Open AccessReconstitution of functional human single-stranded DNA-binding protein from individual subunits expressed by recombinant baculoviruses.
Author(s) -
Evelyn Stigger,
Frank B. Dean,
Jerard Hurwitz,
Suk Hee Lee
Publication year - 1994
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.91.2.579
Subject(s) - recombinant dna , biology , protein subunit , dna , microbiology and biotechnology , dna replication , dna binding protein , in vivo , in vitro , biochemistry , gene , genetics , transcription factor
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.