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PMID: 8290566 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

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

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 91 ·No. 2 ·1994-01-18 ·Pages 579-83

Stigger E, Dean FB, Hurwitz J, Lee SH

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.

MeSH Terms
Animals Baculoviridae/genetics Base Sequence Cell Line DNA Replication DNA, Complementary/genetics DNA, Viral/metabolism DNA-Binding Proteins/chemistry,genetics,metabolism HeLa Cells Humans Molecular Sequence Data Molecular Weight Moths Protein Conformation Recombinant Proteins/chemistry,genetics,metabolism Simian virus 40/metabolism
Chemicals
DNA, Complementary DNA, Viral DNA-Binding Proteins Recombinant Proteins
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Stigger E
Department of Virology and Molecular Biology, St. Jude Children's Research Hospital, Memphis, TN 38101-0318.
Dean F B
Hurwitz J
Lee S H
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Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.91.2.579
Published
1994-01-18
Pages
579-83
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC42992
Subset
IM
Grants
NCI NIH HHS · 5P30 CA 21765-15 · United States
NCI NIH HHS · 5P30 CA 21765-16 · United States
NIGMS NIH HHS · 5R01 GM34559 · United States
External Links
PubMed source DOI: 10.1073/pnas.91.2.579 PMC Full Text View journal details
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