RECONSTITUTION OF DNA-BASE EXCISION-REPAIR WITH PURIFIED HUMAN PROTEINS - INTERACTION BETWEEN DNA-POLYMERASE-BETA AND THE XRCC1 PROTEIN

Repair of a uracil-guanine base pair in DNA has been reconstituted wit h the recombinant human proteins uracil-DNA glycosylase, apurinic/apyr imidinic endonuclease, DNA polymerase beta and DNA ligase III, The XRC C1 protein, which is known to bind DNA ligase III, is not absolutely r equired for the reaction but suppresses strand displacement by DNA pol ymerase beta, allowing for more efficient ligation after filling of a single nucleotide patch, We show that XRCC1 interacts directly with DN A polymerase beta using far Western blotting, affinity precipitation a nd yeast two-hybrid analyses, In addition, a complex formed between DN A polymerase beta and a double-stranded oligonucleotide containing an incised abasic site was supershifted by XRCC1 in a gel retardation ass ay, The region of interaction with DNA polymerase beta is located with in residues 84-183 in the N-terminal half of the XRCC1 protein, wherea s the C-terminal region of XRCC1 is involved in binding DNA ligase III . These data indicate that XRCC1, which has no known catalytic activit y, might serve as a scaffold protein during base excision-repair, DNA strand displacement and excessive gap filling during DNA repair were o bserved in cell-free extracts of an XRCC1-deficient mutant cell line, in agreement with the results from the reconstituted system.