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• W3132816327 abstract "Unrepaired DNA–protein cross-links, due to their bulky nature, can stall replication forks and result in genome instability. Large DNA–protein cross-links can be cleaved into DNA–peptide cross-links, but the extent to which these smaller fragments disrupt normal replication is not clear. Ethylene dibromide (1,2-dibromoethane) is a known carcinogen that can cross-link the repair protein O6-alkylguanine-DNA alkyltransferase (AGT) to the N6 position of deoxyadenosine (dA) in DNA, as well as four other positions in DNA. We investigated the effect of a 15-mer peptide from the active site of AGT, cross-linked to the N6 position of dA, on DNA replication by human translesion synthesis DNA polymerases (Pols) η, ⍳, and κ. The peptide–DNA cross-link was bypassed by the three polymerases at different rates. In steady-state kinetics, the specificity constant (kcat/Km) for incorporation of the correct nucleotide opposite to the adduct decreased by 220-fold with Pol κ, tenfold with pol η, and not at all with Pol ⍳. Pol η incorporated all four nucleotides across from the lesion, with the preference dT > dC > dA > dG, while Pol ⍳ and κ only incorporated the correct nucleotide. However, LC-MS/MS analysis of the primer-template extension product revealed error-free bypass of the cross-linked 15-mer peptide by Pol η. We conclude that a bulky 15-mer peptide cross-linked to the N6 position of dA can retard polymerization and cause miscoding but that overall fidelity is not compromised because only correct pairs are extended. Unrepaired DNA–protein cross-links, due to their bulky nature, can stall replication forks and result in genome instability. Large DNA–protein cross-links can be cleaved into DNA–peptide cross-links, but the extent to which these smaller fragments disrupt normal replication is not clear. Ethylene dibromide (1,2-dibromoethane) is a known carcinogen that can cross-link the repair protein O6-alkylguanine-DNA alkyltransferase (AGT) to the N6 position of deoxyadenosine (dA) in DNA, as well as four other positions in DNA. We investigated the effect of a 15-mer peptide from the active site of AGT, cross-linked to the N6 position of dA, on DNA replication by human translesion synthesis DNA polymerases (Pols) η, ⍳, and κ. The peptide–DNA cross-link was bypassed by the three polymerases at different rates. In steady-state kinetics, the specificity constant (kcat/Km) for incorporation of the correct nucleotide opposite to the adduct decreased by 220-fold with Pol κ, tenfold with pol η, and not at all with Pol ⍳. Pol η incorporated all four nucleotides across from the lesion, with the preference dT > dC > dA > dG, while Pol ⍳ and κ only incorporated the correct nucleotide. However, LC-MS/MS analysis of the primer-template extension product revealed error-free bypass of the cross-linked 15-mer peptide by Pol η. We conclude that a bulky 15-mer peptide cross-linked to the N6 position of dA can retard polymerization and cause miscoding but that overall fidelity is not compromised because only correct pairs are extended. The preservation of genome integrity is vital for the proper development of an organism. Cells are subjected to multiple endogenous and exogenous agents capable of causing lesions and affect multiple DNA transaction processes (e.g., replication, repair, and transcription) (1Colombo C.V. Gnugnoli M. Gobbini E. Longhese M.P. How do cells sense DNA lesions?.Biochem. Soc. Trans. 2020; 48: 677-691Crossref PubMed Scopus (6) Google Scholar). Covalent DNA–protein cross-links are bulky lesions and can be toxic if left unrepaired (2Tretyakova N.Y. Groehler A. Ji S. DNA–protein cross-links: Formation, structural identities, and biological outcomes.Acc. Chem. Res. 2015; 48: 1631-1644Crossref PubMed Scopus (95) Google Scholar); they are formed from both exogenous and endogenous sources. Accumulation of these cross-links has been associated with aging, cancer, neurodegeneration, and Ruijs–Aalfs syndrome (2Tretyakova N.Y. Groehler A. Ji S. DNA–protein cross-links: Formation, structural identities, and biological outcomes.Acc. Chem. Res. 2015; 48: 1631-1644Crossref PubMed Scopus (95) Google Scholar, 3Niedernhofer L.J. Gurkar A.U. Wang Y. Vijg J. Hoeijmakers J.H.J. Robbins P.D. Nuclear genomic instability and aging.Annu. Rev. Biochem. 2018; 87: 295-322Crossref PubMed Scopus (77) Google Scholar, 4Li F. Raczynska J.E. Chen Z. Yu H. Structural insight into DNA-dependent activation of human metalloprotease Spartan.Cell Rep. 2019; 26: 3336-3346.e3334Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 5Vaz B. Popovic M. Ramadan K. DNA-protein crosslink proteolysis repair.Trends Biochem. Sci. 2017; 42: 483-495Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 6Ruggiano A. Ramadan K. DNA–protein crosslink proteases in genome stability.Commun. Biol. 2021; 4: 11Crossref PubMed Scopus (8) Google Scholar), and there has been considerable interest in both DNA–protein cross-links and proteases that can act on them (6Ruggiano A. Ramadan K. DNA–protein crosslink proteases in genome stability.Commun. Biol. 2021; 4: 11Crossref PubMed Scopus (8) Google Scholar). Several laboratories have shown that both reversible and irreversible DNA–protein cross-links can be formed under physiological conditions, that there are DNA-stimulated proteases that can act on these, and that DNA–protein cross-links can be bypassed and can miscode (2Tretyakova N.Y. Groehler A. Ji S. DNA–protein cross-links: Formation, structural identities, and biological outcomes.Acc. Chem. Res. 2015; 48: 1631-1644Crossref PubMed Scopus (95) Google Scholar, 7Wickramaratne S. Boldry E.J. Buehler C. Wang Y.-C. Distefano M.D. Tretyakova N.Y. Error-prone translesion synthesis past DNA-peptide cross-links conjugated to the major groove of DNA via C5 of thymidine.J. Biol. Chem. 2015; 290: 775-787Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 8Groehler A.T. Villalta P.W. Campbell C. Tretyakova N. Covalent DNA-protein cross-linking by phosphoramide mustard and nornitrogen mustard in human cells.Chem. Res. Toxicol. 2016; 29: 190-202Crossref PubMed Scopus (35) Google Scholar, 9Pande P. Ji S. Mukherjee S. Schärer O.D. Tretyakova N.Y. Basu A.K. Mutagenicity of a model DNA-peptide cross-link in human cells: Roles of translesion synthesis DNA polymerases.Chem. Res. Toxicol. 2017; 30: 669-677Crossref PubMed Scopus (16) Google Scholar, 10Ji S. Shao H. Han Q. Seiler C.L. Tretyakova N.Y. Reversible DNA-protein cross-linking at epigenetic DNA marks.Angew. Chem. Int. Ed. Engl. 2017; 56: 14130-14134Crossref PubMed Scopus (47) Google Scholar, 11Ji S. Park D. Kropachev K. Kolbanovskiy M. Fu I. Broyde S. Essawy M. Geacintov N.E. Tretyakova N.Y. 5-Formylcytosine-induced DNA–peptide cross-links reduce transcription efficiency, but do not cause transcription errors in human cells.J. Biol. Chem. 2019; 294: 18387-18397Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar, 12Yang K. Park D. Tretyakova N.Y. Greenberg M.M. Histone tails decrease N7-methyl-2′-deoxyguanosine depurination and yield DNA–protein cross-links in nucleosome core particles and cells.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E11212-E11220Crossref PubMed Scopus (23) Google Scholar, 13Mohni K.N. Wessel S.R. Zhao R. Wojciechowski A.C. Luzwick J.W. Layden H. Eichman B.F. Thompson P.S. Mehta K.P.M. Cortez D. HMCES maintains genome integrity by shielding abasic sites in single-strand DNA.Cell. 2019; 176: 144-153.e113Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 14Thompson P.S. Amidon K.M. Mohni K.N. Cortez D. Eichman B.F. Protection of abasic sites during DNA replication by a stable thiazolidine protein-DNA cross-link.Nat. Struct. Mol. Biol. 2019; 26: 613-618Crossref PubMed Scopus (28) Google Scholar, 15Svoboda M. Konvalinka J. Trempe J.F. Grantz Saskova K. The yeast proteases Ddi1 and Wss1 are both involved in the DNA replication stress response.DNA Repair. 2019; 80: 45-51Crossref PubMed Scopus (18) Google Scholar, 16Stingele J. Bellelli R. Alte F. Hewitt G. Sarek G. Maslen S.L. Tsutakawa S.E. Borg A. Kjær S. Tainer J.A. Skehel J.M. Groll M. Boulton S.J. Mechanism and regulation of DNA-protein crosslink repair by the DNA-dependent metalloprotease SPRTN.Mol. Cell. 2016; 64: 688-703Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 17Vaz B. Popovic M. Newman J.A. Fielden J. Aitkenhead H. Halder S. Singh A.N. Vendrell I. Fischer R. Torrecilla I. Drobnitzky N. Freire R. Amor D.J. Lockhart P.J. Kessler B.M. et al.Metalloprotease SPRTN/DVC1 orchestrates replication-coupled DNA-protein crosslink repair.Mol. Cell. 2016; 64: 704-719Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 18Larsen N.B. Gao A.O. Sparks J.L. Gallina I. Wu R.A. Mann M. Räschle M. Walter J.C. Duxin J.P. Replication-coupled DNA-protein crosslink repair by SPRTN and the proteasome in xenopus egg extracts.Mol. Cell. 2019; 73: 574-588.e577Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 19Enderle J. Dorn A. Beying N. Trapp O. Puchta H. The protease WSS1A, the endonuclease MUS81, and the phosphodiesterase TDP1 are involved in independent pathways of DNA-protein crosslink repair in plants.Plant Cell. 2019; 31: 775-790Crossref PubMed Scopus (12) Google Scholar). The number of cross-links in a cell has been estimated to be high (20Chan W. Ham Y.H. Jin L. Chan H.W. Wong Y.L. Chan C.K. Chung P.Y. Quantification of a novel DNA-protein cross-link product formed by reacting apurinic/apyrimidinic sites in DNA with cysteine residues in protein by liquid chromatography-tandem mass spectrometry coupled with the stable isotope-dilution method.Anal. Chem. 2019; 91: 4987-4994Crossref PubMed Scopus (8) Google Scholar) but the exact number is not known. There is now evidence that DNA–protein adducts may be important in disease states, e.g., cross-linking was reported to be increased following ischemic reperfusion in cardiomyocytes (21Groehler A. Kren S. Li Q. Robledo-Villafane M. Schmidt J. Garry M. Tretyakova N. Oxidative cross-linking of proteins to DNA following ischemia-reperfusion injury.Free Radic. Biol. Med. 2018; 120: 89-101Crossref PubMed Scopus (13) Google Scholar). The list of cross-linked sites includes multiple DNA bases (G, C, T, A) and their modifications (e.g., N7-Me G (12Yang K. Park D. Tretyakova N.Y. Greenberg M.M. Histone tails decrease N7-methyl-2′-deoxyguanosine depurination and yield DNA–protein cross-links in nucleosome core particles and cells.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E11212-E11220Crossref PubMed Scopus (23) Google Scholar), abasic sites (13Mohni K.N. Wessel S.R. Zhao R. Wojciechowski A.C. Luzwick J.W. Layden H. Eichman B.F. Thompson P.S. Mehta K.P.M. Cortez D. HMCES maintains genome integrity by shielding abasic sites in single-strand DNA.Cell. 2019; 176: 144-153.e113Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), and 5-formyl dC (10Ji S. Shao H. Han Q. Seiler C.L. Tretyakova N.Y. Reversible DNA-protein cross-linking at epigenetic DNA marks.Angew. Chem. Int. Ed. Engl. 2017; 56: 14130-14134Crossref PubMed Scopus (47) Google Scholar, 11Ji S. Park D. Kropachev K. Kolbanovskiy M. Fu I. Broyde S. Essawy M. Geacintov N.E. Tretyakova N.Y. 5-Formylcytosine-induced DNA–peptide cross-links reduce transcription efficiency, but do not cause transcription errors in human cells.J. Biol. Chem. 2019; 294: 18387-18397Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar)). The list of proteins in the cross-links includes histones (10Ji S. Shao H. Han Q. Seiler C.L. Tretyakova N.Y. Reversible DNA-protein cross-linking at epigenetic DNA marks.Angew. Chem. Int. Ed. Engl. 2017; 56: 14130-14134Crossref PubMed Scopus (47) Google Scholar, 12Yang K. Park D. Tretyakova N.Y. Greenberg M.M. Histone tails decrease N7-methyl-2′-deoxyguanosine depurination and yield DNA–protein cross-links in nucleosome core particles and cells.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E11212-E11220Crossref PubMed Scopus (23) Google Scholar), HMCES (13Mohni K.N. Wessel S.R. Zhao R. Wojciechowski A.C. Luzwick J.W. Layden H. Eichman B.F. Thompson P.S. Mehta K.P.M. Cortez D. HMCES maintains genome integrity by shielding abasic sites in single-strand DNA.Cell. 2019; 176: 144-153.e113Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), and numerous other proteins (21Groehler A. Kren S. Li Q. Robledo-Villafane M. Schmidt J. Garry M. Tretyakova N. Oxidative cross-linking of proteins to DNA following ischemia-reperfusion injury.Free Radic. Biol. Med. 2018; 120: 89-101Crossref PubMed Scopus (13) Google Scholar, 22Loecken E.M. Guengerich F.P. Reactions of glyceraldehyde 3-phosphate dehydrogenase sulfhydryl groups with bis-electrophiles produce DNA–protein cross-links but not mutations.Chem. Res. Toxicol. 2008; 21: 453-458Crossref PubMed Scopus (26) Google Scholar, 23Loecken E.M. Dasari S. Hill S. Tabb D.L. Guengerich F.P. The bis-electrophile diepoxybutane cross-links DNA to human histones but does not result in enhanced mutagenesis in recombinant systems.Chem. Res. Toxicol. 2009; 22: 1069-1076Crossref PubMed Scopus (19) Google Scholar, 24Loeber R.L. Michaelson-Richie E.D. Codreanu S.G. Liebler D.C. Campbell C.R. Tretyakova N.Y. Proteomic analysis of DNA−protein cross-linking by antitumor nitrogen mustards.Chem. Res. Toxicol. 2009; 22: 1151-1162Crossref PubMed Scopus (64) Google Scholar, 25Ham Y.-H. Chan K.K.J. Madej D. Lam H. Chan W. Proteomics study of DNA–protein crosslinks in methylmethanesulfonate and Fe2+-EDTA-exposed human cells.Chem. Res. Toxicol. 2020; 33: 2739-2744Crossref PubMed Scopus (3) Google Scholar). Some reversible lysine cross-links (Schiff bases) can also destabilize DNA and cause cleavage (12Yang K. Park D. Tretyakova N.Y. Greenberg M.M. Histone tails decrease N7-methyl-2′-deoxyguanosine depurination and yield DNA–protein cross-links in nucleosome core particles and cells.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E11212-E11220Crossref PubMed Scopus (23) Google Scholar, 26Xu W. Boyd R.M. Tree M.O. Samkari F. Zhao L. Mitochondrial transcription factor A promotes DNA strand cleavage at abasic sites.Proc. Natl. Acad. Sci. U. S. A. 2019; 116: 17792-17799Crossref PubMed Scopus (10) Google Scholar). However, the existence of a DNA–protein cross-link is not a priori evidence for miscoding (22Loecken E.M. Guengerich F.P. Reactions of glyceraldehyde 3-phosphate dehydrogenase sulfhydryl groups with bis-electrophiles produce DNA–protein cross-links but not mutations.Chem. Res. Toxicol. 2008; 21: 453-458Crossref PubMed Scopus (26) Google Scholar, 23Loecken E.M. Dasari S. Hill S. Tabb D.L. Guengerich F.P. The bis-electrophile diepoxybutane cross-links DNA to human histones but does not result in enhanced mutagenesis in recombinant systems.Chem. Res. Toxicol. 2009; 22: 1069-1076Crossref PubMed Scopus (19) Google Scholar). Some DNA–protein cross-links are common, and cells have specific enzymes to act on them. One example is tyrosyl-DNA phosphodiesterases (TDP), which are enzymes capable of breaking down the covalent bond between DNA and DNA topoisomerases (27Zhang H. Xiong Y. Chen J. DNA–protein cross-link repair: What do we know now?.Cell Biosci. 2020; 10: 3Crossref PubMed Scopus (12) Google Scholar). The activity of these enzymes is limited by substrate accessibility, suggesting that the adduct needs first to be hydrolyzed to a peptide (28Kühbacher U. Duxin J.P. How to fix DNA-protein crosslinks.DNA Repair. 2020; 94: 102924Crossref PubMed Scopus (8) Google Scholar). Recently DNA-activated proteases (6Ruggiano A. Ramadan K. DNA–protein crosslink proteases in genome stability.Commun. Biol. 2021; 4: 11Crossref PubMed Scopus (8) Google Scholar) and the proteasome have been suggested to be involved in proteolysis by cleaving large DNA–protein cross-links to DNA–peptide cross-links (18Larsen N.B. Gao A.O. Sparks J.L. Gallina I. Wu R.A. Mann M. Räschle M. Walter J.C. Duxin J.P. Replication-coupled DNA-protein crosslink repair by SPRTN and the proteasome in xenopus egg extracts.Mol. Cell. 2019; 73: 574-588.e577Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 29Lopez-Mosqueda J. Maddi K. Prgomet S. Kalayil S. Marinovic-Terzic I. Terzic J. Dikic I. SPRTN is a mammalian DNA-binding metalloprotease that resolves DNA-protein crosslinks.Elife. 2016; 5e21491Crossref PubMed Scopus (70) Google Scholar, 30Reinking H.K. Kang H.-S. Götz M.J. Li H.-Y. Kieser A. Zhao S. Acampora A.C. Weickert P. Fessler E. Jae L.T. Sattler M. Stingele J. DNA structure-specific cleavage of DNA-protein crosslinks by the SPRTN protease.Mol. Cell. 2020; 80: 102-113Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). Some DNA-dependent proteases bind to ubiquitin, or small ubiquitin-like modifier (SUMO), indicative of a role for posttranslational modification in proteolysis (31Borgermann N. Ackermann L. Schwertman P. Hendriks I.A. Thijssen K. Liu J.C. Lans H. Nielsen M.L. Mailand N. SUMOylation promotes protective responses to DNA-protein crosslinks.EMBO J. 2019; 38e101496Crossref PubMed Scopus (40) Google Scholar, 32Vaz B. Ruggiano A. Popovic M. Rodriguez-Berriguete G. Kilgas S. Singh A.N. Higgins G.S. Kiltie A.E. Ramadan K. SPRTN protease and SUMOylation coordinate DNA-protein crosslink repair to prevent genome instability.bioRxiv. 2020; ([preprint])https://doi.org/10.1101/2020.02.14.949289Crossref Scopus (0) Google Scholar, 33Sun Y. Jenkins L.M.M. Su Y.P. Nitiss K.C. Nitiss J.L. Pommier Y. A conserved SUMO-Ubiquitin pathway directed by RNF4/SLX5-SLX8 and PIAS4/SIZ1 drives proteasomal degradation of topoisomerase DNA-protein crosslinks.bioRxiv. 2019; ([preprint])https://doi.org/10.1101/707661Crossref Scopus (0) Google Scholar). Ubiquitination is a key step for proteolysis by the nuclear DNA-dependent metalloprotease SPRTN (18Larsen N.B. Gao A.O. Sparks J.L. Gallina I. Wu R.A. Mann M. Räschle M. Walter J.C. Duxin J.P. Replication-coupled DNA-protein crosslink repair by SPRTN and the proteasome in xenopus egg extracts.Mol. Cell. 2019; 73: 574-588.e577Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). SPRTN uses an accessory process (i.e., ubiquitination) to repair cross-links because the ubiquitin controls its activity (34Zhao S. Kieser A. Li H.-Y. Reinking H.K. Weickert P. Euteneuer S. Yaneva D. Acampora A.C. Götz M.J. Feederle R. Stingele J. A ubiquitin switch controls autocatalytic inactivation of the DNA–protein crosslink repair protease SPRTN.Nucleic Acids Res. 2020; 49: 902-915Crossref Scopus (6) Google Scholar). SPRTN-mediated proteolysis also depends on the location of protein cross-links on the DNA strands (30Reinking H.K. Kang H.-S. Götz M.J. Li H.-Y. Kieser A. Zhao S. Acampora A.C. Weickert P. Fessler E. Jae L.T. Sattler M. Stingele J. DNA structure-specific cleavage of DNA-protein crosslinks by the SPRTN protease.Mol. Cell. 2020; 80: 102-113Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). Thus, SPRTN has various limitations with respect to DNA–protein cross-link proteolysis. FAM111A is also known to act on DNA–protein cross-links and contains a trypsin-like domain (35Kojima Y. Machida Y. Palani S. Caulfield T.R. Radisky E.S. Kaufmann S.H. Machida Y.J. FAM111A protects replication forks from protein obstacles via its trypsin-like domain.Nat. Commun. 2020; 111318Crossref PubMed Scopus (20) Google Scholar). Repair mechanisms can target different components of a DNA–protein cross-link (i.e., the DNA molecule, the cross-link bond, or protein) and may depend on the stage of the cell cycle (27Zhang H. Xiong Y. Chen J. DNA–protein cross-link repair: What do we know now?.Cell Biosci. 2020; 10: 3Crossref PubMed Scopus (12) Google Scholar, 28Kühbacher U. Duxin J.P. How to fix DNA-protein crosslinks.DNA Repair. 2020; 94: 102924Crossref PubMed Scopus (8) Google Scholar, 36Stingele J. Bellelli R. Boulton S.J. Mechanisms of DNA-protein crosslink repair.Nat. Rev. Mol. Cell Biol. 2017; 18: 563-573Crossref PubMed Scopus (111) Google Scholar). Most DNA–protein cross-links are too large for DNA polymerase bypass (37Yudkina A.V. Dvornikova A.P. Zharkov D.O. Variable termination sites of DNA polymerases encountering a DNA-protein cross-link.PLoS One. 2018; 13e0198480Crossref PubMed Scopus (10) Google Scholar), and proteases are believed to cleave the proteins to smaller peptides that can be repaired by nucleotide excision repair (NER) (38Chesner L.N. Campbell C. A quantitative PCR-based assay reveals that nucleotide excision repair plays a predominant role in the removal of DNA-protein crosslinks from plasmids transfected into mammalian cells.DNA repair. 2018; 62: 18-27Crossref PubMed Scopus (10) Google Scholar) or homologous recombination (HR) or bypassed by translesion synthesis (TLS) polymerases (39Yang W. An overview of Y-Family DNA polymerases and a case study of human DNA polymerase η.Biochemistry. 2014; 53: 2793-2803Crossref PubMed Scopus (110) Google Scholar). NER and HR have been shown to remove protein cross-links smaller than 10 kDa (40Nakano T. Morishita S. Katafuchi A. Matsubara M. Horikawa Y. Terato H. Salem A.M.H. Izumi S. Pack S.P. Makino K. Ide H. Nucleotide excision repair and homologous recombination systems commit differentially to the repair of DNA-protein crosslinks.Mol. Cell. 2007; 28: 147-158Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Mammalian TLS polymerases include Y-family Pol η, ⍳, and κ and Rev1 (41Yang W. Gao Y. Translesion and repair DNA polymerases: Diverse structure and mechanism.Annu. Rev. Biochem. 2018; 87: 239-261Crossref PubMed Scopus (81) Google Scholar), which have low processivity and fidelity (39Yang W. An overview of Y-Family DNA polymerases and a case study of human DNA polymerase η.Biochemistry. 2014; 53: 2793-2803Crossref PubMed Scopus (110) Google Scholar). Only in a few cases has the mutagenic potential of a DNA–peptide cross-link been analyzed (42Naldiga S. Ji S. Thomforde J. Nicolae C.M. Lee M. Zhang Z. Moldovan G.-L. Tretyakova N.Y. Basu A.K. Error-prone replication of a 5-formylcytosine-mediated DNA-peptide cross-link in human cells.J. Biol. Chem. 2019; 294: 10619-10627Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar), and essentially all of the work in this area has been done with either uncharacterized cross-links or synthetic models. O6-Alkylguanine DNA-alkyltransferase (AGT, or MGMT), a ∼22 kDa protein, is a crucial repair system for protection from alkylating agents (43Ghodke P.P. Albertolle M.E. Johnson K.M. Guengerich F.P. Synthesis and characterization of site-specific O6-alkylguanine DNA-alkyl transferase-oligonucleotide crosslinks.Curr. Protoc. Nucleic Acid Chem. 2019; 76: e74Crossref PubMed Scopus (3) Google Scholar). It directly removes and transfers alkyl groups from the O6 position on guanine to the Cys-145 located in its active site. Ethylene dibromide (EDB, or 1,2-dibromoethane) is a known carcinogen that had formerly been used as a gasoline additive, soil fumigant, and pesticide (44Huff J.E. 1,2-Dibromoethane (ethylene dibromide).Environ. Health Perspect. 1983; 47: 359-363Crossref PubMed Scopus (27) Google Scholar, 45Rannug U. Beije B. The mutagenic effect of 1,2-dichloroethane on Salmonella typhimurium. II. Activation by the isolated perfused rat liver.Chem. Biol. Interact. 1979; 24: 265-285Crossref PubMed Scopus (69) Google Scholar, 46Rannug U.L.F. Genotoxic effects of 1,2-dibromoethane and 1,2-dichloroethane.Mutat. Res. Rev. Genet. Toxicol. 1980; 76: 269-295Crossref PubMed Scopus (119) Google Scholar, 47Wong L.C.K. Winston J.M. Hong C.B. Plotnick H. Carcinogenicity and toxicity of 1,2-dibromoethane in the rat.Toxicol. Appl. Pharmacol. 1982; 63: 155-165Crossref PubMed Scopus (80) Google Scholar). Overexpression of AGT paradoxically increased EDB toxicity and the incidence of base pair mutations in bacterial and mammalian cells (48Liu L. Pegg A.E. Williams K.M. Guengerich F.P. Paradoxical enhancement of the toxicity of 1,2-dibromoethane by O6-alkylguanine-DNA alkyltransferase.J. Biol. Chem. 2002; 277: 37920-37928Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 49Kalapila A.G. Pegg A.E. Alkyltransferase-mediated toxicity of bis-electrophiles in mammalian cells.Mutat. Res. 2010; 684: 35Crossref PubMed Scopus (9) Google Scholar). Cys-145 of AGT reacts with EDB via a nucleophilic substitution that results in a half-mustard intermediate, which cyclizes to form an unstable episulfonium ion that can form a covalent bond with DNA (Fig. 1). The AGT-EDB episulfonium ion can react with nucleobases at different positions, including the N7, N2, N1, and O6 atoms of dG and the N6 atom of dA (50Chowdhury G. Cho S.-H. Pegg A.E. Guengerich F.P. Detection and characterization of ethylene dibromide-derived DNA-crosslinks formed with O6-alkylguanine-DNA alkyltransferase.Angew. Chem. Int. Ed. 2013; 52: 12879-12882Crossref PubMed Scopus (14) Google Scholar, 51Liu L. Hachey D.L. Valadez G. Williams K.M. Guengerich F.P. Loktionova N.A. Kanugula S. Pegg A.E. Characterization of a mutagenic DNA adduct formed from 1,2-dibromoethane by O6-alkylguanine-DNA alkyltransferase.J. Biol. Chem. 2004; 279: 4250-4259Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The DNA-binding properties of AGT facilitate formation of these specific lesions. EDB is also known to cross-link DNA with the tripeptide glutathione (GSH) in a similar mechanism (52Sedgeman C.A. Su Y. Guengerich F.P. Formation of S-[2-(N6-deoxyadenosinyl)ethyl]glutathione in DNA and replication past the adduct by translesion DNA polymerases.Chem. Res. Toxicol. 2017; 30: 1188-1196Crossref PubMed Scopus (5) Google Scholar). Thus, this DNA–protein cross-link is directly relevant to a practical problem in toxicology, and other 1,2-dihaloalkanes and other bis-functional electrophiles are also relevant (53Valadez J.G. Liu L. Loktionova N.A. Pegg A.E. Guengerich F.P. Activation of bis-electrophiles to mutagenic conjugates by human O6-alkylguanine-DNA alkyltransferase.Chem. Res. Toxicol. 2004; 17: 972-982Crossref PubMed Scopus (39) Google Scholar, 54Liu L. Williams K.M. Guengerich F.P. Pegg A.E. O6-Alkylguanine-DNA alkyltransferase has opposing effects in modulating the genotoxicity of dibromomethane and bromomethyl acetate.Chem. Res. Toxicol. 2004; 17: 742-752Crossref PubMed Scopus (19) Google Scholar). We developed a procedure for the synthesis and characterization of a DNA–peptide cross-link at the N6 position of dA with a 15-mer peptide from the active site of AGT in order to investigate its effect on DNA replication by TLS polymerases. Full-length extension and single nucleotide insertion assays, steady-state kinetics, and LC-ESI-MS/MS analysis were performed to determine the efficiency and fidelity at the N6-dA-adducted peptide. A 15-mer peptide cross-linked to an oligonucleotide (N6-dA) was synthesized using the 15-mer peptide (acyl-PVPILIPCHRVVSSS-amide, AGT residues 138–152, with Cys-150 changed to Ser to permit exclusive modification at Cys-145) and a 6-chloropurine-containing oligonucleotide as outlined in Figure 2 (Scheme S1–S2, See Table S1 for oligonucleotide sequences). The peptide was treated with O-(mesitylsulfonyl)hydroxylamine (MSH), yielding amination of the cysteine to produce a dehydroalanine (dha) residue in its place (Scheme S1). The dha peptide was purified by HPLC (Fig. S1) and characterized by positive ESI-MS/MS (Figs. S2 and S3; Table S2). The 6-chloropurine-containing oligonucleotide was subjected to nucleophilic substitution with cystamine (Scheme S2), and the cystamine-containing oligonucleotide was purified by HPLC (Fig. S4) and characterized by MALDI MS (Fig. S5). Reduction of the cystamine-containing oligonucleotide with DTT yielded N6-(2-thioethyl)dA in the oligonucleotide, used for the next step without any purification (Fig. 2). Finally, the dha peptide was coupled with the thiol-containing oligonucleotide to obtain a 15-mer peptide cross-linked at the N6 position of dA via a two-carbon linker (Fig. 2). Alternatively, the 15-mer peptide cross-linked to DNA was also synthesized by a previously reported method (55Chandrasekar J. Wylder A.C. Silverman S.K. Phosphoserine lyase deoxyribozymes: DNA-catalyzed formation of dehydroalanine residues in peptides.J. Am. Chem. Soc. 2015; 137: 9575-9578Crossref PubMed Scopus (12) Google Scholar). The DNA–peptide cross-link was purified by gel electrophoresis (Fig. S6) and characterized by MALDI MS as well as ESI-MS (Figs. 3 and S7). This structure is analogous to the AGT-DNA cross-link induced by EDB (50Chowdhury G. Cho S.-H. Pegg A.E. Guengerich F.P. Detection and characterization of ethylene dibromide-derived DNA-crosslinks formed with O6-alkylguanine-DNA alkyltransferase.Angew. Chem. Int. Ed. 2013; 52: 12879-12882Crossref PubMed Scopus (14) Google Scholar), but an oxidized sulfur-bearing adduct was observed with an additional mass of 32 a. m. u. (Figs. 2, 3, and S7) which was characterized using LC-MS/MS analysis. The DNA moiety was hydrolyzed with HF to obtain a peptide adducted with adenine (Fig. S8). In mass spectral analysis (positive mode), two major m/z ions were observed, m/z 613.66 (+3) and 919.99 (+2) (Fig. S9), indicative of an oxidized sulfur atom, which was further confirmed from the fragmentation pattern (Fig. S10 and Table S3). Full-length extension and single nucleotide insertion assays were carried out with TLS polymerases. Full-length primer extension reactions (“running start”) were performed in the presence of all four dNTPs using a 12-mer primer (Fig. 4A). The N6-dA-peptide lesion in the template affected each polymerase differently. With the DNA–peptide cross-link, hPol η fully extended the primer with almost similar efficiency as the unmodified template (Fig. 4B, lanes 2–6 and 8–12). hPol ι mainly produced a single nucleotide incorporation product (Fig. 4C, lanes 20–24), similar to the unmodified template. The lesion also affected hPol κ activ" @default.
• W3132816327 created "2021-03-01" @default.
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• W3132816327 date "2021-01-01" @default.
• W3132816327 modified "2023-10-01" @default.
• W3132816327 title "Enzymatic bypass of an N6-deoxyadenosine DNA–ethylene dibromide–peptide cross-link by translesion DNA polymerases" @default.
• W3132816327 cites W1492628327 @default.
• W3132816327 cites W1498147771 @default.
• W3132816327 cites W1657627128 @default.
• W3132816327 cites W1965402781 @default.
• W3132816327 cites W1965416477 @default.
• W3132816327 cites W1967042727 @default.
• W3132816327 cites W1968572877 @default.
• W3132816327 cites W1978753758 @default.
• W3132816327 cites W1979278914 @default.
• W3132816327 cites W1980475249 @default.
• W3132816327 cites W1981926873 @default.
• W3132816327 cites W1983647569 @default.
• W3132816327 cites W1995018740 @default.
• W3132816327 cites W1998798847 @default.
• W3132816327 cites W1998969339 @default.
• W3132816327 cites W2000541687 @default.
• W3132816327 cites W2005084623 @default.
• W3132816327 cites W2005481926 @default.
• W3132816327 cites W2006812263 @default.
• W3132816327 cites W2013386599 @default.
• W3132816327 cites W2019077508 @default.
• W3132816327 cites W2038315508 @default.
• W3132816327 cites W2038507406 @default.
• W3132816327 cites W2046360867 @default.
• W3132816327 cites W2048024112 @default.
• W3132816327 cites W2060167345 @default.
• W3132816327 cites W2065350734 @default.
• W3132816327 cites W2070426133 @default.
• W3132816327 cites W2081477485 @default.
• W3132816327 cites W2085416172 @default.
• W3132816327 cites W2102030344 @default.
• W3132816327 cites W2111591146 @default.
• W3132816327 cites W2117867383 @default.
• W3132816327 cites W2119933989 @default.
• W3132816327 cites W2128988762 @default.
• W3132816327 cites W2145393821 @default.
• W3132816327 cites W2300593191 @default.
• W3132816327 cites W2327611812 @default.
• W3132816327 cites W2341980552 @default.
• W3132816327 cites W2414188270 @default.
• W3132816327 cites W2467588222 @default.
• W3132816327 cites W2519158631 @default.
• W3132816327 cites W2542256116 @default.
• W3132816327 cites W2543333792 @default.
• W3132816327 cites W2550813738 @default.
• W3132816327 cites W2565135425 @default.
• W3132816327 cites W2588973931 @default.
• W3132816327 cites W2606495359 @default.
• W3132816327 cites W2607151306 @default.
• W3132816327 cites W2731454206 @default.
• W3132816327 cites W2752723776 @default.
• W3132816327 cites W2783837646 @default.
• W3132816327 cites W2789435487 @default.
• W3132816327 cites W2794333766 @default.
• W3132816327 cites W2805829106 @default.
• W3132816327 cites W2809067396 @default.
• W3132816327 cites W2901218982 @default.
• W3132816327 cites W2903803657 @default.
• W3132816327 cites W2907856991 @default.
• W3132816327 cites W2908352025 @default.
• W3132816327 cites W2909053754 @default.
• W3132816327 cites W2913115963 @default.
• W3132816327 cites W2920482858 @default.
• W3132816327 cites W2922732417 @default.
• W3132816327 cites W2923574105 @default.
• W3132816327 cites W2925281243 @default.
• W3132816327 cites W2947930642 @default.
• W3132816327 cites W2954867976 @default.
• W3132816327 cites W2961557000 @default.
• W3132816327 cites W2967573610 @default.
• W3132816327 cites W2978199079 @default.
• W3132816327 cites W2979591665 @default.
• W3132816327 cites W2994811156 @default.
• W3132816327 cites W2996384555 @default.
• W3132816327 cites W3003171911 @default.
• W3132816327 cites W3011781484 @default.
• W3132816327 cites W3013513430 @default.
• W3132816327 cites W3013887952 @default.
• W3132816327 cites W3040903653 @default.
• W3132816327 cites W3080105230 @default.
• W3132816327 cites W3093856511 @default.
• W3132816327 cites W3118073226 @default.
• W3132816327 cites W3118677209 @default.
• W3132816327 cites W4233259050 @default.
• W3132816327 cites W4248361105 @default.
• W3132816327 doi "https://doi.org/10.1016/j.jbc.2021.100444" @default.
• W3132816327 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/8024977" @default.

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