Abstract
Replication protein A (RPA) is a multisubunit, single-stranded DNA-binding protein that is absolutely required for replication of SV40 DNA. The three cDNAs encoding the subunits of human replication protein A (70, 32, and 14 kDa) have been expressed individually and in combination in Escherichia coli. When subunits were expressed individually, appropriately sized polypeptides were synthesized, but were found to be either insoluble or aggregated with other proteins. We examined the interactions between individual RPA subunits by expressing pairs of subunits and determining if they formed stable complexes. Only the 32- and 14-kDa subunits formed a soluble complex when coexpressed. This complex was purified and characterized. The 32-14 kDa subcomplex did not have any effect on DNA replication and was not phosphorylated efficiently in vitro. We believe that the 32.14-kDa subcomplex may be a precursor in the assembly of the complete RPA complex. Coexpression of all three subunits of RPA resulted in a significant portion of each polypeptide forming a soluble complex. We have purified recombinant RPA complex from E. coli and demonstrated that it has properties similar to those of human RPA. Recombinant human RPA has the same subunit composition and the same activities as the authentic complex from human cells. Recombinant human RPA binds single-stranded DNA and is capable of supporting SV40 DNA replication in vitro. In addition, recombinant RPA became phosphorylated when incubated under replication conditions.
Highlights
From the Wepartment of Biochemistry, University of Zowa, Iowa City, Iowa 52242 and the §Department of Molecular Biology and Genetics, The JohnsHopkins University, School of Medicine, Baltimore, Maryland 21205
Characterization of the function of these proteins in this polypeptides were synthesized, but were found toei-be cell-free replication reactionhas led to a general understanding ther insolubleor aggregated with other proteinWse. ex- of the mechanism of SV40 DNA replication [7,8,9, 11, 24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41]. atsTuhumnhebiiyitusnsnfecoidobtrsmymtfhopeeerldexmixnpsettrawdeebrsaaalsseicnstcopigooluumnrpbsipfalioebleiefredcxstoeswamus.enbpeOdunlenncxiilnhtyswadtrihhavaeneicnddt3euc2rado-izleeaeRxtdnepPr.drAmeT31sish24nsu.e-ei1kbnd4D-g. aiogfsSiriVtnieg4-iss0nepTqeoucfaiernfneitcpcigelDi.ecNnaTAthido-iisbnriernacedntaisdcntgitcohpaneruosriteeneqsiilntuoiaictrhtaeilaosiotznfbeidDontNuthenArAwarTicenptPdsliiancwnagdtiitoohfant.shtIihSenteVgoilr4seia0-kDa subcomplex did not have any effect on DNA repli- stranded DNA-binding protein
It is believed that DNA polymerase significantportion of eachpolypeptideforminga a is involved in lagging strand synthesis
Summary
Stimulates the enzymatic activities ofDNA polymerases a , p, Materials and E on primed templates [18, 32, 51, 66]. P3d-RPA14/32-~3a-RPA32 was digested with XbuI and BumHI, and the3'-overhangs were filledin with T4 DNA polymerase, generating a 0.94-kb DNA fragment containing a Shine-Dalgamo ribosomebinding site followed by the cDNA sequence for the 32-kDa subunit. Plld-tRPA-p3d-RPA14/32was digested with XbaI and EspI and treated with T4 DNA polymerase to generate a blunt-ended 1.6-kb fragment containing the coding sequences for both the 14- and 32-kDa subunits. Each cDNA was cloned into plasmids containing theT7 RNA polymerase promoter described by Studier et al [87] (Fig. 1).The 70-kDa subunit coding sequence was cloned using naturally occurring. Ion-exchange resins were alcohol)and analyzedby electrophoresis on 1%agarose gels in1x TAE buffer.Assays performed with partially purified protein fractions contained the indicated amountsof hRPA, rhRPA, or rhRPA32.14; 0.64 pg used to tryto solubilize the 70-kDa subunit in lysates [94].
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