Biology subjects

Caglayan, M.

Publications and source records attributed to Caglayan, M..

10 recordsLinked to original sources

bioRxiv2025

Single-molecule analysis of gap and nick binding by LIG1 and LIG3α at the final step of DNA repair

DNA ligase (LIG) 1 and LIG3 repair broken single-strand breaks in the phosphodiester backbone at the final ligation step of DNA excision repair pathways, and complement each other during nuclear replication in case of unligated Okazaki fragments. We previously reported that both ligases discriminate against nicks containing non-canonical ends and ligate gap intermediate if left unfilled by DNA polymerases. However, it remains unknown how the dynamics of DNA binding differ for gap versus nick substrates by LIG1 and LIG3 at single-molecule level. Here, using total internal reflection fluorescence (TIRF) and ligation assays, we showed that LIG3 binds less frequently but forms longer-lived complex than LIG1 for nicks containing canonical A:T, mismatch G:T, and damaged 8oxoG:A, and they exhibit subtle differences in discriminating unusual ends. Moreover, our results identified gap DNA as a new target to which LIG1 and LIG3 can bind as efficient as their preferential nick sites. We showed gap ligation and observed that more percentage of LIG1 molecules form stable long-lived complex on DNA containing one nucleotide gap, whereas LIG3 forms short-lived gap complex without any differences in the percentage of molecule forming gap-bound complex. Finally, our findings demonstrated that LIG1 can still stably bind to larger gaps with better recognition, whereas LIG3 binding becomes further infrequent and shorter-lived. Overall, our study provides single-molecule insights into intricate differences between LIG1 and LIG3 for binding to a range of mutagenic and deleterious DNA repair and replication intermediates that could be a threat for maintaining genome stability at the final step.

biochemistry↗

bioRxiv2025

Characterization of nick binding and sealing by LIG1 Huntington's disease-asssociated K845N variant at biochemical, structural, and single-molecule levels

DNA ligase 1 (LIG1) joins broken strand breaks and discriminates against nicks containing mismatch or oxidative damage. Huntingtons disease (HD)-associated mutation K845N in LIG1 gene has been predicted to be onset delaying and suppresses CAG repeat expansion. Yet, how this mutation impacts faithful nick sealing and efficient DNA binding by LIG1 remains unknown. Here, using biochemistry, X-ray crystallography, and total internal reflection fluorescence microscopy, we comprehensively characterized the impact of LIG1 HD-associated mutation at biochemical, structural, and single-molecule levels. Our results showed a reduced ligation efficiency by LIG1 K845N variant in the presence of nick substrates containing all possible 12 mismatches, 8-oxoG, and ribonucleotides at the 3-end when compared with the wild-type enzyme. Furthermore, our structures provided an atomic insight into differences in distances between the functional groups of K/N845 and DNA ends, demonstrating similar conformation and a lack of large scale alternations at the ligase active site. Finally, our single-molecule measurements in real-time revealed that K845N mutant binds less frequently for shorter life-time to nick DNA than wild-type protein. Overall findings contribute to understand the mechanism by which LIG1 ensures fidelity and nick binding to maintain genome stability at the final ligation step in normal versus disease states.

biochemistry↗

bioRxiv2024

Biochemical, structural, and single-molecule characterization of LIG1 active site mutants demonstrate role of F635 and F872 residues for faithful ligation

Human DNA ligase 1 (LIG1) finalizes DNA repair pathways by an ultimate ligation step and discriminates against nicks containing unusual ends, yet the contribution of the conserved active site residues for faithful end joining remains unknown. Here, using biochemistry, X-ray crystallography, and single-molecule approaches, we comprehensively characterized LIG1 mutants carrying Ala(A) and Leu(L) substitutions at the active site residues Phe(F)635 and Phe(F)872. Our results showed an abolished ligation of nick DNA substrates with all 12 non-canonical mismatches, while the mutagenic nick sealing of oxidatively damaged ends by wild-type enzyme is significantly reduced by F635A/L and F872A/L substitutions. Furthermore, sugar discrimination against a single ribonucleotide at 3- or 5-end of nick DNA is distinctly affected depending on architecture of 3-terminus:template base pairing. Finally, our LIG1 structures demonstrated the importance of DNA end alignment governed by the distance to nick site through F635 and F872 residues, and single-molecule measurements showed similar nick DNA binding modes for LIG1 wild-type and active site mutants in real-time. Overall, our study provides a mechanistic insight into the mechanism by which conserved F635 and F872 residues contribute to ligation efficiency of nick repair intermediates that mimic DNA polymerase-mediated mismatch, damaged, or ribonucleotide insertion products and how LIG1 ensures faithful end joining at the final step of DNA repair to maintain genome integrity.

biochemistry↗

bioRxiv2024

Mutagenic ligation of polβ mismatch insertion products during 8-oxoG bypass by LIG1 and LIG3α at the downstream steps of base excision repair pathway

Base excision repair (BER) maintains genome integrity by fixing oxidized bases that could be formed when reactive oxygen species attack directly on the DNA. We previously reported the importance of a proper coordination at the downstream steps involving gap filling by DNA polymerase (pol) {beta} and subsequent nick sealing by DNA ligase (LIG) 1 or 3. Yet, how the fidelity of 8-oxoG bypass by pol{beta} affects the efficiency of ligation remains unclear. Here, we show that LIG1 can seal nick products of pol{beta} after both dATP and dCTP insertions during 8- oxoG bypass, while ribonucleotide insertions completely diminish the repair coordination with both ligases, highlighting a critical role for nucleotide selectivity in maintaining BER accuracy. Furthermore, our results demonstrate that LIG3 exhibits an inability to ligate nicks of pol{beta} dCTP:8-oxoG insertion or with preinserted 3-dC:8-oxoG. Finally, AP-Endonuclease 1 (APE1) proofreads nick repair intermediates containing 3-dA/rA and 3-dC/rC mismatches templating 8-oxoG. Overall, our findings provide a mechanistic insight into how the dual coding potential of the oxidative lesion and identity of BER ligase govern mutagenic versus error-free repair outcomes at the final steps and how the ribonucleotide challenge compromises the BER coordination leading to the formation of deleterious repair intermediates.

biochemistry↗

bioRxiv2024

Structural and biochemical characterization of LIG1 during mutagenic nick sealing of oxidatively damaged ends at the final step of DNA repair

DNA ligase 1 (LIG1) joins broken strand-breaks in the phosphodiester backbone to finalize DNA repair pathways. We previously reported that LIG1 fails on nick repair intermediate with 3-oxidative damage incorporated by DNA polymerase (pol) {beta} at the downstream steps of base excision repair (BER) pathway. Here, we determined X-ray structures of LIG1/nick DNA complexes containing 3-8oxodG and 3-8oxorG opposite either a templating Cytosine or Adenine and demonstrated that the ligase active site engages with mutagenic repair intermediates during steps 2 and 3 of the ligation reaction referring to the formation of DNA-AMP intermediate and a final phosphodiester bond, respectively. Furthermore, we showed the mutagenic nick sealing of DNA substrates with 3-8oxodG:A and 3-8oxorG:A by LIG1 wild-type, immunodeficiency disease-associated variants, and DNA ligase 3 (LIG3) in vitro. Finally, we observed that LIG1 and LIG3 seal resulting nick after an incorporation of 8oxorGTP:A by pol{beta} and AP-Endonuclease 1 (APE1) can clean oxidatively damaged ends at the final steps. Overall, our findings uncover a mechanistic insight into how LIG1 discriminates DNA or DNA/RNA junctions including oxidative damage and a functional coordination between the downstream enzymes, pol{beta}, APE1, and BER ligases, to process mutagenic repair intermediates to maintain repair efficiency.

biochemistry↗

bioRxiv2024

Uncovering nick DNA binding by LIG1 at the single-molecule level

DNA ligases repair the strand breaks are made continually and naturally throughout the genome, if left unrepaired and allowed to persist, they can lead to genome instability in the forms of lethal double-strand (ds) breaks, deletions, and duplications. DNA ligase 1 (LIG1) joins Okazaki fragments during the replication machinery and seals nicks at the end of most DNA repair pathways. Yet, how LIG1 recognizes its target substrate is entirely missing. Here, we uncover the dynamics of nick DNA binding by LIG1 at the single-molecule level. Our findings reveal that LIG1 binds to dsDNA both specifically and non-specifically and exhibits diffusive behavior to form a stable complex at the nick. Furthermore, by comparing with the LIG1 C-terminal protein, we demonstrate that the N-terminal non-catalytic region promotes binding enriched at nick sites and facilitates an efficient nick search process by promoting 1D diffusion along the DNA. Our findings provide a novel single-molecule insight into the nick binding by LIG1, which is critical to repair broken phosphodiester bonds in the DNA backbone to maintain genome integrity.

biochemistry↗

bioRxiv2023

Structures of LIG1 active site mutants reveal the importance of DNA end rigidity for mismatch discrimination

ATP-dependent DNA ligases catalyze phosphodiester bond formation in the conserved three-step chemical reaction of nick sealing. Human DNA ligase I (LIG1) finalizes almost all DNA repair pathways following DNA polymerase-mediated nucleotide insertion. We previously reported that LIG1 discriminates mismatches depending on the architecture of the 3-terminus at a nick, however the contribution of conserved active site residues to faithful ligation remains unknown. Here, we comprehensively dissect the nick DNA substrate specificity of LIG1 active site mutants carrying Ala(A) and Leu(L) substitutions at Phe(F)635 and Phe(F)F872 residues and show completely abolished ligation of nick DNA substrates with all 12 non-canonical mismatches. LIG1EE/AA structures of F635A and F872A mutants in complex with nick DNA containing A:C and G:T mismatches demonstrate the importance of DNA end rigidity, as well as uncover a shift in a flexible loop near 5-end of the nick, which causes an increased barrier to adenylate transfer from LIG1 to the 5-end of the nick. Furthermore, LIG1EE/AA/8oxoG:A structures of both mutants demonstrated that F635 and F872 play critical roles during steps 1 or 2 of the ligation reaction depending on the position of the active site residue near the DNA ends. Overall, our study contributes towards a better understanding of the substrate discrimination mechanism of LIG1 against mutagenic repair intermediates with mismatched or damaged ends and reveals the importance of conserved ligase active site residues to maintain ligation fidelity.

biochemistry↗

bioRxiv2022

Structures of LIG1 uncover a lack of sugar discrimination against a ribonucleotide at the 3'-end of nick DNA

Human DNA ligase I (LIG1) is the main replicative ligase that seals Okazaki fragments and finalizes DNA repair pathways by joining canonical 3-OH and 5-P ends of the nick DNA in a three-step ligation reaction. Ribonucleotides can be misincorporated by DNA polymerases resulting in a nick with 3-ribonucleotide while RNase H2 mediated cleavage leaves a nick harboring 5-ribonucleotide during ribonucleotide excision repair. However, how LIG1 surveils DNA ends with a "wrong" sugar at atomic resolution is unknown. Here, we determine X-ray structures of LIG1/nick DNA complexes with 3- or 5-single ribonucleotide during different stages of the ligation reaction. Our LIG1/5-rG:C structure reveals a global conformational change, which discriminates against 5-RNA/DNA junctions at the initial step when the ligase-AMP intermediate is formed. Furthermore, we capture LIG1/3-RNA-DNA heteroduplexes that are tolerated at the active site where AMP is transferred to nick DNA (step 2) and final phosphodiester bond formation occurs (step 3). Finally, we demonstrate the mutagenic and defective ligation of the nick DNA with 3- and 5-ribonucleotide, respectively, in vitro. Together, these results uncover how LIG1 encounters ribonucleotides embedded into genome during nuclear replication and the last step of DNA repair pathways to maintain genome integrity.

biochemistry↗

bioRxiv2022

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

DNA ligase I (LIG1) catalyzes final ligation step following DNA polymerase (pol) {beta} gap filling and an incorrect nucleotide insertion by pol{beta} 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 pol{beta}, LIG1, and APE1 can maintain accurate repair.

biochemistry↗

bioRxiv2020

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

DNA ligase I (LIG1) completes base excision repair (BER) pathway at the last nick sealing step following DNA polymerase (pol) {beta} gap filling DNA synthesis. We previously reported that pol {beta} 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 {beta} 8-oxodGTP insertion products and an inefficient substrate-product channeling from pol {beta} 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 {beta}, 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.

biochemistry↗