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1
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Trasviña-Arenas CH, Dissanayake UC, Tamayo N, Hashemian M, Lin WJ, Demir M, Hoyos-Gonzalez N, Fisher AJ, Cisneros GA, Horvath MP, David SS. Structure of human MUTYH and functional profiling of cancer-associated variants reveal an allosteric network between its [4Fe-4S] cluster cofactor and active site required for DNA repair. Nat Commun 2025; 16:3596. [PMID: 40234396 PMCID: PMC12000561 DOI: 10.1038/s41467-025-58361-w] [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: 10/04/2024] [Accepted: 03/20/2025] [Indexed: 04/17/2025] Open
Abstract
MUTYH is a clinically important DNA glycosylase that thwarts mutations by initiating base-excision repair at 8-oxoguanine (OG):A lesions. The roles for its [4Fe-4S] cofactor in DNA repair remain enigmatic. Functional profiling of cancer-associated variants near the [4Fe-4S] cofactor reveals that most variations abrogate both retention of the cofactor and enzyme activity. Surprisingly, R241Q and N238S retained the metal cluster and bound substrate DNA tightly, but were completely inactive. We determine the crystal structure of human MUTYH bound to a transition state mimic and this shows that Arg241 and Asn238 build an H-bond network connecting the [4Fe-4S] cluster to the catalytic Asp236 that mediates base excision. The structure of the bacterial MutY variant R149Q, along with molecular dynamics simulations of the human enzyme, support a model in which the cofactor functions to position and activate the catalytic Asp. These results suggest that allosteric cross-talk between the DNA binding [4Fe-4S] cofactor and the base excision site of MUTYH regulate its DNA repair function.
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Affiliation(s)
- Carlos H Trasviña-Arenas
- Department of Chemistry, University of California, Davis, CA, USA
- Research Center on Aging, Center for Research and Advanced Studies (CINVESTAV), Mexico City, Mexico
| | - Upeksha C Dissanayake
- Department of Chemistry and Biochemistry, University of Texas at Dallas, Richardson, TX, USA
| | - Nikole Tamayo
- Department of Chemistry, University of California, Davis, CA, USA
- Chemistry and Chemical Biology Graduate Program, University of California, Davis, CA, USA
| | - Mohammad Hashemian
- Department of Chemistry, University of California, Davis, CA, USA
- Chemistry and Chemical Biology Graduate Program, University of California, Davis, CA, USA
| | - Wen-Jen Lin
- Department of Chemistry, University of California, Davis, CA, USA
- Chemistry and Chemical Biology Graduate Program, University of California, Davis, CA, USA
| | - Merve Demir
- Department of Chemistry, University of California, Davis, CA, USA
- Chemistry and Chemical Biology Graduate Program, University of California, Davis, CA, USA
| | | | - Andrew J Fisher
- Department of Chemistry, University of California, Davis, CA, USA
- Chemistry and Chemical Biology Graduate Program, University of California, Davis, CA, USA
- Department of Molecular and Cellular Biology, University of California, Davis, CA, USA
| | - G Andrés Cisneros
- Department of Chemistry and Biochemistry, University of Texas at Dallas, Richardson, TX, USA.
- Department of Physics, University of Texas at Dallas, Richardson, TX, USA.
| | - Martin P Horvath
- School of Biological Sciences, University of Utah, Salt Lake City, UT, USA.
| | - Sheila S David
- Department of Chemistry, University of California, Davis, CA, USA.
- Chemistry and Chemical Biology Graduate Program, University of California, Davis, CA, USA.
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2
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Hemker SL, Marsh A, Hernandez F, Glick E, Clark G, Bashir A, Jiang K, Kitzman JO. Saturation mapping of MUTYH variant effects using DNA repair reporters. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2025:2025.03.01.640912. [PMID: 40093110 PMCID: PMC11908140 DOI: 10.1101/2025.03.01.640912] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 03/19/2025]
Abstract
Variants of uncertain significance (VUS) limit the actionability of genetic testing. A prominent example is MUTYH, a base excision repair factor associated with polyposis and colorectal cancer, which has a pathogenic variant carrier rate approaching 1 in 50 individuals in some populations. To systematically interrogate variant function in MUTYH, we coupled deep mutational scanning with a DNA repair reporter containing its lesion substrate, 8OG:A. Our variant-to-function map covers >97% of all possible MUTYH point variants (n=10,941) and achieves 100% accuracy classifying the pathogenicity of known clinical variants (n=247). Leveraging a large clinical registry, we observe significant associations with colorectal polyps and cancer, with more severely impaired missense variants conferring greater risk. We recapitulate known functional differences between pathogenic founder alleles, and highlight sites of complete missense intolerance, including residues that intercalate DNA and coordinate essential Zn2+ or Fe-S clusters. This map provides a resource to resolve the 1,032 existing missense VUS and 90 variants with conflicting interpretations in MUTYH, and demonstrates a scalable strategy to interrogate other clinically relevant DNA repair factors.
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Affiliation(s)
- Shelby L. Hemker
- Department of Human Genetics, University of Michigan Medical School, Ann Arbor, MI 48109, USA
| | | | | | - Elena Glick
- Department of Human Genetics, University of Michigan Medical School, Ann Arbor, MI 48109, USA
| | - Grace Clark
- Department of Human Genetics, University of Michigan Medical School, Ann Arbor, MI 48109, USA
| | - Alyssa Bashir
- Department of Human Genetics, University of Michigan Medical School, Ann Arbor, MI 48109, USA
| | - Krystal Jiang
- Department of Human Genetics, University of Michigan Medical School, Ann Arbor, MI 48109, USA
| | - Jacob O. Kitzman
- Department of Human Genetics, University of Michigan Medical School, Ann Arbor, MI 48109, USA
- Gilbert S. Omenn Department of Computational Medicine & Bioinformatics, University of Michigan Medical School, Ann Arbor, MI 48109, USA
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3
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Wang R, Li M, Li G, Pu S. High-contrast multicolour photoswitching based on dithienylethene Schiff base with a hydrazinylquinoline moiety. SPECTROCHIMICA ACTA. PART A, MOLECULAR AND BIOMOLECULAR SPECTROSCOPY 2024; 308:123679. [PMID: 38039644 DOI: 10.1016/j.saa.2023.123679] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/23/2023] [Revised: 10/29/2023] [Accepted: 11/21/2023] [Indexed: 12/03/2023]
Abstract
A new asymmetrical photochromic diarylethene DTE-HQo composed of a 2-hydrazinoquinoline moiety as the binding unit for ions and dithenylethene as a photoswitching trigger was reported. DTE-HQo displayed favourable photochromism upon irradiation with UV/vis light. Its fluorescent behaviour could be efficiently modulated by light, Zn2+, Cd2+ and HSO4-. The binding of Zn2+ induced a strong fluorescence peak at 510 nm in DTE-HQo due to the formation of a 1:2 complex [Zn2+ + 2DTE-HQo], resulting in a notable colour change from dark to intense white emission. Triggered by Cd2+, DTE-HQo formed a 1:1 complex [Cd2+ + DTE-HQo], leading to an enhanced emission intensity by 21-fold with an emission peak red-shifted from 461 nm to 514 nm. Unexpectedly, [Zn2+ + 2DTE-HQo] underwent hydrolysis when stimulated with water, generating a yellow-emitting complex [Zn2+ + DTE-HQo]. This color change easily distinguishes it from Cd2+ complex. Additionally, DTE-HQo showed high selectivity towards HSO4- and exhibited distinct "turn-on" fluorescence with a colour change from dark to bright blue upon stimulation. Moreover, the strong emission complexes of DTE-HQo with Zn2+, Cd2+ and HSO4- could be effectively quenched during the photocyclization process. Therefore, DTE-HQo can serve as an unimolecular multicolour photoswitching chemosensor, offering potential applications as a multifunctional probe for detecting Zn2+, Cd2+ and HSO4-.
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Affiliation(s)
- Renjie Wang
- Department of Ecology and Environment, Yuzhang Normal University, Nanchang 330031, China; Jiangxi Key Laboratory of Organic Chemistry, Jiangxi Science and Technology Normal University, Nanchang 330013, China.
| | - Mengyuan Li
- Jiangxi Key Laboratory of Organic Chemistry, Jiangxi Science and Technology Normal University, Nanchang 330013, China
| | - Gang Li
- Department of Ecology and Environment, Yuzhang Normal University, Nanchang 330031, China
| | - Shouzhi Pu
- Department of Ecology and Environment, Yuzhang Normal University, Nanchang 330031, China; Jiangxi Key Laboratory of Organic Chemistry, Jiangxi Science and Technology Normal University, Nanchang 330013, China.
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4
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Sun J, Wang J, Chen X. Functionalization of Mesoporous Silica with a G-A-Mismatched dsDNA Chain for Efficient Identification and Selective Capturing of the MutY Protein. ACS APPLIED MATERIALS & INTERFACES 2023; 15:8884-8894. [PMID: 36757327 DOI: 10.1021/acsami.2c19257] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/18/2023]
Abstract
MUTYH adenine DNA glycosylase and its homologous protein (collectively MutY) are typical DNA glycosylases with a [4Fe4S] cluster and a helix-hairpin-helix (HhH) motif in its structure. In the present work, the binding behaviors of the MutY protein to dsDNA containing different base mismatches were investigated. The type and distribution of base mismatch in the dsDNA chain were found to influence the DNA-protein binding interaction greatly. The [4Fe4S] cluster of the MutY protein is able to identify a G-A mismatch in the dsDNA chain specifically by monitoring the anomalies of charge transport in the dsDNA chain, allowing the entrance of the identified dsDNA chain into the internal cavity of the MutY protein and the strong DNA-protein binding at the HhH motif of the protein through multiple H-bonds. The dsDNA chain with a centrally located G-A mismatch is thus functionalized on mesoporous silica (MSN) via amination reaction, and the obtained dsDNA(G-A)@MSN is used as a powerful sorbent for the selective capturing of the MutY protein from complex samples. By using 0.5% NH3·H2O (m/v) as a stripping reagent, efficient isolation of the MutY protein from different cell lines and bacteria is achieved and the recovered MutY protein is demonstrated to maintain favorable DNA adenine glycosylase activity.
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Affiliation(s)
- Jingqi Sun
- Research Center for Analytical Sciences, Department of Chemistry, College of Sciences, Northeastern University, Box 332, Shenyang, Liaoning 110819, China
| | - Jianhua Wang
- Research Center for Analytical Sciences, Department of Chemistry, College of Sciences, Northeastern University, Box 332, Shenyang, Liaoning 110819, China
| | - Xuwei Chen
- Research Center for Analytical Sciences, Department of Chemistry, College of Sciences, Northeastern University, Box 332, Shenyang, Liaoning 110819, China
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Torgasheva NA, Diatlova EA, Grin IR, Endutkin AV, Mechetin GV, Vokhtantsev IP, Yudkina AV, Zharkov DO. Noncatalytic Domains in DNA Glycosylases. Int J Mol Sci 2022; 23:ijms23137286. [PMID: 35806289 PMCID: PMC9266487 DOI: 10.3390/ijms23137286] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2022] [Revised: 06/28/2022] [Accepted: 06/29/2022] [Indexed: 02/04/2023] Open
Abstract
Many proteins consist of two or more structural domains: separate parts that have a defined structure and function. For example, in enzymes, the catalytic activity is often localized in a core fragment, while other domains or disordered parts of the same protein participate in a number of regulatory processes. This situation is often observed in many DNA glycosylases, the proteins that remove damaged nucleobases thus initiating base excision DNA repair. This review covers the present knowledge about the functions and evolution of such noncatalytic parts in DNA glycosylases, mostly concerned with the human enzymes but also considering some unique members of this group coming from plants and prokaryotes.
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Affiliation(s)
- Natalia A. Torgasheva
- SB RAS Institute of Chemical Biology and Fundamental Medicine, 8 Lavrentieva Avenue, 630090 Novosibirsk, Russia; (N.A.T.); (E.A.D.); (I.R.G.); (A.V.E.); (G.V.M.); (I.P.V.); (A.V.Y.)
| | - Evgeniia A. Diatlova
- SB RAS Institute of Chemical Biology and Fundamental Medicine, 8 Lavrentieva Avenue, 630090 Novosibirsk, Russia; (N.A.T.); (E.A.D.); (I.R.G.); (A.V.E.); (G.V.M.); (I.P.V.); (A.V.Y.)
- Department of Natural Sciences, Novosibirsk State University, 2 Pirogova Street, 630090 Novosibirsk, Russia
| | - Inga R. Grin
- SB RAS Institute of Chemical Biology and Fundamental Medicine, 8 Lavrentieva Avenue, 630090 Novosibirsk, Russia; (N.A.T.); (E.A.D.); (I.R.G.); (A.V.E.); (G.V.M.); (I.P.V.); (A.V.Y.)
| | - Anton V. Endutkin
- SB RAS Institute of Chemical Biology and Fundamental Medicine, 8 Lavrentieva Avenue, 630090 Novosibirsk, Russia; (N.A.T.); (E.A.D.); (I.R.G.); (A.V.E.); (G.V.M.); (I.P.V.); (A.V.Y.)
| | - Grigory V. Mechetin
- SB RAS Institute of Chemical Biology and Fundamental Medicine, 8 Lavrentieva Avenue, 630090 Novosibirsk, Russia; (N.A.T.); (E.A.D.); (I.R.G.); (A.V.E.); (G.V.M.); (I.P.V.); (A.V.Y.)
| | - Ivan P. Vokhtantsev
- SB RAS Institute of Chemical Biology and Fundamental Medicine, 8 Lavrentieva Avenue, 630090 Novosibirsk, Russia; (N.A.T.); (E.A.D.); (I.R.G.); (A.V.E.); (G.V.M.); (I.P.V.); (A.V.Y.)
- Department of Natural Sciences, Novosibirsk State University, 2 Pirogova Street, 630090 Novosibirsk, Russia
| | - Anna V. Yudkina
- SB RAS Institute of Chemical Biology and Fundamental Medicine, 8 Lavrentieva Avenue, 630090 Novosibirsk, Russia; (N.A.T.); (E.A.D.); (I.R.G.); (A.V.E.); (G.V.M.); (I.P.V.); (A.V.Y.)
| | - Dmitry O. Zharkov
- SB RAS Institute of Chemical Biology and Fundamental Medicine, 8 Lavrentieva Avenue, 630090 Novosibirsk, Russia; (N.A.T.); (E.A.D.); (I.R.G.); (A.V.E.); (G.V.M.); (I.P.V.); (A.V.Y.)
- Department of Natural Sciences, Novosibirsk State University, 2 Pirogova Street, 630090 Novosibirsk, Russia
- Correspondence:
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6
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Trasviña-Arenas CH, Demir M, Lin WJ, David SS. Structure, function and evolution of the Helix-hairpin-Helix DNA glycosylase superfamily: Piecing together the evolutionary puzzle of DNA base damage repair mechanisms. DNA Repair (Amst) 2021; 108:103231. [PMID: 34649144 DOI: 10.1016/j.dnarep.2021.103231] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/25/2021] [Revised: 09/20/2021] [Accepted: 09/23/2021] [Indexed: 10/20/2022]
Abstract
The Base Excision Repair (BER) pathway is a highly conserved DNA repair system targeting chemical base modifications that arise from oxidation, deamination and alkylation reactions. BER features lesion-specific DNA glycosylases (DGs) which recognize and excise modified or inappropriate DNA bases to produce apurinic/apyrimidinic (AP) sites and coordinate AP-site hand-off to subsequent BER pathway enzymes. The DG superfamilies identified have evolved independently to cope with a wide variety of nucleobase chemical modifications. Most DG superfamilies recognize a distinct set of structurally related lesions. In contrast, the Helix-hairpin-Helix (HhH) DG superfamily has the remarkable ability to act upon structurally diverse sets of base modifications. The versatility in substrate recognition of the HhH-DG superfamily has been shaped by motif and domain acquisitions during evolution. In this paper, we review the structural features and catalytic mechanisms of the HhH-DG superfamily and draw a hypothetical reconstruction of the evolutionary path where these DGs developed diverse and unique enzymatic features.
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Affiliation(s)
| | - Merve Demir
- Department of Chemistry, University of California, Davis, CA 95616, U.S.A
| | - Wen-Jen Lin
- Department of Chemistry, University of California, Davis, CA 95616, U.S.A
| | - Sheila S David
- Department of Chemistry, University of California, Davis, CA 95616, U.S.A..
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7
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Keith N, Jackson CE, Glaholt SP, Young K, Lynch M, Shaw JR. Genome-Wide Analysis of Cadmium-Induced, Germline Mutations in a Long-Term Daphnia pulex Mutation-Accumulation Experiment. ENVIRONMENTAL HEALTH PERSPECTIVES 2021; 129:107003. [PMID: 34623885 PMCID: PMC8500294 DOI: 10.1289/ehp8932] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 01/08/2021] [Revised: 09/15/2021] [Accepted: 09/21/2021] [Indexed: 06/13/2023]
Abstract
BACKGROUND Germline mutations provide the raw material for all evolutionary processes and contribute to the occurrence of spontaneous human diseases and disorders. Yet despite the daily interaction of humans and other organisms with an increasing number of chemicals that are potentially mutagenic, precise measurements of chemically induced changes to the genome-wide rate and spectrum of germline mutation are lacking. OBJECTIVES A large-scale Daphnia pulex mutation-accumulation experiment was propagated in the presence and absence of an environmentally relevant cadmium concentration to quantify the influence of cadmium on germline mutation rates and spectra. RESULTS Cadmium exposure dramatically changed the genome-wide rates and regional spectra of germline mutations. In comparison with those in control conditions, Daphnia exposed to cadmium had a higher overall A : T → G : C mutation rates and a lower overall C : G → G : C mutation rate. Daphnia exposed to cadmium had a higher intergenic mutation rate and a lower exonic mutation rate. The higher intergenic mutation rate under cadmium exposure was the result of an elevated intergenic A : T → G : C rate, whereas the lower exon mutation rate in cadmium was the result of a complete loss of exonic C : G → G : C mutations-mutations that are known to be enriched at 5-hydroxymethylcytosine. We experimentally show that cadmium exposure significantly reduced 5-hydroxymethylcytosine levels. DISCUSSION These results provide evidence that cadmium changes regional mutation rates and can influence regional rates by interfering with an epigenetic process in the Daphnia pulex germline. We further suggest these observed cadmium-induced changes to the Daphnia germline mutation rate may be explained by cadmium's inhibition of zinc-containing domains. The cadmium-induced changes to germline mutation rates and spectra we report provide a comprehensive view of the mutagenic perils of cadmium and give insight into its potential impact on human population health. https://doi.org/10.1289/EHP8932.
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Affiliation(s)
- Nathan Keith
- O’Neill School of Public and Environmental Affairs, Indiana University, Bloomington, Indiana, USA
| | - Craig E. Jackson
- O’Neill School of Public and Environmental Affairs, Indiana University, Bloomington, Indiana, USA
| | - Stephen P. Glaholt
- O’Neill School of Public and Environmental Affairs, Indiana University, Bloomington, Indiana, USA
| | - Kimberly Young
- Department of Biology, Indiana University, Bloomington, Indiana, USA
| | - Michael Lynch
- Biodesign Center for Mechanisms of Evolution, Arizona State University, Tempe, Arizona, USA
| | - Joseph R. Shaw
- O’Neill School of Public and Environmental Affairs, Indiana University, Bloomington, Indiana, USA
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8
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Nakamura T, Okabe K, Hirayama S, Chirifu M, Ikemizu S, Morioka H, Nakabeppu Y, Yamagata Y. Structure of the mammalian adenine DNA glycosylase MUTYH: insights into the base excision repair pathway and cancer. Nucleic Acids Res 2021; 49:7154-7163. [PMID: 34142156 PMCID: PMC8266592 DOI: 10.1093/nar/gkab492] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/02/2021] [Revised: 05/17/2021] [Accepted: 05/22/2021] [Indexed: 11/17/2022] Open
Abstract
Mammalian MutY homologue (MUTYH) is an adenine DNA glycosylase that excises adenine inserted opposite 8-oxoguanine (8-oxoG). The inherited variations in human MUTYH gene are known to cause MUTYH-associated polyposis (MAP), which is associated with colorectal cancer. MUTYH is involved in base excision repair (BER) with proliferating cell nuclear antigen (PCNA) in DNA replication, which is unique and critical for effective mutation-avoidance. It is also reported that MUTYH has a Zn-binding motif in a unique interdomain connector (IDC) region, which interacts with Rad9–Rad1–Hus1 complex (9–1–1) in DNA damage response, and with apurinic/apyrimidinic endonuclease 1 (APE1) in BER. However, the structural basis for the BER pathway by MUTYH and its interacting proteins is unclear. Here, we determined the crystal structures of complexes between mouse MUTYH and DNA, and between the C-terminal domain of mouse MUTYH and human PCNA. The structures elucidated the repair mechanism for the A:8-oxoG mispair including DNA replication-coupled repair process involving MUTYH and PCNA. The Zn-binding motif was revealed to comprise one histidine and three cysteine residues. The IDC, including the Zn-binding motif, is exposed on the MUTYH surface, suggesting its interaction modes with 9–1–1 and APE1, respectively. The structure of MUTYH explains how MAP mutations perturb MUTYH function.
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Affiliation(s)
- Teruya Nakamura
- Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oehonmachi, Chuo-ku, Kumamoto, 862-0973 Kumamoto, Japan.,Priority Organization for Innovation and Excellence, Kumamoto University, 5-1 Oehonmachi, Chuo-ku, Kumamoto, 862-0973 Kumamoto, Japan
| | - Kohtaro Okabe
- Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oehonmachi, Chuo-ku, Kumamoto, 862-0973 Kumamoto, Japan
| | - Shogo Hirayama
- Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oehonmachi, Chuo-ku, Kumamoto, 862-0973 Kumamoto, Japan
| | - Mami Chirifu
- Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oehonmachi, Chuo-ku, Kumamoto, 862-0973 Kumamoto, Japan
| | - Shinji Ikemizu
- Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oehonmachi, Chuo-ku, Kumamoto, 862-0973 Kumamoto, Japan
| | - Hiroshi Morioka
- Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oehonmachi, Chuo-ku, Kumamoto, 862-0973 Kumamoto, Japan
| | - Yusaku Nakabeppu
- Division of Neurofunctional Genomics, Department of Immunobiology and Neuroscience, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
| | - Yuriko Yamagata
- Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oehonmachi, Chuo-ku, Kumamoto, 862-0973 Kumamoto, Japan.,Shokei University and Shokei University Junior College, 2-6-78, Kuhonji, Chuo-ku, Kumamoto, 862-8678 Kumamoto, Japan
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9
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Curia MC, Catalano T, Aceto GM. MUTYH: Not just polyposis. World J Clin Oncol 2020; 11:428-449. [PMID: 32821650 PMCID: PMC7407923 DOI: 10.5306/wjco.v11.i7.428] [Citation(s) in RCA: 45] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/28/2020] [Revised: 05/08/2020] [Accepted: 05/27/2020] [Indexed: 02/06/2023] Open
Abstract
MUTYH is a base excision repair enzyme, it plays a crucial role in the correction of DNA errors from guanine oxidation and may be considered a cell protective factor. In humans it is an adenine DNA glycosylase that removes adenine misincorporated in 7,8-dihydro-8-oxoguanine (8-oxoG) pairs, inducing G:C to T:A transversions. MUTYH functionally cooperates with OGG1 that eliminates 8-oxodG derived from excessive reactive oxygen species production. MUTYH mutations have been linked to MUTYH associated polyposis syndrome (MAP), an autosomal recessive disorder characterized by multiple colorectal adenomas. MAP patients show a greatly increased lifetime risk for gastrointestinal cancers. The cancer risk in mono-allelic carriers associated with one MUTYH mutant allele is controversial and it remains to be clarified whether the altered functions of this protein may have a pathophysiological involvement in other diseases besides familial gastrointestinal diseases. This review evaluates the role of MUTYH, focusing on current studies of human neoplastic and non-neoplastic diseases different to colon polyposis and colorectal cancer. This will provide novel insights into the understanding of the molecular basis underlying MUTYH-related pathogenesis. Furthermore, we describe the association between MUTYH single nucleotide polymorphisms (SNPs) and different cancer and non-cancer diseases. We address the utility to increase our knowledge regarding MUTYH in the light of recent advances in the literature with the aim of a better understanding of the potential for identifying new therapeutic targets. Considering the multiple functions and interactions of MUTYH protein, its involvement in pathologies based on oxidative stress damage could be hypothesized. Although the development of extraintestinal cancer in MUTYH heterozygotes is not completely defined, the risk for malignancies of the duodenum, ovary, and bladder is also increased as well as the onset of benign and malignant endocrine tumors. The presence of MUTYH pathogenic variants is an independent predictor of poor prognosis in sporadic gastric cancer and in salivary gland secretory carcinoma, while its inhibition has been shown to reduce the survival of pancreatic ductal adenocarcinoma cells. Furthermore, some MUTYH SNPs have been associated with lung, hepatocellular and cervical cancer risk. An additional role of MUTYH seems to contribute to the prevention of numerous other disorders with an inflammatory/degenerative basis, including neurological and ocular diseases. Finally, it is interesting to note that MUTYH could be a new therapeutic target and future studies will shed light on its specific functions in the prevention of diseases and in the improvement of the chemo-sensitivity of cancer cells.
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Affiliation(s)
- Maria Cristina Curia
- Department of Medical, Oral and Biotechnological Sciences, “G. d'Annunzio” University of Chieti-Pescara, Chieti, Via dei Vestini 66100, Italy
| | - Teresa Catalano
- Department of Clinical and Experimental Medicine, University of Messina, Messina, Via Consolare Valeria 98125, Italy
| | - Gitana Maria Aceto
- Department of Medical, Oral and Biotechnological Sciences, “G. d'Annunzio” University of Chieti-Pescara, Chieti, Via dei Vestini 66100, Italy
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10
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Ren G, Ha Y, Liu YS, Feng X, Zhang N, Yu P, Zhang L, Yang W, Feng J, Guo J, Liu X. Deciphering the Solvent Effect for the Solvation Structure of Ca 2+ in Polar Molecular Liquids. J Phys Chem B 2020; 124:3408-3417. [PMID: 32223137 DOI: 10.1021/acs.jpcb.0c02437] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
Although the crystal structures for many inorganic compounds are readily available, researchers are still working hard to understand the relations between the structures and chemical properties of solutions because most of the chemical reactions take place in solutions. A huge amount of effort has been put toward modeling the ion solvation structure from the perspectives of both experiments and theories. In this study, the solvation structures of Ca2+ ions in aqueous and alcoholic solutions at different concentrations were carefully evaluated by Ca K-edge X-ray absorption near-edge structure (XANES) and extended X-ray absorption fine structure (EXAFS) analyses. Density functional theory (DFT) calculations were also performed to correlate the results with the experimental data and then further extended to other similar systems. It was found that the number of coordinating solvent molecules decreases with increasing Ca2+ concentration and increasing solvent molecule sizes. From the EXAFS data, it was observed that the first solvation shell of Ca2+ splits into two Ca-O distances in a methanol solution and the counter ion Cl- might also be within the first shell at high concentrations. For the first time, the effects of solvents with different polarities and sizes on the ion solvation environment were systematically evaluated.
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Affiliation(s)
- Guoxi Ren
- State Key Laboratory of Functional Materials for Informatics, Shanghai Institute of Microsystem and Information Technology, Chinese Academy of Sciences, 865 Changning Road, Shanghai 200050, China.,CAS Center for Excellence in Superconducting Electronics (CENSE), Chinese Academy of Sciences, Shanghai 200050, China.,University of Chinese Academy of Sciences, Beijing 100049, China
| | - Yang Ha
- Advanced Light Source, Lawrence Berkeley National Laboratory, Berkeley, California 94720, United States
| | - Yi-Sheng Liu
- Advanced Light Source, Lawrence Berkeley National Laboratory, Berkeley, California 94720, United States
| | - Xuefei Feng
- Advanced Light Source, Lawrence Berkeley National Laboratory, Berkeley, California 94720, United States
| | - Nian Zhang
- State Key Laboratory of Functional Materials for Informatics, Shanghai Institute of Microsystem and Information Technology, Chinese Academy of Sciences, 865 Changning Road, Shanghai 200050, China.,CAS Center for Excellence in Superconducting Electronics (CENSE), Chinese Academy of Sciences, Shanghai 200050, China
| | - Pengfei Yu
- State Key Laboratory of Functional Materials for Informatics, Shanghai Institute of Microsystem and Information Technology, Chinese Academy of Sciences, 865 Changning Road, Shanghai 200050, China.,CAS Center for Excellence in Superconducting Electronics (CENSE), Chinese Academy of Sciences, Shanghai 200050, China
| | - Liang Zhang
- Jiangsu Key Laboratory for Carbon-Based Functional Materials & Devices, Institute of Functional Nano & Soft Materials (FUNSOM), Soochow University, 199 Ren'ai Road, Suzhou 215123, Jiangsu, China
| | - Wanli Yang
- Advanced Light Source, Lawrence Berkeley National Laboratory, Berkeley, California 94720, United States
| | - Jun Feng
- Advanced Light Source, Lawrence Berkeley National Laboratory, Berkeley, California 94720, United States
| | - Jinghua Guo
- Advanced Light Source, Lawrence Berkeley National Laboratory, Berkeley, California 94720, United States
| | - Xiaosong Liu
- State Key Laboratory of Functional Materials for Informatics, Shanghai Institute of Microsystem and Information Technology, Chinese Academy of Sciences, 865 Changning Road, Shanghai 200050, China.,CAS Center for Excellence in Superconducting Electronics (CENSE), Chinese Academy of Sciences, Shanghai 200050, China.,School of Physical Science and Technology, Shanghai Tech University, Shanghai 201210, China
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11
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Gumkowski JD, Martinie RJ, Corrigan PS, Pan J, Bauerle MR, Almarei M, Booker SJ, Silakov A, Krebs C, Boal AK. Analysis of RNA Methylation by Phylogenetically Diverse Cfr Radical S-Adenosylmethionine Enzymes Reveals an Iron-Binding Accessory Domain in a Clostridial Enzyme. Biochemistry 2019; 58:3169-3184. [PMID: 31246421 DOI: 10.1021/acs.biochem.9b00197] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]
Abstract
Cfr is a radical S-adenosylmethionine (SAM) RNA methylase linked to multidrug antibiotic resistance in bacterial pathogens. It catalyzes a chemically challenging C-C bond-forming reaction to methylate C8 of A2503 (Escherichia coli numbering) of 23S rRNA during ribosome assembly. The cfr gene has been identified as a mobile genetic element in diverse bacteria and in the genome of select Bacillales and Clostridiales species. Despite the importance of Cfr, few representatives have been purified and characterized in vitro. Here we show that Cfr homologues from Bacillus amyloliquefaciens, Enterococcus faecalis, Paenibacillus lautus, and Clostridioides difficile act as C8 adenine RNA methylases in biochemical assays. C. difficile Cfr contains an additional Cys-rich C-terminal domain that binds a mononuclear Fe2+ ion in a rubredoxin-type Cys4 motif. The C-terminal domain can be truncated with minimal impact on C. difficile Cfr activity, but the rate of turnover is decreased upon disruption of the Fe2+-binding site by Zn2+ substitution or ligand mutation. These findings indicate an important purpose for the observed C-terminal iron in the native fusion protein. Bioinformatic analysis of the C. difficile Cfr Cys-rich domain shows that it is widespread (∼1400 homologues) as a stand-alone gene in pathogenic or commensal Bacilli and Clostridia, with >10% encoded adjacent to a predicted radical SAM RNA methylase. Although the domain is not essential for in vitro C. difficile Cfr activity, the genomic co-occurrence and high abundance in the human microbiome suggest a possible functional role for a specialized rubredoxin in certain radical SAM RNA methylases that are relevant to human health.
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Affiliation(s)
- James D Gumkowski
- Department of Chemistry , The Pennsylvania State University , University Park , Pennsylvania 16802 , United States
| | - Ryan J Martinie
- Department of Chemistry , The Pennsylvania State University , University Park , Pennsylvania 16802 , United States
| | - Patrick S Corrigan
- Department of Chemistry , The Pennsylvania State University , University Park , Pennsylvania 16802 , United States
| | - Juan Pan
- Department of Chemistry , The Pennsylvania State University , University Park , Pennsylvania 16802 , United States
| | - Matthew R Bauerle
- Department of Chemistry , The Pennsylvania State University , University Park , Pennsylvania 16802 , United States
| | - Mohamed Almarei
- Department of Biochemistry and Molecular Biology , The Pennsylvania State University , University Park , Pennsylvania 16802 , United States
| | - Squire J Booker
- Department of Chemistry , The Pennsylvania State University , University Park , Pennsylvania 16802 , United States.,Department of Biochemistry and Molecular Biology , The Pennsylvania State University , University Park , Pennsylvania 16802 , United States.,Howard Hughes Medical Institute , The Pennsylvania State University , University Park , Pennsylvania 16802 , United States
| | - Alexey Silakov
- Department of Chemistry , The Pennsylvania State University , University Park , Pennsylvania 16802 , United States
| | - Carsten Krebs
- Department of Chemistry , The Pennsylvania State University , University Park , Pennsylvania 16802 , United States.,Department of Biochemistry and Molecular Biology , The Pennsylvania State University , University Park , Pennsylvania 16802 , United States
| | - Amie K Boal
- Department of Chemistry , The Pennsylvania State University , University Park , Pennsylvania 16802 , United States.,Department of Biochemistry and Molecular Biology , The Pennsylvania State University , University Park , Pennsylvania 16802 , United States
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12
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Raetz AG, David SS. When you're strange: Unusual features of the MUTYH glycosylase and implications in cancer. DNA Repair (Amst) 2019; 80:16-25. [PMID: 31203172 DOI: 10.1016/j.dnarep.2019.05.005] [Citation(s) in RCA: 27] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/23/2019] [Revised: 05/23/2019] [Accepted: 05/29/2019] [Indexed: 02/06/2023]
Abstract
MUTYH is a base-excision repair glycosylase that removes adenine opposite 8-oxoguanine (OG). Variants of MUTYH defective in functional activity lead to MUTYH-associated polyposis (MAP), which progresses to cancer with very high penetrance. Whole genome and whole exome sequencing studies have found MUTYH deficiencies in an increasing number of cancer types. While the canonical OG:A repair activity of MUTYH is well characterized and similar to bacterial MutY, here we review more recent evidence that MUTYH has activities independent of OG:A repair and appear centered on the interdomain connector (IDC) region of MUTYH. We summarize evidence that MUTYH is involved in rapid DNA damage response (DDR) signaling, including PARP activation, 9-1-1 and ATR signaling, and SIRT6 activity. MUTYH alters survival and DDR to a wide variety of DNA damaging agents in a time course that is not consistent with the formation of OG:A mispairs. Studies that suggest MUTYH inhibits the repair of alkyl-DNA damage and cyclopyrimidine dimers (CPDs) is reviewed, and evidence of a synthetic lethal interaction with mismatch repair (MMR) is summarized. Based on these studies we suggest that MUTYH has evolved from an OG:A mispair glycosylase to a multifunctional scaffold for DNA damage response signaling.
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Affiliation(s)
- Alan G Raetz
- Department of Chemistry, University of California, Davis, Davis, CA, USA.
| | - Sheila S David
- Department of Chemistry, University of California, Davis, Davis, CA, USA.
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13
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Tse EM, Zwang TJ, Bedoya S, Barton JK. Effective Distance for DNA-Mediated Charge Transport between Repair Proteins. ACS CENTRAL SCIENCE 2019; 5:65-72. [PMID: 30693326 PMCID: PMC6346725 DOI: 10.1021/acscentsci.8b00566] [Citation(s) in RCA: 23] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/14/2018] [Indexed: 05/23/2023]
Abstract
The stacked aromatic base pairs within the DNA double helix facilitate charge transport down its length in the absence of lesions, mismatches, and other stacking perturbations. DNA repair proteins containing [4Fe4S] clusters can take advantage of DNA charge transport (CT) chemistry to scan the genome for mistakes more efficiently. Here we examine the effective length over which charge can be transported along DNA between these repair proteins. We define the effective CT distance as the length of DNA within which two proteins are able to influence their ensemble affinity to the DNA duplex via CT. Endonuclease III, a DNA repair glycosylase containing a [4Fe4S] cluster, was incubated with DNA duplexes of different lengths (1.5-9 kb), and atomic force microscopy was used to quantify the binding of proteins to these duplexes to determine how the relative protein affinity changes with increasing DNA length. A sharp change in binding slope is observed at 3509 base pairs, or about 1.2 μm, that supports the existence of two regimes for protein binding, one within the range for DNA CT, one outside of the range for CT; DNA CT between the redox proteins bound to DNA effectively decreases the ensemble binding affinity of oxidized and reduced proteins to DNA. Utilizing an Endonuclease III mutant Y82A, which is defective in carrying out DNA CT, shows only one regime for protein binding. Decreasing the temperature to 4 °C or including metallointercalators on the duplex, both of which should enhance base stacking and decrease DNA floppiness, leads to extending the effective length for DNA charge transport to ∼5300 bp or 1.8 μm. These results thus support DNA charge transport between repair proteins over kilobase distances. The results furthermore highlight the ability of DNA repair proteins to search the genome quickly and efficiently using DNA charge transport chemistry.
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14
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Nuñez NN, Khuu C, Babu CS, Bertolani SJ, Rajavel AN, Spear JE, Armas JA, Wright JD, Siegel JB, Lim C, David SS. The Zinc Linchpin Motif in the DNA Repair Glycosylase MUTYH: Identifying the Zn 2+ Ligands and Roles in Damage Recognition and Repair. J Am Chem Soc 2018; 140:13260-13271. [PMID: 30208271 PMCID: PMC6443246 DOI: 10.1021/jacs.8b06923] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
The DNA base excision repair (BER) glycosylase MUTYH prevents DNA mutations by catalyzing adenine (A) excision from inappropriately formed 8-oxoguanine (8-oxoG):A mismatches. The importance of this mutation suppression activity in tumor suppressor genes is underscored by the association of inherited variants of MUTYH with colorectal polyposis in a hereditary colorectal cancer syndrome known as MUTYH-associated polyposis, or MAP. Many of the MAP variants encompass amino acid changes that occur at positions surrounding the two-metal cofactor-binding sites of MUTYH. One of these cofactors, found in nearly all MUTYH orthologs, is a [4Fe-4S]2+ cluster coordinated by four Cys residues located in the N-terminal catalytic domain. We recently uncovered a second functionally relevant metal cofactor site present only in higher eukaryotic MUTYH orthologs: a Zn2+ ion coordinated by three Cys residues located within the extended interdomain connector (IDC) region of MUTYH that connects the N-terminal adenine excision and C-terminal 8-oxoG recognition domains. In this work, we identified a candidate for the fourth Zn2+ coordinating ligand using a combination of bioinformatics and computational modeling. In addition, using in vitro enzyme activity assays, fluorescence polarization DNA binding assays, circular dichroism spectroscopy, and cell-based rifampicin resistance assays, the functional impact of reduced Zn2+ chelation was evaluated. Taken together, these results illustrate the critical role that the "Zn2+ linchpin motif" plays in MUTYH repair activity by providing for proper engagement of the functional domains on the 8-oxoG:A mismatch required for base excision catalysis. The functional importance of the Zn2+ linchpin also suggests that adjacent MAP variants or exposure to environmental chemicals may compromise Zn2+ coordination, and ability of MUTYH to prevent disease.
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Affiliation(s)
- Nicole N. Nuñez
- Department of Chemistry, University of California, Davis, One Shields Avenue, Davis, California, 95616, USA
| | - Cindy Khuu
- Department of Chemistry, University of California, Davis, One Shields Avenue, Davis, California, 95616, USA
- Biochemistry, Molecular, Cellular and Developmental Graduate Group, University of California, Davis, 95616, USA
| | - C. Satheesan Babu
- Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan R. O. C
| | - Steve J. Bertolani
- Department of Chemistry, University of California, Davis, One Shields Avenue, Davis, California, 95616, USA
- Genome Center, University of California, Davis, One Shields Avenue, Davis, California, 95616, USA
| | - Anisha N. Rajavel
- Department of Chemistry, University of California, Davis, One Shields Avenue, Davis, California, 95616, USA
| | - Jensen E. Spear
- Department of Chemistry, University of California, Davis, One Shields Avenue, Davis, California, 95616, USA
| | - Jeremy A. Armas
- Department of Chemistry, University of California, Davis, One Shields Avenue, Davis, California, 95616, USA
| | - Jon D. Wright
- Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan R. O. C
| | - Justin B. Siegel
- Department of Chemistry, University of California, Davis, One Shields Avenue, Davis, California, 95616, USA
- Genome Center, University of California, Davis, One Shields Avenue, Davis, California, 95616, USA
- Department of Biochemistry and Molecular Medicine, University of California, Davis, One Shields Avenue, Davis, California, 95616, USA
| | - Carmay Lim
- Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan R. O. C
| | - Sheila S. David
- Department of Chemistry, University of California, Davis, One Shields Avenue, Davis, California, 95616, USA
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15
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Majumdar C, Nuñez NN, Raetz AG, Khuu C, David SS. Cellular Assays for Studying the Fe-S Cluster Containing Base Excision Repair Glycosylase MUTYH and Homologs. Methods Enzymol 2018; 599:69-99. [PMID: 29746250 DOI: 10.1016/bs.mie.2017.12.006] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Many DNA repair enzymes, including the human adenine glycosylase MUTYH, require iron-sulfur (Fe-S) cluster cofactors for DNA damage recognition and subsequent repair. MUTYH prokaryotic and eukaryotic homologs are a family of adenine (A) glycosylases that cleave A when mispaired with the oxidatively damaged guanine lesion, 8-oxo-7,8-dihydroguanine (OG). Faulty OG:A repair has been linked to the inheritance of missense mutations in the MUTYH gene. These inherited mutations can result in the onset of a familial colorectal cancer disorder known as MUTYH-associated polyposis (MAP). While in vitro studies can be exceptional at unraveling how MutY interacts with its OG:A substrate, cell-based assays are needed to provide a cellular context to these studies. In addition, strategic comparison of in vitro and in vivo studies can provide exquisite insight into the search, selection, excision process, and the coordination with protein partners, required to mediate full repair of the lesion. A commonly used assay is the rifampicin resistance assay that provides an indirect evaluation of the intrinsic mutation rate in Escherichia coli (E. coli or Ec), read out as antibiotic-resistant cell growth. Our laboratory has also developed a bacterial plasmid-based assay that allows for direct evaluation of repair of a defined OG:A mispair. This assay provides a means to assess the impact of catalytic defects in affinity and excision on overall repair. Finally, a mammalian GFP-based reporter assay has been developed that more accurately models features of mammalian cells. Taken together, these assays provide a cellular context to the repair activity of MUTYH and its homologs that illuminates the role these enzymes play in preventing mutations and disease.
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Affiliation(s)
| | - Nicole N Nuñez
- University of California, Davis, Davis, CA, United States
| | - Alan G Raetz
- University of California, Davis, Davis, CA, United States
| | - Cindy Khuu
- University of California, Davis, Davis, CA, United States
| | - Sheila S David
- University of California, Davis, Davis, CA, United States.
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16
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Nuñez NN, Majumdar C, Lay KT, David SS. Fe-S Clusters and MutY Base Excision Repair Glycosylases: Purification, Kinetics, and DNA Affinity Measurements. Methods Enzymol 2018; 599:21-68. [PMID: 29746241 DOI: 10.1016/bs.mie.2017.11.035] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023]
Abstract
A growing number of iron-sulfur (Fe-S) cluster cofactors have been identified in DNA repair proteins. MutY and its homologs are base excision repair (BER) glycosylases that prevent mutations associated with the common oxidation product of guanine (G), 8-oxo-7,8-dihydroguanine (OG) by catalyzing adenine (A) base excision from inappropriately formed OG:A mispairs. The finding of an [4Fe-4S]2+ cluster cofactor in MutY, Endonuclease III, and structurally similar BER enzymes was surprising and initially thought to represent an example of a purely structural role for the cofactor. However, in the two decades subsequent to the initial discovery, purification and in vitro analysis of bacterial MutYs and mammalian homologs, such as human MUTYH and mouse Mutyh, have demonstrated that proper Fe-S cluster coordination is required for OG:A substrate recognition and adenine excision. In addition, the Fe-S cluster in MutY has been shown to be capable of redox chemistry in the presence of DNA. The work in our laboratory aimed at addressing the importance of the MutY Fe-S cluster has involved a battery of approaches, with the overarching hypothesis that understanding the role(s) of the Fe-S cluster is intimately associated with understanding the biological and chemical properties of MutY and its unique damaged DNA substrate as a whole. In this chapter, we focus on methods of enzyme expression and purification, detailed enzyme kinetics, and DNA affinity assays. The methods described herein have not only been leveraged to provide insight into the roles of the MutY Fe-S cluster but have also been provided crucial information needed to delineate the impact of inherited variants of the human homolog MUTYH associated with a colorectal cancer syndrome known as MUTYH-associated polyposis or MAP. Notably, many MAP-associated variants have been found adjacent to the Fe-S cluster further underscoring the intimate relationship between the cofactor, MUTYH-mediated DNA repair, and disease.
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Affiliation(s)
| | | | - Kori T Lay
- University of California, Davis, CA, United States
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17
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Banda DM, Nuñez NN, Burnside MA, Bradshaw KM, David SS. Repair of 8-oxoG:A mismatches by the MUTYH glycosylase: Mechanism, metals and medicine. Free Radic Biol Med 2017; 107:202-215. [PMID: 28087410 PMCID: PMC5457711 DOI: 10.1016/j.freeradbiomed.2017.01.008] [Citation(s) in RCA: 77] [Impact Index Per Article: 9.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/17/2016] [Revised: 01/01/2017] [Accepted: 01/04/2017] [Indexed: 12/12/2022]
Abstract
Reactive oxygen and nitrogen species (RONS) may infringe on the passing of pristine genetic information by inducing DNA inter- and intra-strand crosslinks, protein-DNA crosslinks, and chemical alterations to the sugar or base moieties of DNA. 8-Oxo-7,8-dihydroguanine (8-oxoG) is one of the most prevalent DNA lesions formed by RONS and is repaired through the base excision repair (BER) pathway involving the DNA repair glycosylases OGG1 and MUTYH in eukaryotes. MUTYH removes adenine (A) from 8-oxoG:A mispairs, thus mitigating the potential of G:C to T:A transversion mutations from occurring in the genome. The paramount role of MUTYH in guarding the genome is well established in the etiology of a colorectal cancer predisposition syndrome involving variants of MUTYH, referred to as MUTYH-associated polyposis (MAP). In this review, we highlight recent advances in understanding how MUTYH structure and related function participate in the manifestation of human disease such as MAP. Here we focus on the importance of MUTYH's metal cofactor sites, including a recently discovered "Zinc linchpin" motif, as well as updates to the catalytic mechanism. Finally, we touch on the insight gleaned from studies with MAP-associated MUTYH variants and recent advances in understanding the multifaceted roles of MUTYH in the cell, both in the prevention of mutagenesis and tumorigenesis.
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Affiliation(s)
- Douglas M Banda
- Department of Chemistry, University of California, Davis, One Shields Avenue, Davis, CA 95616, United States
| | - Nicole N Nuñez
- Department of Chemistry, University of California, Davis, One Shields Avenue, Davis, CA 95616, United States
| | - Michael A Burnside
- Department of Chemistry, University of California, Davis, One Shields Avenue, Davis, CA 95616, United States
| | - Katie M Bradshaw
- Department of Chemistry, University of California, Davis, One Shields Avenue, Davis, CA 95616, United States
| | - Sheila S David
- Department of Chemistry, University of California, Davis, One Shields Avenue, Davis, CA 95616, United States.
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18
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Wickramaratne S, Banda DM, Ji S, Manlove AH, Malayappan B, Nuñez NN, Samson L, Campbell C, David SS, Tretyakova N. Base Excision Repair of N 6-Deoxyadenosine Adducts of 1,3-Butadiene. Biochemistry 2016; 55:6070-6081. [PMID: 27552084 DOI: 10.1021/acs.biochem.6b00553] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
The important industrial and environmental carcinogen 1,3-butadiene (BD) forms a range of adenine adducts in DNA, including N6-(2-hydroxy-3-buten-1-yl)-2'-deoxyadenosine (N6-HB-dA), 1,N6-(2-hydroxy-3-hydroxymethylpropan-1,3-diyl)-2'-deoxyadenosine (1,N6-HMHP-dA), and N6,N6-(2,3-dihydroxybutan-1,4-diyl)-2'-deoxyadenosine (N6,N6-DHB-dA). If not removed prior to DNA replication, these lesions can contribute to A → T and A → G mutations commonly observed following exposure to BD and its metabolites. In this study, base excision repair of BD-induced 2'-deoxyadenosine (BD-dA) lesions was investigated. Synthetic DNA duplexes containing site-specific and stereospecific (S)-N6-HB-dA, (R,S)-1,N6-HMHP-dA, and (R,R)-N6,N6-DHB-dA adducts were prepared by a postoligomerization strategy. Incision assays with nuclear extracts from human fibrosarcoma (HT1080) cells have revealed that BD-dA adducts were recognized and cleaved by a BER mechanism, with the relative excision efficiency decreasing in the following order: (S)-N6-HB-dA > (R,R)-N6,N6-DHB-dA > (R,S)-1,N6-HMHP-dA. The extent of strand cleavage at the adduct site was decreased in the presence of BER inhibitor methoxyamine and by competitor duplexes containing known BER substrates. Similar strand cleavage assays conducted using several eukaryotic DNA glycosylases/lyases (AAG, Mutyh, hNEIL1, and hOGG1) have failed to observe correct incision products at the BD-dA lesion sites, suggesting that a different BER enzyme may be involved in the removal of BD-dA adducts in human cells.
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Affiliation(s)
- Susith Wickramaratne
- Masonic Cancer Center and Departments of Chemistry and Medicinal Chemistry, University of Minnesota , Minneapolis, Minnesota 55455, United States
| | - Douglas M Banda
- Department of Chemistry, University of California , Davis, California 95616, United States
| | - Shaofei Ji
- Masonic Cancer Center and Departments of Chemistry and Medicinal Chemistry, University of Minnesota , Minneapolis, Minnesota 55455, United States
| | - Amelia H Manlove
- Department of Chemistry, University of California , Davis, California 95616, United States
| | - Bhaskar Malayappan
- Masonic Cancer Center and Departments of Chemistry and Medicinal Chemistry, University of Minnesota , Minneapolis, Minnesota 55455, United States
| | - Nicole N Nuñez
- Department of Chemistry, University of California , Davis, California 95616, United States
| | - Leona Samson
- Division of Biological Engineering, Massachusetts Institute of Technology , Cambridge, Massachusetts 02139, United States
| | - Colin Campbell
- Department of Pharmacology, University of Minnesota , Minneapolis, Minnesota 55455, United States
| | - Sheila S David
- Department of Chemistry, University of California , Davis, California 95616, United States
| | - Natalia Tretyakova
- Masonic Cancer Center and Departments of Chemistry and Medicinal Chemistry, University of Minnesota , Minneapolis, Minnesota 55455, United States
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19
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Tomar JS, Narwal M, Kumar P, Peddinti RK. Characterization of substrate binding and enzymatic removal of a 3-methyladenine lesion from genomic DNA with TAG of MDR A. baumannii. MOLECULAR BIOSYSTEMS 2016; 12:3259-3265. [PMID: 27714027 DOI: 10.1039/c6mb00517a] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
The rise of multiple-drug resistance in bacterial pathogens imposes a serious public health concern and has led to increased interest in studying various pathways as well as enzymes. Different DNA glycosylases collaborate during bacterial infection and disease by overcoming the effects of ROS- and RNS-mediated host innate immunity response. 3-Methyladenine DNA glycosylase I, an essential DNA repair enzyme, was chosen for the present study from the MDR species of A. baumannii. The enzyme was especially chosen because of its functional significance in A. baumannii and due to its structural variation from its human homologue. MDR strains such as A. baumannii are interesting targets owing to their evolved mechanisms of evading a host defence. In the absence of any structural information, the enzyme was characterized biophysically and biochemically. Binding studies with 3mA and Zn2+ indicated that the activity of TAG-Ab is an enthalpy-driven process. Fluorescence thermal denaturation studies described that the denaturation of TAG-Ab is a two-step process. Modified RP-HPLC-based glycosylase assay attested that the heterologously expressed and purified TAG-Ab enzyme is active and catalyses the removal of 3mA. Other binding parameters and the effect of adenine on substrate binding are also discussed in detail.
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Affiliation(s)
- Jyoti Singh Tomar
- Department of Chemistry, Indian Institute of Technology Roorkee, Roorkee-247667, India.
| | - Manju Narwal
- Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee-247667, India
| | - Pravindra Kumar
- Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee-247667, India
| | - Rama Krishna Peddinti
- Department of Chemistry, Indian Institute of Technology Roorkee, Roorkee-247667, India.
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20
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Distinct functional consequences of MUTYH variants associated with colorectal cancer: Damaged DNA affinity, glycosylase activity and interaction with PCNA and Hus1. DNA Repair (Amst) 2015; 34:39-51. [PMID: 26377631 DOI: 10.1016/j.dnarep.2015.08.001] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2015] [Accepted: 08/03/2015] [Indexed: 12/13/2022]
Abstract
MUTYH is a base excision repair (BER) enzyme that prevents mutations in DNA associated with 8-oxoguanine (OG) by catalyzing the removal of adenine from inappropriately formed OG:A base-pairs. Germline mutations in the MUTYH gene are linked to colorectal polyposis and a high risk of colorectal cancer, a syndrome referred to as MUTYH-associated polyposis (MAP). There are over 300 different MUTYH mutations associated with MAP and a large fraction of these gene changes code for missense MUTYH variants. Herein, the adenine glycosylase activity, mismatch recognition properties, and interaction with relevant protein partners of human MUTYH and five MAP variants (R295C, P281L, Q324H, P502L, and R520Q) were examined. P281L MUTYH was found to be severely compromised both in DNA binding and base excision activity, consistent with the location of this variation in the iron-sulfur cluster (FCL) DNA binding motif of MUTYH. Both R295C and R520Q MUTYH were found to have low fractions of active enzyme, compromised affinity for damaged DNA, and reduced rates for adenine excision. In contrast, both Q324H and P502L MUTYH function relatively similarly to WT MUTYH in both binding and glycosylase assays. However, P502L and R520Q exhibited reduced affinity for PCNA (proliferation cell nuclear antigen), consistent with their location in the PCNA-binding motif of MUTYH. Whereas, only Q324H, and not R295C, was found to have reduced affinity for Hus1 of the Rad9-Hus1-Rad1 complex, despite both being localized to the same region implicated for interaction with Hus1. These results underscore the diversity of functional consequences due to MUTYH variants that may impact the progression of MAP.
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21
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Fuss JO, Tsai CL, Ishida JP, Tainer JA. Emerging critical roles of Fe-S clusters in DNA replication and repair. BIOCHIMICA ET BIOPHYSICA ACTA 2015; 1853:1253-71. [PMID: 25655665 PMCID: PMC4576882 DOI: 10.1016/j.bbamcr.2015.01.018] [Citation(s) in RCA: 178] [Impact Index Per Article: 17.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/23/2014] [Revised: 01/13/2015] [Accepted: 01/26/2015] [Indexed: 10/24/2022]
Abstract
Fe-S clusters are partners in the origin of life that predate cells, acetyl-CoA metabolism, DNA, and the RNA world. The double helix solved the mystery of DNA replication by base pairing for accurate copying. Yet, for genome stability necessary to life, the double helix has equally important implications for damage repair. Here we examine striking advances that uncover Fe-S cluster roles both in copying the genetic sequence by DNA polymerases and in crucial repair processes for genome maintenance, as mutational defects cause cancer and degenerative disease. Moreover, we examine an exciting, controversial role for Fe-S clusters in a third element required for life - the long-range coordination and regulation of replication and repair events. By their ability to delocalize electrons over both Fe and S centers, Fe-S clusters have unbeatable features for protein conformational control and charge transfer via double-stranded DNA that may fundamentally transform our understanding of life, replication, and repair. This article is part of a Special Issue entitled: Fe/S proteins: Analysis, structure, function, biogenesis and diseases.
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Affiliation(s)
- Jill O Fuss
- Life Sciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA 94720, USA.
| | - Chi-Lin Tsai
- Life Sciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA 94720, USA
| | - Justin P Ishida
- Life Sciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA 94720, USA
| | - John A Tainer
- Life Sciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA 94720, USA; Department of Molecular and Cellular Oncology, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030, USA.
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Paul VD, Lill R. Biogenesis of cytosolic and nuclear iron-sulfur proteins and their role in genome stability. BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH 2015; 1853:1528-39. [PMID: 25583461 DOI: 10.1016/j.bbamcr.2014.12.018] [Citation(s) in RCA: 176] [Impact Index Per Article: 17.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/02/2014] [Revised: 12/08/2014] [Accepted: 12/12/2014] [Indexed: 01/09/2023]
Abstract
Iron-sulfur (Fe-S) clusters are versatile protein cofactors that require numerous components for their synthesis and insertion into apoproteins. In eukaryotes, maturation of cytosolic and nuclear Fe-S proteins is accomplished by cooperation of the mitochondrial iron-sulfur cluster (ISC) assembly and export machineries, and the cytosolic iron-sulfur protein assembly (CIA) system. Currently, nine CIA proteins are known to specifically assist the two major steps of the biogenesis reaction. They are essential for cell viability and conserved from yeast to man. The essential character of this biosynthetic process is explained by the involvement of Fe-S proteins in central processes of life, e.g., protein translation and numerous steps of nuclear DNA metabolism such as DNA replication and repair. Malfunctioning of these latter Fe-S enzymes leads to genome instability, a hallmark of cancer. This review is focused on the maturation and biological function of cytosolic and nuclear Fe-S proteins, a topic of central interest for both basic and medical research. This article is part of a Special Issue entitled: Fe/S proteins: Analysis, structure, function, biogenesis and diseases.
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Affiliation(s)
- Viktoria Désirée Paul
- Institut für Zytobiologie und Zytopathologie, Philipps-Universität Marburg, Robert-Koch-Straße 6, 35032 Marburg, Germany
| | - Roland Lill
- Institut für Zytobiologie und Zytopathologie, Philipps-Universität Marburg, Robert-Koch-Straße 6, 35032 Marburg, Germany; LOEWE Zentrum für Synthetische Mikrobiologie SynMikro, Hans-Meerwein-Str., 35043 Marburg, Germany.
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Khrenova M, Savitsky AP, Topol IA, Nemukhin AV. Exploration of the zinc finger motif in controlling activity of matrix metalloproteinases. J Phys Chem B 2014; 118:13505-12. [PMID: 25375834 PMCID: PMC4254000 DOI: 10.1021/jp5088702] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2014] [Revised: 11/05/2014] [Indexed: 12/19/2022]
Abstract
Discovering ways to control the activity of matrix metalloproteinases (MMPs), zinc-dependent enzymes capable of degrading extracellular matrix proteins, is an important field of cancer research. We report here a novel strategy for assembling MMP inhibitors on the basis of oligopeptide ligands by exploring the pattern known as the zinc finger motif. Advanced molecular modeling tools were used to characterize the structural binding motifs of experimentally tested MMP inhibitors, as well as those of newly proposed peptidomimetics, in their zinc-containing active sites. The results of simulations based on the quantum mechanics/molecular mechanics (QM/MM) approach and Car-Parrinello molecular dynamics with QM/MM potentials demonstrate that, upon binding of Regasepin1, a known MMP-9 inhibitor, the Zn(2+)(His3) structural element is rearranged to the Zn(2+)(Cys2His2) zinc finger motif, in which two Cys residues are borrowed from the ligand. Following consideration of the crystal structure of MMP-2 with its inhibitor, the oligopeptide APP-IP, we proposed a new peptidomimetic with two replacements in the substrate, Tyr3Cys and Asp6Cys. Simulations show that this peptide variant blocks an enzyme active site by the Zn(2+)(Cys2His2) zinc finger construct. Similarly, a natural substrate of MMP-2, Ace-Gln-Gly ∼ Ile-Ala-Gly-Nme, can be converted to an inhibiting compound by two replacements, Ile by Cys and Gly by the d isomer of Cys, favoring formation of the zinc finger motif.
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Affiliation(s)
- Maria
G. Khrenova
- A.N. Bach Institute
of Biochemistry of the Russian Academy of Science, Leninsky Prospect, 33, Moscow 119071, Russian Federation
- Chemistry
Department, M.V. Lomonosov Moscow State
University, Leninskie
Gory 1/3, Moscow, 119991, Russian Federation
| | - Alexander P. Savitsky
- A.N. Bach Institute
of Biochemistry of the Russian Academy of Science, Leninsky Prospect, 33, Moscow 119071, Russian Federation
| | - Igor A. Topol
- Advanced
Biomedical Computing Center, Information Systems Program, Leidos Biomedical
Research Inc., Frederick National Laboratory
for Cancer Research, Frederick, Maryland 21702, United States
| | - Alexander V. Nemukhin
- Chemistry
Department, M.V. Lomonosov Moscow State
University, Leninskie
Gory 1/3, Moscow, 119991, Russian Federation
- N.M.
Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Kosygina 4, Moscow, 119334, Russian Federation
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