FUNCTIONAL-ANALYSIS OF HUMAN REPLICATION PROTEIN-A IN NUCLEOTIDE EXCISION-REPAIR

Human replication protein A (RPA) is a three-subunit protein complex ( 70-, 34-, and 11-kDa subunits) involved in DNA replication, repair, an d recombination. Both the 70- (p70) and 34-kDa (p34) subunits interact with Xeroderma pigmentosum group A complementing protein (XPA), a key protein involved in nucleotide excision repair. Our deletion analysis indicated that no particular domain(s) of RPA p70 was essential for i ts interaction with XPA, whereas 33 amino acids from the C terminus of p34 (p34 Delta 33C) were necessary for the XPA interaction. Furthermo re, mutant RPA lacking the p34 C terminus failed to interact with XPA, suggesting that p34, not p70, is primarily responsible for the intera ction of RPA with XPA. RPA stimulated the interaction of XPA with W-da maged DNA through an RPA-XPA complex on damaged DNA sites because (i) the RPA mutant lacking the C terminus of p34 failed to stimulate an XP A-DNA interaction, and (ii) the ssDNA binding domain of RPA (amino aci ds 296-458) was necessary for the stimulation of the XPA-DNA interacti on. Two separate domains of p70, a single-stranded DNA binding domain and a zinc-finger domain, were necessary for RPA function in nucleotid e excision repair. The mutant RPA (RPA: p34 Delta 33C), which lacks it s stimulatory effect on the XPA-DNA interaction, also poorly supported nucleotide excision repair, suggesting that the XPA-RPA interaction o n damaged DNA is necessary for DNA repair activity.