flexible and writhed helices. The formation of stable unusual structures within the cell is likely to disturb DNA metabolism and be a critical intermediate in the molecular mechanism(s) leading to genetic instabilities of DNA repeats and, hence, to disease pathogenesis.
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Affiliation(s)
- R P Bowater
- Molecular Biology Sector, School of Biological Sciences, University of East Anglia, Norwich, United Kingdom
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17
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da Silva EF, Reha-Krantz LJ. Dinucleotide repeat expansion catalyzed by bacteriophage T4 DNA polymerase in vitro. J Biol Chem 2000; 275:31528-35. [PMID: 10924513 DOI: 10.1074/jbc.m004594200] [Citation(s) in RCA: 21] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
DNA replication normally occurs with high fidelity, but certain "slippery" regions of DNA with tracts of mono-, di-, and trinucleotide repeats are frequently mutation hot spots. We have developed an in vitro assay to study the mechanism of dinucleotide repeat expansion. The primer-template resembles a base excision repair substrate with a single nucleotide gap centered opposite a tract of nine CA repeats; nonrepeat sequences flank the dinucleotide repeats. DNA polymerases are expected to repair the gap, but further extension is possible if the DNA polymerase can displace the downstream oligonucleotide. We report here that the wild type bacteriophage T4 DNA polymerase carries out gap and strand displacement replication and also catalyzes a dinucleotide expansion reaction. Repeat expansion was not detected for an exonuclease-deficient T4 DNA polymerase or for Escherichia coli DNA polymerase I. The dinucleotide repeat expansion reaction catalyzed by wild type T4 DNA polymerase required a downstream oligonucleotide to "stall" replication and 3' --> 5' exonuclease activity to remove the 3'-nonrepeat sequence adjacent to the repeat tract in the template strand. These results suggest that dinucleotide repeat expansion may be stimulated in vivo during DNA repair or during processing of Okazaki fragments.
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Affiliation(s)
- E F da Silva
- Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6G 2E9, Canada
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18
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Nozawa K, Suzuki M, Takemura M, Yoshida S. In vitro expansion of mammalian telomere repeats by DNA polymerase alpha-primase. Nucleic Acids Res 2000; 28:3117-24. [PMID: 10931927 PMCID: PMC108427 DOI: 10.1093/nar/28.16.3117] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
Among the polymerases, DNA polymerase alpha-primase is involved in lagging strand DNA synthesis. A previous report indicated that DNA polymerase alpha-primase initiates primer RNA synthesis with purine bases on a single-stranded G-rich telomere repeat. In this study, we found that DNA polymerase alpha-primase precisely initiated with adenosine opposite the 3'-side thymidine in the G-rich telomere repeat 5'-(TTAGGG)(n)-3' under rATP-rich conditions. Then, DNA polymerase alpha-primase synthesized the nascent DNA fragments by extending the primer. It was remarkable that DNA polymerase alpha-primase further expanded the product DNA far beyond the length of the template DNA, as ladders of multiple hexanucleotides on polyacrylamide gel electrophoresis. Using an oligomer duplex 5'-A(GGGTTA)(5)-3'/5'-(TAACCC)(5)T-3' as a template-primer, we show that both the Klenow fragment of Escherichia coli DNA polymerase I and HIV reverse transcriptase could expand telomere DNA sequences as well, giving products greater than the size of the template DNA. The maximum product lengths with these polymerases were approximately 40-90 nt longer than the template length. Our data imply that DNA polymerases have an intrinsic activity to expand the hexanucleotide repeats of the telomere sequence by a slippage mechanism and that DNA polymerase alpha uses both the repeat DNA primers and the de novo RNA primers for expansion. On the other hand, a plasmid harboring a eukaryotic telomere repeat showed remarkable genetic instability in E.coli. The telomere repeats exhibited either expansions or deletions by multiple hexanucleotide repeats during culture for a number of generations, suggesting involvement of the slippage mechanism in the instability of telomeric DNA in vivo.
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Affiliation(s)
- K Nozawa
- Laboratory of Cancer Cell Biology, Research Institute for Disease Mechanism and Control, Nagoya University School of Medicine, Tsurumai-cho 65, Showa-ku, Nagoya 466-8550, Japan
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19
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Lyons-Darden T, Topal MD. Abasic sites induce triplet-repeat expansion during DNA replication in vitro. J Biol Chem 1999; 274:25975-8. [PMID: 10473539 DOI: 10.1074/jbc.274.37.25975] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The occurrence of triplet-repeat expansion (TRE) during transmission of genetic information is involved in many neurological and neuromuscular diseases including Fragile X syndrome and myotonic dystrophy. DNA slippage during replicative synthesis appears to cause TRE. The causes of DNA slippage, however, remain mostly unknown. We investigated the effects of abasic sites on the occurrence of TRE during DNA replication in vitro using Escherichia coli Klenow polymerase I as the model polymerase. Here we show that a single abasic site analog, synthesized in the triplet-repeat tract at the 5' end of the template strand, induced dramatic TRE during DNA synthesis. The amount of TRE induced decreased when the abasic site was moved to the middle of the repeat tract, consistent with effectively decreasing the length of the repeat tract. Placing the abasic site in the primer did not induce TRE. TRE was sequence-dependent. The damage-induced increase in growing strand TRE depended on the sequence of the growing strand repeat as AAT approximately ATT > CAG > CTG. The expansions required replication from a primer complementary to the repeat tract. The expanded tracts were sequenced and contained multiple additions of the original repeat. The results imply that DNA damage can play a significant role in generating TRE in vivo.
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Affiliation(s)
- T Lyons-Darden
- Lineberger Comprehensive Cancer Center and Department of Pathology, University of North Carolina Medical School, Chapel Hill, North Carolina 27599-7295, USA
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