Thermodynamics of human DNA ligase I trimerization and association with DNA polymerase β

Abstract

The interaction between human DNA polymerase β (pol β) and DNA ligase I, which appear to be responsible for the gap filling and nick ligation steps in short patch or simple base excision repair, has been examined by affinity chromatography and analytical ultracentrifugation. Domain mapping studies revealed that complex formation is mediated through the non-catalytic N- terminal domain of DNA ligase I and the N-terminal 8-kDa domain of pol β that interacts with the DNA template and excises 5'-deoxyribose phosphate residue. Intact pol β, a 39-kDa bi-domain enzyme, undergoes indefinite self- association, forming oligomers of many sizes. The binding sites for self- association reside within the C-terminal 31-kDa domain. DNA ligase I undergoes self-association to form a homotrimer. At temperatures over 18 °C, three pol β monomers attached to the DNA ligase I trimer, forming a stable heterohexamer. In contrast, at lower temperatures (<18°C), pol β and DNA ligase I formed a stable 1:1 binary complex only. In agreement with the domain mapping studies, the 8-kDa domain of pol β interacted with DNA ligase I, forming a stable 3:3 complex with DNA ligase I at all temperatures, whereas the 31-kDa domain of pol β did not. Our results indicate that the association between pol β and DNA ligase I involves both electrostatic binding and an entropy-driven process. Electrostatic binding dominates the interaction mediated by the 8-kDa domain of pol β, whereas the entropy- driven aspect of interprotein binding appears to be contributed by the 31- kDa domain.