Molecular basis of nick ligation in the nucleosome by DNA Ligase IIIα

Eukaryotic genomic DNA is packaged into chromatin through a fundamental repeating unit known as the nucleosome core particle. Within this chromatin context, genomic DNA is constantly exposed to endogenous and exogenous stress that result in the formation of DNA damage, which must be effectively repaired to maintain genome stability. Single-strand breaks (SSBs) are among the most prevalent forms of DNA damage that arise via the oxidation-induced disintegration of the sugar-phosphate backbone or as repair intermediates during base excision repair. DNA ligase IIIα (LigIIIα) is one of the primary enzymes responsible for repairing SSBs containing an intact 5'-phosphate and 3'-OH (nick) during the terminal step of single-strand break repair (SSBR) and base excision repair (BER) pathways. To date, a complete mechanistic description for how LigIIIα processes nicks within chromatin remains elusive. Here, we use a combination of biochemical assays, molecular dynamics simulations, and cryogenic electron microscopy (cryo-EM) to define the molecular basis of nick ligation in the nucleosome by LigIIIα. Quantitative enzyme kinetics reveal that the LigIIIα ligation rate is highly dependent on the translational position of the nick in the nucleosome, where nicks near the nucleosome entry/exit site are ligated with moderate efficiency and nicks near the nucleosome dyad are refractory to ligation. Cryo-EM structures of LigIIIα bound to nicks at four unique translational positions in the nucleosome reveal the structural basis for this position-dependent catalytic activity, identifying that local steric constraints imposed by the histone octamer prevent LigIIIα from readily adopting a ligation-competent conformation. Further biochemical and structural analysis demonstrates that the scaffolding protein XRCC1, which forms a heterodimer with LigIIIα, does not substantially alter the ability of LigIIIα to bind or ligate nicks in the nucleosome. Together, this work provides foundational insight into the processing of nicks in the nucleosome during the terminal step of SSBR/BER.