Recombinant replication protein A: expression, complex formation, and functional characterization.
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.
Henricksen
L A
LA
Department of Biochemistry, University of Iowa, Iowa City 52242.
Umbricht
C B
CB
Wold
M S
MS
D001483:Base Sequence; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D017931:DNA Primers; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005822:Genetic Vectors; D006801:Humans; D015151:Immunoblotting; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D051716:Replication Protein A; D013539:Simian virus 40 / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; C497167:RPA1 protein, human; D011994:Recombinant Proteins; D051716:Replication Protein A