An interaction between replication protein A and SV40 T antigen appears essential for primosome assembly during SV40 DNA replication.
Replication protein A from human cells (hRPA) is a multisubunit single-stranded DNA-binding protein (ssb) and is essential for SV40 DNA replication in vitro. The related RPA from Saccharomyces cerevisiae (scRPA) is unable to substitute for hRPA in SV40 DNA replication. To understand this species specificity, we evaluated human and yeast RPA in enzymatic assays with SV40 T antigen (TAg) and human DNA polymerase alpha/primase, the factors essential for initiation of SV40 DNA replication. Both human and yeast RPA stimulated the polymerase and (at subsaturating levels of RPA) the primase activities of human DNA polymerase alpha/primase on homopolymer DNA templates. In contrast, both human and yeast RPA inhibited synthesis by DNA polymerase alpha/primase on natural single-stranded DNA (ssDNA) templates. T antigen reversed the inhibition of DNA polymerase alpha/primase activity on hRPA-coated natural ssDNA, as previously described, but was unable to reverse the inhibition on scRPA or Escherichia coli ssb-coated templates. Therefore, the ability of an ssb to reconstitute SV40 DNA replication correlated with its ability to allow the TAg stimulation of polymerase alpha/primase in this assay. Enzyme-linked immunoassays demonstrated that hRPA interacts with TAg, as previously described; however, scRPA does not bind to TAg in this assay. These and other recent results suggest that T antigen contains a function analogous to some prokaryotic DNA replication proteins that facilitate primosome assembly on ssb-coated template DNAs.
Melendy
T
T
Cold Spring Harbor Laboratory, New York 11724-2206.
Stillman
B
B
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000378:metabolism*; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D019915:DNA Primase; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007313:Insecta; D007700:Kinetics; D008954:Models, Biological; D012316:RNA Nucleotidyltransferases / Q000378:metabolism*; D051716:Replication Protein A; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013539:Simian virus 40 / Q000235:genetics* / Q000378:metabolism*
D000952:Antigens, Polyomavirus Transforming; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; C497167:RPA1 protein, human; D051716:Replication Protein A; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D004259:DNA-Directed DNA Polymerase