RECOMBINANT REPLICATION PROTEIN-A - EXPRESSION, COMPLEX-FORMATION, AND FUNCTIONAL-CHARACTERIZATION

Replication protein A (RPA) is a multisubunit, single-stranded DNA-bin ding 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 combinatio n in Escherichia coli. When subunits were expressed individually, appr opriately sized polypeptides were synthesized, but were found to be ei ther insoluble or aggregated with other proteins. We examined the inte ractions between individual RPA subunits by expressing pairs of subuni ts and determining if they formed stable complexes. Only the 32- and 1 4-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 f rom E. coli and demonstrated that it has properties similar to those o f human RPA. Recombinant human RPA has the same subunit composition an d the same activities as the authentic complex from human cells. Recom binant human RPA binds single-stranded DNA and is capable of supportin g SV40 DNA replication in vitro. In addition, recombinant RPA became p hosphorylated when incubated under replication conditions.