RECONSTITUTION OF FUNCTIONAL HUMAN SINGLE-STRANDED DNA-BINDING PROTEIN FROM INDIVIDUAL SUBUNITS EXPRESSED BY RECOMBINANT BACULOVIRUSES

Human single stranded DNA binding protein (HSSB), also known as replic ation protein A, is composed of a 70-kDa single-stranded DNA-binding s ubunit (p70) and 34-kDa and 11-kDa (p34 and p11, respectively) subunit s of unknown functions. We have examined interactions among the HSSB s ubunits in vivo by coinfecting insect cells with different combination s of recombinant baculoviruses encoding p70, p34, or p11. In vivo, coe xpressed p34 and p11 subunits formed stable complexes, whereas neither p34 nor p11 formed stable complexes with p70. In cells coinfected wit h viruses expressing all three subunits, the stable heterotrimer forme d, which, when purified, replaced HSSB isolated from HeLa cells in var ious assays, including simian virus 40 DNA replication in vitro. These data suggest that, in the assembly of functionally active HSSB, forma tion of the p34-p11 complex precedes p70 addition to the complex.