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 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 c...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 1994-01, Vol.91 (2), p.579-583
Main Authors: Stigger, Evelyn, Dean, Frank B., Hurwitz, Jerard, Lee, Suk-Hee
Format: Article
Language:English
Subjects:
Animals
Antibodies
Baculoviridae
Baculoviridae - genetics
Base Sequence
Biochemistry
Biological and medical sciences
Cell Line
Deoxyribonucleic acid
DNA
DNA Replication
DNA, Complementary - genetics
DNA, Viral - metabolism
DNA-Binding Proteins - chemistry
DNA-Binding Proteins - genetics
DNA-Binding Proteins - metabolism
Fundamental and applied biological sciences. Psychology
HeLa Cells
Humans
Insect genetics
Molecular and cellular biology
Molecular genetics
Molecular Sequence Data
Molecular Weight
Moths
Phosphorylation
Protein Conformation
Proteins
Recombinant Proteins - chemistry
Recombinant Proteins - genetics
Recombinant Proteins - metabolism
Replication
Simian virus 40 - metabolism
Single stranded DNA
Viral tumor antigens
Viruses
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Summary: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.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.91.2.579