Reconstitution of DNA base excision-repair with purified human proteins: Interaction between DNA polymerase β and the XRCC1 protein

Abstract

Repair of a uracil-guanine base pair in DNB has been reconstituted with the recombinant human proteins uracil-DNA glycosylase, apurinic/apyrimidinic endonuclease, DNA polymerase β and DNA ligase III. The XRCC1 protein, which is known to bind DNA ligase III, is not absolutely required for the reaction but suppresses strand displacement by DNA polymerase β, allowing for more efficient ligation after filling of a single nucleotide patch. We show that XRCC1 interacts directly with DNA polymerase β using far estern blotting, affinity precipitation and yeast two-hybrid analyses. In addition, a complex formed between DNA polymerase β and a double-stranded oligonucleotide containing an incised abasic site was supershifted by XRCC1 in a gel retardation assay. The region of interaction with DNA polymerase β is located within residues 84-183 in the N-terminal half of the XRCC1 protein, whereas the C-terminal region of XRCC1 is involved in binding DNA ligase III. These data indicate that XRCC1, which has no known catalytic activity, might serve as a scaffold protein during base excision-repair. DNA strand displacement and excessive gap filling during DNA repair were observed in cell-free extracts of an XRCC1-deficient mutant cell line, in agreement with the results from the reconstituted system.