Replication protein A [RPA; also known as replication factor A (RFA) and human single-stranded DNA-binding protein] is a single-stranded DNA-binding protein that is required for multiple processes in eukaryotic DNA metabolism, including DNA replication, DNA repair, and recombination. RPA homologues have been identified in all eukaryotic organisms examined and are all abundant heterotrimeric proteins composed of subunits of approximately 70, 30, and 14 kDa. Members of this family bind nonspecifically to single-stranded DNA and interact with and/or modify the activities of multiple proteins. In cells, RPA is phosphorylated by DNA-dependent protein kinase when RPA is bound to single-stranded DNA (during S phase and after DNA damage). Phosphorylation of RPA may play a role in coordinating DNA metabolism in the cell. RPA may also have a role in modulating gene expression.

Replication protein A (RPA) is a heterotrimeric, single-stranded DNA binding protein that is essential for eukaryotic DNA replication. In order to gain a better understanding of the interactions between RPA and DNA, we have examined the interactions of human RPA with single-stranded oligonucleotides. Our analysis of RPA.DNA complexes demonstrated that RPA binds as a heterotrimer. Stoichiometric binding reactions monitored by fluorescence quenching indicated that the binding site size of human RPA is 30 nucleotides and that between 20-30 nucleotides of DNA directly interact with RPA. The binding of RPA to DNA of different lengths was systematically examined using deoxythymidine-containing oligonucleotides. We found that the binding affinity of RPA for short oligonucleotides was length dependent. The apparent association constant of RPA varied over 200-fold from approximately 7 x 10(7) M-1 for oligo(dT)10 to approximately 1.5 x 10(10) M-1 for oligo(dT)50. Human RPA binds to oligonucleotides with low cooperativity; the cooperativity parameter (omega) for RPA binding was estimated to be approximately 15.

Human replication protein (RPA) functions in DNA replication, homologous recombination and nucleotide excision repair. This multisubunit single-stranded DNA-binding protein may be required to make unique protein-protein contacts because heterologous single-stranded binding proteins cannot substitute for RPA in these diverse DNA transactions. We report here that, by using affinity chromatography and immunoprecipitation, we found that human RPA bound specifically and directly to two excision repair proteins, the xeroderma pigmentosum damage-recognition protein XPA (refs 8, 9) and the endonuclease XPG (refs 10-13). Although it had been suggested that RPA might function before the DNA synthesis repair stage, our finding that a complex of RPA and XPA showed a striking cooperativity in binding to DNA lesions indicates that RPA may function at the very earliest stage of excision repair. In addition, by binding XPG, RPA may target this endonuclease to damaged DNA.

Replication protein A (RP-A; also known as replication factor A and human SSB), is a single-stranded DNA-binding protein that is required for simian virus 40 DNA replication in vitro. RP-A isolated from both human and yeast cells is a very stable complex composed of 3 subunits (70,32, and 14 kDa Characterization of the mechanism of chromosomal DNA replication is essential for understanding the process of cell growth. Eukaryotic chromosomes are very large and complex; therefore, the study of model systems has been vital to reach our current understanding of DNA replication. One such model system, the papovavirus simian virus 40 (SV40), has properties very similar to those of cellular chromosomes (see recent reviews in references 7, 27, and 46). The SV40 genome exists in the cell in an authentic chromatin structure. Replication of SV40 DNA is dependent upon cellular replication proteins and a single virally encoded protein, large T antigen. SV40 DNA replication initiates at a specific DNA sequence and then proceeds bidirectionally, as does chromosomal replication. The development of a cell-free replication reaction capable of replicating SV40 DNA (34) led to the identification and purification of seven cellular proteins required for catalyzing DNA synthesis (36, 47, 52). Studies from several laboratories have led to a basic understanding of the mechanism of SV40 DNA replication (36, 47, 52). Initiation of replication occurs at a well-defined sequence, the SV40 origin of replication (Fig. 1)

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