THERMODYNAMICS OF HUMAN DNA-LIGASE-I TRIMERIZATION AND ASSOCIATION WITH DNA-POLYMERASE-BETA

The interaction between human DNA polymerase beta (pol beta) and DNA L igase I, which appear to be responsible for the gap filling and nick l igation 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 t hrough the non-catalytic N-terminal domain of DNA ligase I and the N-t erminal 8-kDa domain of pol beta that interacts with the DNA template and excises 5'-deoxyribose phosphate residue. Intact pol beta, a 39-kD a bi-domain enzyme, undergoes indefinite self-association, forming oli gomers of many sizes. The binding sites for self-association reside wi thin the C-terminal 31-kDa domain. DNA ligase I undergoes self-associa tion to form a homotrimer, At temperatures over 18 degrees C, three po l beta monomers attached to the DNA ligase I trimer, forming a stable heterohexamer, In contrast, at lower temperatures (<18 degrees C), pol beta and DNA ligase I formed a stable 1:1 binary complex only. In agr eement with the domain mapping studies, the 8-kDa domain of pol beta i nteracted with DNA ligase I, forming a stable 3:3 complex with DNA lig ase I at all temperatures, whereas the 31-kDa domain of pol beta did n ot. Our results indicate that the association between pol beta and DNA ligase I involves both electrostatic binding and an entropy-driven pr ocess. Electrostatic binding dominates the interaction mediated by the 8-kDa domain of pol beta, whereas the entropy-driven aspect of interp rotein binding appears to be contributed by the 31-kDa domain.