DNA ligase I fidelity mediates the mutagenic ligation of pol β oxidized and mismatch nucleotide insertion products in base excision repair
2021; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1016/j.jbc.2021.100427
ISSN1083-351X
AutoresPradnya Kamble, Kalen Hall, Mahesh Chandak, Qun Tang, Melike Çağlayan,
Tópico(s)Carcinogens and Genotoxicity Assessment
ResumoDNA ligase I (LIG1) completes the base excision repair (BER) pathway at the last nick-sealing step after DNA polymerase (pol) β gap-filling DNA synthesis. However, the mechanism by which LIG1 fidelity mediates the faithful substrate–product channeling and ligation of repair intermediates at the final steps of the BER pathway remains unclear. We previously reported that pol β 8-oxo-2'-deoxyribonucleoside 5'-triphosphate insertion confounds LIG1, leading to the formation of ligation failure products with a 5'-adenylate block. Here, using reconstituted BER assays in vitro, we report the mutagenic ligation of pol β 8-oxo-2'-deoxyribonucleoside 5'-triphosphate insertion products and an inefficient ligation of pol β Watson–Crick–like dG:T mismatch insertion by the LIG1 mutant with a perturbed fidelity (E346A/E592A). Moreover, our results reveal that the substrate discrimination of LIG1 for the nicked repair intermediates with preinserted 3'-8-oxodG or mismatches is governed by mutations at both E346 and E592 residues. Finally, we found that aprataxin and flap endonuclease 1, as compensatory DNA-end processing enzymes, can remove the 5'-adenylate block from the abortive ligation products harboring 3'-8-oxodG or the 12 possible noncanonical base pairs. These findings contribute to the understanding of the role of LIG1 as an important determinant in faithful BER and how a multiprotein complex (LIG1, pol β, aprataxin, and flap endonuclease 1) can coordinate to prevent the formation of mutagenic repair intermediates with damaged or mismatched ends at the downstream steps of the BER pathway. DNA ligase I (LIG1) completes the base excision repair (BER) pathway at the last nick-sealing step after DNA polymerase (pol) β gap-filling DNA synthesis. However, the mechanism by which LIG1 fidelity mediates the faithful substrate–product channeling and ligation of repair intermediates at the final steps of the BER pathway remains unclear. We previously reported that pol β 8-oxo-2'-deoxyribonucleoside 5'-triphosphate insertion confounds LIG1, leading to the formation of ligation failure products with a 5'-adenylate block. Here, using reconstituted BER assays in vitro, we report the mutagenic ligation of pol β 8-oxo-2'-deoxyribonucleoside 5'-triphosphate insertion products and an inefficient ligation of pol β Watson–Crick–like dG:T mismatch insertion by the LIG1 mutant with a perturbed fidelity (E346A/E592A). Moreover, our results reveal that the substrate discrimination of LIG1 for the nicked repair intermediates with preinserted 3'-8-oxodG or mismatches is governed by mutations at both E346 and E592 residues. Finally, we found that aprataxin and flap endonuclease 1, as compensatory DNA-end processing enzymes, can remove the 5'-adenylate block from the abortive ligation products harboring 3'-8-oxodG or the 12 possible noncanonical base pairs. These findings contribute to the understanding of the role of LIG1 as an important determinant in faithful BER and how a multiprotein complex (LIG1, pol β, aprataxin, and flap endonuclease 1) can coordinate to prevent the formation of mutagenic repair intermediates with damaged or mismatched ends at the downstream steps of the BER pathway. Human DNA ligases (LIGs) (LIG1, LIG3, and LIG4) catalyze the formation of a phosphodiester bond between the 5'-phosphate (P) and 3'-hydroxyl (OH) termini of a DNA intermediate during DNA replication, repair, and genetic recombination (1Ellenberger T. Tomkinson A.E. Eukaryotic DNA ligases: Structural and functional insights.Annu. Rev. Biochem. 2008; 77: 313-338Crossref PubMed Scopus (241) Google Scholar, 2Timson D.J. Singleton M.R. Wigley D.B. DNA ligases in the repair and replication of DNA.Mutat. Res. 2000; 460: 301-318Crossref PubMed Scopus (127) Google Scholar, 3Tomkinson A.E. Mackey Z.B. Structure and function of mammalian DNA ligases.Mutat. Res. 1998; 407: 1-9Crossref PubMed Scopus (174) Google Scholar, 4Tomkinson A.E. Vijayakumar S. Pascal J.M. Ellenberger T. DNA ligases: Structure, reaction mechanism, and function.Chem. Rev. 2006; 106: 687-699Crossref PubMed Scopus (197) Google Scholar). The DNA ligation reaction by a LIG includes three chemical sequential steps and requires ATP and a divalent metal ion (Mg2+) (5Taylor M.R. Conrad J.A. Wahl D. O'Brien P.J. Kinetic mechanism of human DNA ligase I reveals magnesium-dependent changes in the rate-limiting step that compromise ligation efficiency.J. Biol. Chem. 2011; 286: 23054-23062Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). In the first step, ATP is hydrolyzed to produce an adenylate (AMP) that is then covalently linked to the ligase active site lysine, forming the adenylated ligase (6Cherepanov A.V. de Vries S. Dynamic mechanism of nick recognition by DNA ligase.Eur. J. Biochem. 2002; 269: 5993-5999Crossref PubMed Scopus (27) Google Scholar). Next, after binding of the adenylated ligase to a nicked DNA substrate, the AMP group is transferred to the 5'-P end of the nick, forming an adenylated DNA intermediate (7Yang S.W. Chan J.Y. Analysis of the formation of AMP-DNA intermediate and the successive reaction by human DNA ligases I and II.J. Biol. Chem. 1992; 267: 8117-8122Abstract Full Text PDF PubMed Google Scholar). In the final step, LIG catalyzes a nucleophilic attack of the 3'-OH in the DNA nick on the adenylated 5'-P to form a phosphodiester bond (8Dickson K.S. Burns C.M. Richardson J.P. Determination of the free-energy change for repair of a DNA phosphodiester bond.J. Biol. Chem. 2000; 275: 15828-15831Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Despite the fact that the ligation mechanism is a universally conserved process, we still lack the understanding of how LIG recognizes and processes damaged or mismatched DNA ends. Successful DNA ligation relies on the formation of a Watson–Crick base pair of the nicked DNA that is formed during prior gap-filling DNA synthesis by a DNA polymerase (9Çağlayan M. Interplay between DNA polymerases and DNA ligases: Influence on substrate channeling and the fidelity of DNA ligation.J. Mol. Biol. 2019; 431: 2068-2081Crossref PubMed Scopus (9) Google Scholar, 10Çağlayan M. Wilson S.H. Oxidant and environmental toxicant-induced effects compromise DNA ligation during base excision DNA repair.DNA Repair (Amst.). 2015; 35: 85-89Crossref PubMed Scopus (24) Google Scholar). Human DNA polymerases and LIGs have been considered as key determinants of genome integrity (11Showalter A.K. Lamarche B.J. Bakhtina M. Su M.I. Tang K.H. Tsai M.D. Mechanistic comparison of high-fidelity and error-prone DNA polymerases and ligases involved in DNA repair.Chem. Rev. 2006; 106: 340-360Crossref PubMed Scopus (56) Google Scholar). In our previous studies, we demonstrated the importance of the coordination between DNA polymerase (pol) β and LIG1 during the repair of single DNA base lesions through base excision repair (BER) (9Çağlayan M. Interplay between DNA polymerases and DNA ligases: Influence on substrate channeling and the fidelity of DNA ligation.J. Mol. Biol. 2019; 431: 2068-2081Crossref PubMed Scopus (9) Google Scholar, 10Çağlayan M. Wilson S.H. Oxidant and environmental toxicant-induced effects compromise DNA ligation during base excision DNA repair.DNA Repair (Amst.). 2015; 35: 85-89Crossref PubMed Scopus (24) Google Scholar, 12Çağlayan M. Wilson S.H. Role of DNA polymerase β oxidized nucleotide insertion in DNA ligation failure.J. Radiat. Res. 2017; 58: 603-607Crossref PubMed Scopus (5) Google Scholar, 13Çağlayan M. Horton J.K. Dai D.P. Stefanick D.F. Wilson S.H. Oxidized nucleotide insertion by pol β confounds ligation during base excision repair.Nat. Commun. 2017; 8: 14045Crossref PubMed Scopus (31) Google Scholar, 14Tang Q. Kamble P. Çağlayan M. DNA ligase I variants fail in the ligation of mutagenic repair intermediates with mismatches and oxidative DNA damage.Mutagenesis. 2020; 35: 391-404Crossref PubMed Scopus (2) Google Scholar, 15Çağlayan M. The ligation of pol β mismatch insertion products governs the formation of promutagenic base excision DNA repair intermediates.Nucleic Acids Res. 2020; 48: 3708-3721Crossref PubMed Scopus (0) Google Scholar, 16Çağlayan M. Pol β gap filling, DNA ligation and substrate-product channeling during base excision repair opposite oxidized 5-methylcytosine modifications.DNA Repair (Amst.). 2020; 95: 102945Crossref PubMed Scopus (5) Google Scholar). BER is a critical process for preventing the mutagenic and lethal consequences of DNA damage that arises from endogenous and environmental agents and underlies disease and aging (17Lindahl T. Keynote: Past, present, and future aspects of base excision repair.Prog. Nucleic Acid Res. Mol. Biol. 2001; 68: xvii-xxxCrossref PubMed Google Scholar, 18Beard W.A. Horton J.K. Prasad R. Wilson S.H. Eukaryotic base excision repair: New approaches shine light on mechanism.Annu. Rev. Biochem. 2019; 88: 137-162Crossref PubMed Scopus (67) Google Scholar). The repair pathway involves a series of sequential enzymatic steps that are tightly coordinated through protein–protein interactions in a process referred to as passing-the-baton or substrate-product channeling (19Wilson S.H. Kunkel T.A. Passing the baton in base excision repair.Nat. Struct. Biol. 2000; 7: 176-178Crossref PubMed Scopus (315) Google Scholar, 20Prasad R. Shock D.D. Beard W.A. Wilson S.H. Substrate channeling in mammalian base excision repair pathways: Passing the baton.J. Biol. Chem. 2010; 285: 40479-40488Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 21Prasad R. Beard W.A. Batra V.K. Liu Y. Shock D.D. Wilson S.H. A review of recent experiments on step-to-step "hand-off" of the DNA intermediates in mammalian base excision repair pathways.Mol. Biol. (Mosk.). 2011; 45: 586-600Crossref PubMed Scopus (8) Google Scholar). This mechanism includes hand off of repair intermediates from gap-filling DNA synthesis by pol β to the DNA ligation reaction by LIG1 at the downstream steps of the BER pathway (9Çağlayan M. Interplay between DNA polymerases and DNA ligases: Influence on substrate channeling and the fidelity of DNA ligation.J. Mol. Biol. 2019; 431: 2068-2081Crossref PubMed Scopus (9) Google Scholar). However, how deviations in the functional coordination between pol β and LIG1 affect the BER process remains largely unknown. In particular, abnormalities created due to incorporation of damaged or mismatch nucleotides by pol β could lead to the formation of toxic and mutagenic repair intermediates that can drive genome instability or cell death. For example, endogenous and exogenous oxidative stress can oxidize guanine in the nucleotide pool (dGTP), resulting in the formation of the most abundant form of oxidative DNA damage, that is, the nucleotide triphosphate, 2'-deoxyribonucleoside 5'-triphosphate (8-oxodGTP) (22Ventura I. Russo M.T. De Luca G. Bignami M. Oxidized purine nucleotides, genome instability and neurodegeneration.Mutat. Res. 2010; 703: 59-65Crossref PubMed Scopus (19) Google Scholar, 23Nakabeppu Y. Sakumi K. Sakamoto K. Tsuchimoto D. Tsuzuki T. Nakatsu Y. Mutagenesis and carcinogenesis caused by the oxidation of nucleic acids.Biol. Chem. 2006; 387: 373-379Crossref PubMed Scopus (201) Google Scholar). The deleterious effect of 8-oxodGTP is mediated through its incorporation into the genome by repair or replication DNA polymerases (24Katafuchi A. Nohmi T. DNA polymerases involved in the incorporation of oxidized nucleotides into DNA: Their efficiency and template base preference.Mutat. Res. 2010; 703: 24-31Crossref PubMed Scopus (35) Google Scholar). For example, pol β performs mutagenic repair by inserting 8-oxodGTP opposite adenine within an active site that exhibits a frayed structure because of the lack of base pairing with a template base after the oxidized nucleotide insertion (25Freudenthal B.D. Beard W.A. Perera L. Shock D.D. Kim T. Schlick T. Wilson S.H. Uncovering the polymerase-induced cytotoxicity of an oxidized nucleotide.Nature. 2016; 517: 635-639Crossref Scopus (107) Google Scholar). In our prior studies, we demonstrated that pol β 8-oxodGTP insertion confounds the DNA ligation step of the BER pathway and leads to the formation of a ligation failure product with a 5'-adenylate (AMP) block (12Çağlayan M. Wilson S.H. Role of DNA polymerase β oxidized nucleotide insertion in DNA ligation failure.J. Radiat. Res. 2017; 58: 603-607Crossref PubMed Scopus (5) Google Scholar, 13Çağlayan M. Horton J.K. Dai D.P. Stefanick D.F. Wilson S.H. Oxidized nucleotide insertion by pol β confounds ligation during base excision repair.Nat. Commun. 2017; 8: 14045Crossref PubMed Scopus (31) Google Scholar, 14Tang Q. Kamble P. Çağlayan M. DNA ligase I variants fail in the ligation of mutagenic repair intermediates with mismatches and oxidative DNA damage.Mutagenesis. 2020; 35: 391-404Crossref PubMed Scopus (2) Google Scholar). We also reported the effect of pol β mismatch nucleotide insertion on the substrate-product channeling to LIG1 during the final steps of BER and the fidelity of this mechanism in the presence of the epigenetically important 5-methylcytosine base modifications (15Çağlayan M. The ligation of pol β mismatch insertion products governs the formation of promutagenic base excision DNA repair intermediates.Nucleic Acids Res. 2020; 48: 3708-3721Crossref PubMed Scopus (0) Google Scholar, 16Çağlayan M. Pol β gap filling, DNA ligation and substrate-product channeling during base excision repair opposite oxidized 5-methylcytosine modifications.DNA Repair (Amst.). 2020; 95: 102945Crossref PubMed Scopus (5) Google Scholar). The X-ray crystal structures of the three human LIGs in complex with DNA have revealed a conserved three-domain architecture that encircles the nicked DNA and induce partial unwinding and alignment of the 3'- and 5'-DNA ends (26Pascal J.M. O'Brien P.J. Tomkinson A.E. Ellenberger T. Human DNA ligase I completely encircles and partially unwinds nicked DNA.Nature. 2004; 432: 473-478Crossref PubMed Scopus (251) Google Scholar, 27Cotner-Gohara E. Kim I.K. Hammel M. Tainer J.A. Tomkinson A.E. Ellenberger T. Human DNA ligase III recognizes DNA ends by dynamic switching between two DNA-bound states.Biochemistry. 2010; 49: 6165-6176Crossref PubMed Scopus (79) Google Scholar, 28Conlin M.P. Reid D.A. Small G.W. Chang H.H. Watanabe G. Lieber M.R. Ramsden D.A. Rothenberg E. DNA Ligase IV guides end-processing choice during nonhomologous end joining.Cell Rep. 2017; 20: 2810-2819Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 29Ochi T. Gu X. Blundell T.L. Structure of the catalytic region of DNA ligase IV in complex with an Artemis fragment sheds light on double-strand break repair.Structure. 2013; 21: 672-679Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 30Ochi T. Wu Q. Chirgadze D.Y. Grossmann J.G. Bolanos-Garcia V.M. Blundell T.L. Structural insights into the role of domain flexibility in human DNA ligase IV.Structure. 2012; 20: 1212-1222Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 31Kaminski A.M. Tumbale P.P. Schellenberg M.J. Williams R.S. Williams J.G. Kunkel T.A. Pedersen L.C. Bebenek K. Structures of DNA-bound human ligase IV catalytic core reveal insights into substrate binding and catalysis.Nat. Commun. 2018; 9: 2642Crossref PubMed Scopus (23) Google Scholar, 32Tumbale P.P. Jurkiw T.J. Schellenberg M.J. Riccio A.A. O'Brien P.J. Williams R.S. Two-tiered enforcement of high-fidelity DNA ligation.Nat. Commun. 2019; 10: 5431Crossref PubMed Scopus (11) Google Scholar). Recently, the crystal structures of LIG1 revealed that the enzyme's high fidelity is mediated by Mg2+-dependent DNA binding (referred as MgHiFi metal site), a strategy that the enzyme uses during the adenyl transfer and nick-sealing steps of the ligation reaction, and is scaffolded by the two conserved amino acid residues (E346 and E592) (32Tumbale P.P. Jurkiw T.J. Schellenberg M.J. Riccio A.A. O'Brien P.J. Williams R.S. Two-tiered enforcement of high-fidelity DNA ligation.Nat. Commun. 2019; 10: 5431Crossref PubMed Scopus (11) Google Scholar). Moreover, mutations at these glutamate residues to alanine (E346A/E592A or EE/AA) lead to an LIG1 enzyme with lower fidelity and create an open cavity that accommodates a damaged DNA terminus at the protein active site (32Tumbale P.P. Jurkiw T.J. Schellenberg M.J. Riccio A.A. O'Brien P.J. Williams R.S. Two-tiered enforcement of high-fidelity DNA ligation.Nat. Commun. 2019; 10: 5431Crossref PubMed Scopus (11) Google Scholar). However, how such a distinct environment, which the staggered LIG1 conformation creates due to mutations at the MgHiFi metal site, affects ligase substrate discrimination and coordination with pol β during substrate–product channeling of repair intermediates with damaged or mismatched DNA ends at the downstream steps of BER remains entirely undefined. Aprataxin (APTX), a member of the histidine triad superfamily, hydrolyzes an adenylate (AMP) moiety from the 5'-end of DNA ligation failure products and allows further attempts at completing repair (33Tumbale P. Schellenberg M.J. Mueller G.A. Fairweather E. Watson M. Little J.N. Krahn J. Waddell I. London R.E. Williams R.S. Mechanism of APTX nicked DNA sensing and pleiotropic inactivation in neurodegenerative disease.EMBO J. 2018; 37e98875Crossref PubMed Scopus (8) Google Scholar). In our previous studies, we reported a role of flap endonuclease 1 (FEN1) for the processing of blocked repair intermediates with 5'-AMP (34Çağlayan M. Batra V.K. Sassa A. Prasad R. Wilson S.H. Role of polymerase β in complementing aprataxin deficiency during abasic-site base excision repair.Nat. Struct. Mol. Biol. 2014; 21: 497-499Crossref PubMed Scopus (27) Google Scholar, 35Çağlayan M. Horton J.K. Prasad R. Wilson S.H. Complementation of aprataxin deficiency by base excision repair enzymes.Nucleic Acids Res. 2015; 43: 2271-2281Crossref PubMed Scopus (18) Google Scholar, 36Çağlayan M. Prasad R. Krasich R. Longley M.J. Kadoda K. Tsuda M. Sasanuma H. Takeda S. Tano K. Copeland W.C. Wilson S.H. Complementation of aprataxin deficiency by base excision repair enzymes in mitochondrial extracts.Nucleic Acids Res. 2015; 45: 10079-10088Crossref Scopus (13) Google Scholar). This compensatory mechanism is important to complement a deficiency in APTX activity, which is associated with mutations in the aptx gene and linked to the autosomal recessive neurodegenerative disorder ataxia with oculomotor apraxia type 1 (AOA1) (37Ahel I. Rass U. El-Khamisy S.F. Katyal S. Clements P.M. McKinnon P.J. Caldecott K.W. West S.C. The neurodegenerative disease protein aprataxin resolves abortive DNA ligation intermediates.Nature. 2006; 443: 713-716Crossref PubMed Scopus (278) Google Scholar). Recently, the process by which APTX removes 5'-AMP during the ligation of oxidative DNA damage-containing DNA ends has been suggested as a surveillance mechanism that protects LIG1 from ligation failure (32Tumbale P.P. Jurkiw T.J. Schellenberg M.J. Riccio A.A. O'Brien P.J. Williams R.S. Two-tiered enforcement of high-fidelity DNA ligation.Nat. Commun. 2019; 10: 5431Crossref PubMed Scopus (11) Google Scholar). Yet, the specificity of APTX and FEN1 for the repair intermediates including 5'-AMP and 3'-damaged or mismatched ends that mimic the ligation failure products after pol β–mediated mutagenic 8-oxodGTP or mismatch insertion is still unknown. In this study, we examined the importance of LIG1 fidelity for faithful substrate–product channeling and ligation of repair intermediates at the final steps of the BER pathway. For this purpose, we evaluated the LIG1 mutants with lower fidelity (E346A, E592A, and EE/AA) for the ligation of pol β nucleotide insertion products and the nicked DNA substrates with 3'-preinserted damaged or mismatched bases in vitro. Our findings revealed the mutagenic ligation of pol β 8-oxodGTP insertion products and an inefficient DNA ligation after pol β Watson–Crick–like dGTP:T mismatch insertion by the LIG1 EE/AA mutant in a reconstituted BER reaction. Moreover, the ligation efficiency of the nicked repair intermediates with preinserted 3'-8-oxodG or mismatches was found to be dependent on the type of the template base and requires the presence of a double mutation at the E346 and E592 residues that typically ensure high fidelity. Finally, our findings demonstrated the compensatory roles of the DNA-end trimming enzymes, APTX and FEN1, for the processing of the ligation failure products with a 5'-AMP in the presence of 8-oxodG or all 12 possible mismatched bases at the 3'-end of the mutagenic repair intermediate. The findings herein contribute to our understanding of the efficiency and fidelity of substrate–product channeling during the final steps of BER in situations involving aberrant LIG1 fidelity and provide novel insight into the importance of the coordinated repair by a multiprotein complex for faithful BER. We previously demonstrated that the nicked repair product of pol β 8-oxodGTP insertion cannot be used as a DNA substrate by LIG1 during substrate–product channeling at the downstream steps of BER (12Çağlayan M. Wilson S.H. Role of DNA polymerase β oxidized nucleotide insertion in DNA ligation failure.J. Radiat. Res. 2017; 58: 603-607Crossref PubMed Scopus (5) Google Scholar, 13Çağlayan M. Horton J.K. Dai D.P. Stefanick D.F. Wilson S.H. Oxidized nucleotide insertion by pol β confounds ligation during base excision repair.Nat. Commun. 2017; 8: 14045Crossref PubMed Scopus (31) Google Scholar, 14Tang Q. Kamble P. Çağlayan M. DNA ligase I variants fail in the ligation of mutagenic repair intermediates with mismatches and oxidative DNA damage.Mutagenesis. 2020; 35: 391-404Crossref PubMed Scopus (2) Google Scholar, 15Çağlayan M. The ligation of pol β mismatch insertion products governs the formation of promutagenic base excision DNA repair intermediates.Nucleic Acids Res. 2020; 48: 3708-3721Crossref PubMed Scopus (0) Google Scholar, 16Çağlayan M. Pol β gap filling, DNA ligation and substrate-product channeling during base excision repair opposite oxidized 5-methylcytosine modifications.DNA Repair (Amst.). 2020; 95: 102945Crossref PubMed Scopus (5) Google Scholar). In the present study, we first evaluated the effect of low-fidelity LIG1 for the ligation of pol β 8-oxodGTP insertion products in vitro. For this purpose, we used the coupled assay that measures the activities of pol β and LIG1 simultaneously in a reaction mixture that includes LIG1 WT or EE/AA mutant, pol β, 8-oxodGTP, and one-nucleotide-gap DNA substrate with template base A or C (Fig. 1A). For the one-nucleotide-gap DNA substrate with template A, consistent with our previous studies (13Çağlayan M. Horton J.K. Dai D.P. Stefanick D.F. Wilson S.H. Oxidized nucleotide insertion by pol β confounds ligation during base excision repair.Nat. Commun. 2017; 8: 14045Crossref PubMed Scopus (31) Google Scholar, 15Çağlayan M. The ligation of pol β mismatch insertion products governs the formation of promutagenic base excision DNA repair intermediates.Nucleic Acids Res. 2020; 48: 3708-3721Crossref PubMed Scopus (0) Google Scholar), we observed that WT LIG1 fails after pol β 8-oxodGTP insertion opposite A (Fig. 1B, lanes 2–5). This feature of LIG1 results in ligation failure and accumulation of abortive ligation products with 5'-adenylate (5'-AMP). The observed ligation failure was accompanied by the formation of mutagenic ligation (i.e., sealing of the 3'-damage-containing nick intermediate, in this case, 8-oxodG) over the time of reaction incubation (Fig. 1C). Conversely, there was no ligation failure after pol β oxidized nucleotide insertions in the presence of the LIG1 mutant EE/AA that impairs the ligase fidelity. In this case, we only observed the products of mutagenic ligation after the pol β 8-oxodGTP insertion opposite A (Fig. 1B, lanes 6–9). The amount of this mutagenic ligation product was ∼10-fold higher than that of the product obtained with WT LIG1 (Fig. 1C). For the one-nucleotide-gap DNA substrate with template C, we also obtained the products of mutagenic ligation and ligation failure by WT LIG1 after pol β 8-oxodGTP insertion opposite C (Fig. 2A, lanes 2–8). However, due to the pol β weak insertion efficiency of 8-oxodGTP:C as reported previously (13Çağlayan M. Horton J.K. Dai D.P. Stefanick D.F. Wilson S.H. Oxidized nucleotide insertion by pol β confounds ligation during base excision repair.Nat. Commun. 2017; 8: 14045Crossref PubMed Scopus (31) Google Scholar, 15Çağlayan M. The ligation of pol β mismatch insertion products governs the formation of promutagenic base excision DNA repair intermediates.Nucleic Acids Res. 2020; 48: 3708-3721Crossref PubMed Scopus (0) Google Scholar), the mutagenic ligation and 5'-adenylate products accumulated at later time points of the reaction (2–6 min) compared with the products obtained at earlier time points (10–60 s) in the case of pol β 8-oxodGTP:A insertion (Fig. 1B versus Fig. 2B). Similarly, we observed only the products of mutagenic ligation in the coupled reactions that include the LIG1 low-fidelity mutant EE/AA (Fig. 2A, lanes 9–15). The amount of this mutagenic ligation product was also higher than that of the WT protein (Fig. 2B). Yet, the EE/AA mutant was ∼80-fold more efficient for ligation of the pol β product after 8-oxodGMP insertion opposite A relative to C for the initial time points (30 s and 60 s) that are common to both pol β insertion reactions (Fig. S1). In the control coupled reactions that include pol β, dGTP, and one-nucleotide-gap DNA substrate with template C, we evaluated the ligation of pol β dGTP:C insertion products in the presence of either WT protein or the LIG1 mutant EE/AA (Fig. 3A). The results demonstrated a complete ligation product over the time of reaction for both WT and the low-fidelity LIG1 mutant (Fig. 3B, lanes 2–8 and 9–15, respectively). In this case, we did not observe a significant difference in the amount of ligation products between WT and the EE/AA mutant (Fig. 3C). We then compared the efficiency of pol β nucleotide insertion and its conversion to the ligation products by WT or EE/AA mutant in the insertion (pol β alone) and coupled (pol β and LIG1) reactions separately (Fig. S2). The experiments demonstrated a more efficient conversion of pol β dGTP:C insertions to ligated repair products in the presence of the EE/AA mutant as shown by a faster decrease in the amount of the insertion products at the same time points of both reactions (Fig. S2, A and B). Overall results indicate that (i) the functional coordination between pol β and LIG1 is sensitive to the ligase fidelity and (ii) the cavity that is formed due to the EE/AA mutation at the MgHiFi of the LIG1 active site facilitates sealing of the nicked pol β repair product including an inserted 8-oxodGMP, while showing slight differences in the efficiency of mutagenic ligation depending on the type of template base to which pol β inserts the damaged nucleotide (Scheme S1). The mutations (P529L, E566K, R641L, and R771W) in the LIG1 gene have been identified in the patients with LIG1-deficiency syndrome that exhibit immunodeficiency and cancer predisposition (38Barnes D.E. Tomkinson A.E. Lehmann A.R. Webster A.D. Lindahl T. Mutations in the DNA ligase I gene of an individual with immunodeficiencies and cellular hypersensitivity to DNA-damaging agents.Cell. 1992; 69: 495-503Abstract Full Text PDF PubMed Scopus (224) Google Scholar, 39Webster A.D. Barnes D.E. Arlett C.F. Lehmann A.R. Lindahl T. Growth retardation and immunodeficiency in a patient with mutations in the DNA ligase I gene.Lancet. 1992; 339: 1508-1509Abstract PubMed Scopus (81) Google Scholar). These amino acid residues residing in the different domains of the protein are located apart from the MgHiFi (E346 and E592) at the LIG1 active site (Fig. S3). Recently, we reported that the LIG1 variants associated with LIG1-deficiency disease exhibit altered ligation fidelity for pol β–promoted mutagenesis products (14Tang Q. Kamble P. Çağlayan M. DNA ligase I variants fail in the ligation of mutagenic repair intermediates with mismatches and oxidative DNA damage.Mutagenesis. 2020; 35: 391-404Crossref PubMed Scopus (2) Google Scholar). In the present study, in addition to the LIG1 low-fidelity mutants, we evaluated the effect of the LIG1 variants for the ligation of pol β 8-oxodGTP insertion products in vitro using the same coupled assay as described above. In contrast to the mutagenic ligation observed with the LIG1 EE/AA mutant (Figs. 1 and 2), we obtained the ligation failure after the pol β 8-oxodGTP insertions by all LIG1 variants tested in this study (Fig. 4). For example, the LIG1 variants P529L, R771W, and R641L fail on the pol β repair product with an inserted 8-oxodGMP opposite C (Fig. 4A, lanes 6–9, 10–13, and 14–17, respectively), which yielded the ligation failure products in a BER reaction as also shown for WT LIG1 (Fig. 4A, lanes 2–5). The amounts of ligation failure products by the variants were higher than that of the WT protein and exhibit slight differences between the disease mutations (Fig. 4B). These findings suggest that the disease-associated LIG1 variants (i.e., P529L, R641L, and R771W) do not interfere with the ligase fidelity and exhibit WT level of ligation efficiency in preventing formation of mutagenic repair intermediates after pol β oxidized nucleotide insertions (Scheme S1). In the recently published study (40Jurkiw T.J. Tumbale P.P. Schellenberg M.J. Cunningham-Rundles C. Williams R.S. O'Brien P.J. LIG1 syndrome mutations remodel a cooperative network of ligand binding interactions to compromise ligation efficiency.Nucleic Acids Res. 2021; 49: 1619-1630Crossref PubMed Scopus (3) Google Scholar), the role of destabilized Mg2+ cofactor binding in the ligation failure by the LIG1 deficiency disease mutants that could contribute to the development of the disease pathology has been sugges
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