A SEQUENCE IN THE N-TERMINAL REGION OF HUMAN URACIL-DNA GLYCOSYLASE WITH HOMOLOGY TO XPA INTERACTS WITH THE C-TERMINAL PART OF THE 34-KDA SUBUNIT OF REPLICATION PROTEIN-A

Uracil-DNA glycosylase releases free uracil from DNA and initiates bas e excision repair for removal of this potentially mutagenic DNA lesion , Using the yeast two-hybrid system, human uracil-DNA glycosylase enco ded by the UNG gene (UNG) was found to interact with the C-terminal pa rt of the 34-kDa subunit of replication protein A (RPA2). No interacti on with RPA4 (a homolog of RPA2), RPA1, or RPA3 was observed, A sandwi ch enzyme-linked immunosorbent assay with trimeric RPA and the two-hyb rid system both demonstrated that the interaction depends on a region in UNG localized between amino acids 28 and 79 in the open reading fra me. In this part of UNG a 23-amino acid sequence has a significant hom ology to the RPA2-binding region of XPA, a protein involved in damage recognition in nucleotide excision repair, Trimeric RPA did not enhanc e the activity of UNG in vitro on single- or double-stranded DNA, A pa rt of the N-terminal region of UNG corresponding in size to the comple te presequence was efficiently removed by proteinase K, leaving the pr oteinase K-resistant compact catalytic domain intact and fully active. These results indicate that the N-terminal part constitutes a separat e structural domain required for RPA binding and suggest a possible fu nction for RPA in base excision repair.