Biology subjects

Ratcliffe, J. E.

Publications and source records attributed to Ratcliffe, J. E..

3 recordsLinked to original sources

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↗