Reconstitution of functional human single-stranded DNA-binding protein from individual subunits expressed by recombinant baculoviruses

Abstract

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