Abstract
Human replication protein A (RPA) is a three-subunit protein complex (70-, 34-, and 11-kDa subunits) involved in DNA replication, repair, and 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 its interaction with XPA, whereas 33 amino acids from the C terminus of p34 (p34Delta33C) were necessary for the XPA interaction. Furthermore, mutant RPA lacking the p34 C terminus failed to interact with XPA, suggesting that p34, not p70, is primarily responsible for the interaction of RPA with XPA. RPA stimulated the interaction of XPA with UV-damaged 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 XPA-DNA interaction, and (ii) the ssDNA binding domain of RPA (amino acids 296-458) was necessary for the stimulation of the XPA-DNA interaction. Two separate domains of p70, a single-stranded DNA binding domain and a zinc-finger domain, were necessary for RPA function in nucleotide excision repair. The mutant RPA (RPA:p34Delta33C), which lacks its stimulatory effect on the XPA-DNA interaction, also poorly supported nucleotide excision repair, suggesting that the XPA-RPA interaction on damaged DNA is necessary for DNA repair activity.
Highlights
Human replication protein A is composed of three tightly associated polypeptides (70, 34, and 11-kDa subunits)
The C Terminus of p34 Is Primarily Responsible for Interaction of replication protein A (RPA) with Xeroderma pigmentosum group A complementing protein (XPA)—RPA interacts with SV40 Tag, XPA, and human Rad52, key proteins involved in replication, repair, and recombination, respectively
To identify the domain(s) of RPA p70 and p34 involved in these protein-protein interactions, we examined p70 and p34 deletion mutants for their ability to interact with XPA
Summary
Human replication protein A ( known as human singlestranded DNA-binding protein) is composed of three tightly associated polypeptides (70-, 34-, and 11-kDa subunits) It was originally identified as a factor required for in vitro SV40 DNA replication [1,2,3]. Its role in the early stage of repair was suggested because the RPA requirement can be bypassed by the presence of Escherichia coli UvrABC, which incises damaged DNA [14]. This early stage involvement includes the interaction of RPA with the UV damage recognition factor XPA [17,18,19,20]. The p70 subunit has multiple functional domains, an N-terminal domain for interacting with pol ␣ [12, 35], p53 [36], two middle subdomains with DNA binding activity [12, 37], four cysteine-type zinc finger domains (amino acids 481–503), This paper is available on line at http://www.jbc.org
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
