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. Huntington’s 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.