Abstract
The interaction between human DNA polymerase beta (pol beta) 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 beta that interacts with the DNA template and excises 5'-deoxyribose phosphate residue. Intact pol beta, 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 degreesC, three pol beta monomers attached to the DNA ligase I trimer, forming a stable heterohexamer. In contrast, at lower temperatures (<18 degreesC), pol beta 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 beta 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 beta did not. Our results indicate that the association between pol beta 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 beta, whereas the entropy-driven aspect of interprotein binding appears to be contributed by the 31-kDa domain.
Highlights
The DNA repair pathway termed base excision repair (BER)1 purifies genomic DNA of damaged nucleotides and abasic sites arising from a variety of exogenous and endogenous sources [1]
BER can be distinguished from other DNA excision repair pathways by the relatively small repair patch produced in double-stranded DNA after incision at the abasic site and by the fact that base lesions repaired by the BER pathway are generally limited to modifications that are less bulky than those lesions repaired by the nucleotide excision repair pathway
Identification of the Regions Required for Complex Formation between DNA Ligase I and pol  by Affinity Chromatography—Analysis of the structure of pol , initially by controlled proteolysis and more recently by x-ray crystallography, has shown that this enzyme is composed of an 8-kDa N-terminal domain that binds to DNA template and carries dRP lyase activity and a 31-kDa C-terminal domain that contains the DNA polymerase active site
Summary
The DNA repair pathway termed base excision repair (BER)1 purifies genomic DNA of damaged nucleotides and abasic sites arising from a variety of exogenous and endogenous sources [1]. Where association was found to take place, the corresponding equilibrium constants were estimated by least-squares fitting of appropriate mathematical models to both absorbency and transmitted light intensity data.
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