Pol μ ribonucleotide insertion opposite 8-oxodG facilitates the ligation of premutagenic DNA repair intermediate

Çağlayan, Melike

doi:10.1038/s41598-020-57886-y

Cited by 11 publications

(31 citation statements)

References 48 publications

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“…Oligodeoxyribonucleotides with and without a 6-carboxyfluorescein label and the 5'-adenylate (AMP) were obtained from Integrated DNA Technologies. The DNA substrates used in this study were prepared as described previously ( 12 , 13 , 14 , 15 , 16 , 34 , 35 , 36 , 71 , 72 ). The one-nucleotide-gap DNA substrates were used for coupled assays ( Table S1 ).…”

Section: Methodsmentioning

confidence: 99%

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DNA ligase I fidelity mediates the mutagenic ligation of pol β oxidized and mismatch nucleotide insertion products in base excision repair

Kamble

Chandak

et al. 2021

Journal of Biological Chemistry

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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.

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Section: Methodsmentioning

confidence: 99%

“…The ligation assays were performed to analyze the substrate specificity of LIG1 as described previously ( 12 , 13 , 14 , 15 , 16 , 71 , 72 ). For this purpose, we used the nicked DNA substrates with 3'-preinserted 8-oxodG or mismatched bases ( Table S2 ).…”

Section: Methodsmentioning

confidence: 99%

DNA ligase I fidelity mediates the mutagenic ligation of pol β oxidized and mismatch nucleotide insertion products in base excision repair

Kamble

Chandak

et al. 2021

Journal of Biological Chemistry

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“…Oligodeoxyribonucleotides with and without a 6-carboxyfluorescein (FAM) label and the 5'adenylate (AMP) were obtained from Integrated DNA Technologies. The DNA substrates used in this study were prepared as described previously (12)(13)(14)(15)(16)(34)(35)(36)38,39). The one nucleotide gapped DNA substrates were used for coupled assays (Supplementary Table 1).…”

Section: Preparation Of Dna Substratesmentioning

confidence: 99%

“…The coupled assays were performed to measure pol β and LIG1 activities simultaneously in the same reaction mixture as described previously (12)(13)(14)(15)(16)38,39). For this purpose, we used the one nucleotide gap DNA substrates (Supplementary Table 1).…”

Section: Coupled Assaysmentioning

confidence: 99%

DNA ligase I fidelity mediates the mutagenic ligation of pol β oxidized nucleotide insertion products and base excision repair intermediates with mismatches

Kamble

Chandak

et al. 2020

Preprint

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DNA ligase I (LIG1) completes base excision repair (BER) pathway at the last nick sealing step following DNA polymerase (pol) β gap filling DNA synthesis. We previously reported that pol β 8-oxo-2'-deoxyribonucleoside 5'-triphosphate (8-oxodGTP) insertion confounds LIG1 leading to the formation of ligation failure products with 5'-adenylate (AMP) block. Here, we report the mutagenic ligation of pol β 8-oxodGTP insertion products and an inefficient substrate-product channeling from pol β Watson-Crick like dG:T mismatch insertion to DNA ligation by LIG1 mutant with perturbed fidelity (E346A/E592A) in vitro. Moreover, our results revealed that the substrate discrimination of LIG1 for the nicked repair intermediates with preinserted 3'-8-oxodG or mismatches is governed by the mutations at both E346 and E592 residues. Finally, we found that Aprataxin (APTX) and Flap Endonuclease 1 (FEN1), as compensatory DNA-end processing enzymes, can remove 5'-AMP block from the abortive ligation products with 3'-8-oxodG or all possible 12 non-canonical base pairs. These findings contribute to understand the role of LIG1 as an important determinant of faithful BER, and how a multi-protein complex (LIG1, pol β, APTX and FEN1) can coordinate to hinder the formation of mutagenic repair intermediates with damaged or mismatched ends at the downstream steps of the BER pathway.

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“…In our previous studies, we demonstrated the importance of polβ and DNA ligase I (LIG1) coordination for the accurate channeling of repair intermediates to maintain BER efficiency at the downstream steps of the repair pathway (23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36). For example, we reported that the incorporation of oxidized nucleotide 7,8-dihydro-8'-oxo-dGTP (8-oxodGTP) by polβ confounds LIG1, leading to the formation of abortive repair intermediates (26,27).…”

Section: Introductionmentioning

confidence: 99%

Structures of LIG1 engaging with mutagenic mismatches inserted by polβ in base excision repair

Tang

McKenna

Çağlayan

2022

Preprint

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DNA ligase I (LIG1) catalyzes final ligation step following DNA polymerase (pol) β gap filling and an incorrect nucleotide insertion by polβ creates a nick repair intermediate with mismatched end at the downstream steps of base excision repair (BER) pathway. Yet, how LIG1 discriminates against the mutagenic 3'-mismatches at atomic resolution remains undefined. Here, we determined X-ray structures of LIG1/nick DNA complexes with G:T and A:C mismatches and uncovered the ligase strategies that favor or deter ligation of base substitution errors. Our structures revealed that LIG1 active site can accommodate G:T mismatch in a similar conformation with A:T base pairing, while it stays in the LIG1-adenylate intermediate during initial step of ligation reaction in the presence of A:C mismatch at 3'-strand. Moreover, we showed mutagenic ligation and aberrant nick sealing of the nick DNA substrates with 3'-preinserted dG:T and dA:C mismatches, respectively. Finally, we demonstrated that AP-Endonuclease 1 (APE1), as a compensatory proofreading enzyme, interacts and coordinates with LIG1 during mismatch removal and DNA ligation. Our overall findings and ligase/nick DNA structures provide the features of accurate versus mutagenic outcomes at the final BER steps where a multi-protein complex including polβ, LIG1, and APE1 can maintain accurate repair.

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Pol μ ribonucleotide insertion opposite 8-oxodG facilitates the ligation of premutagenic DNA repair intermediate

Cited by 11 publications

References 48 publications

DNA ligase I fidelity mediates the mutagenic ligation of pol β oxidized and mismatch nucleotide insertion products in base excision repair

DNA ligase I fidelity mediates the mutagenic ligation of pol β oxidized and mismatch nucleotide insertion products in base excision repair

DNA ligase I fidelity mediates the mutagenic ligation of pol β oxidized nucleotide insertion products and base excision repair intermediates with mismatches

Structures of LIG1 engaging with mutagenic mismatches inserted by polβ in base excision repair

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