Recombinant replication protein A : expression, complex formation, and functional characterization

Leigh A Henricksen; Christopher B Umbricht; M. S WOLD

doi:10.1016/S0021-9258(19)78100-9

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Recombinant replication protein A : expression, complex formation, and functional characterization

Journal article

Open access

Peer reviewed

Recombinant replication protein A : expression, complex formation, and functional characterization

Leigh A Henricksen, Christopher B Umbricht and M. S WOLD

The Journal of biological chemistry, Vol.269(15), pp.11121-11132

1994

DOI: 10.1016/S0021-9258(19)78100-9

PMID: 8157639

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Abstract

Replication protein A (RPA) is a multisubunit, single-stranded DNA-binding protein that is absolutely required for replication of SV40 DNA. The three cDNAs encoding the subunits of human replication protein A (70, 32, and 14 kDa) have been expressed individually and in combination in Escherichia coli. When subunits were expressed individually, appropriately sized polypeptides were synthesized, but were found to be either insoluble or aggregated with other proteins. We examined the interactions between individual RPA subunits by expressing pairs of subunits and determining if they formed stable complexes. Only the 32- and 14-kDa subunits formed a soluble complex when coexpressed. This complex was purified and characterized. The 32-14 kDa subcomplex did not have any effect on DNA replication and was not phosphorylated efficiently in vitro. We believe that the 32.14-kDa subcomplex may be a precursor in the assembly of the complete RPA complex. Coexpression of all three subunits of RPA resulted in a significant portion of each polypeptide forming a soluble complex. We have purified recombinant RPA complex from E. coli and demonstrated that it has properties similar to those of human RPA. Recombinant human RPA has the same subunit composition and the same activities as the authentic complex from human cells. Recombinant human RPA binds single-stranded DNA and is capable of supporting SV40 DNA replication in vitro. In addition, recombinant RPA became phosphorylated when incubated under replication conditions.

Molecular Genetics

Fundamental and applied biological sciences. Psychology

Biological and medical sciences

Replication

Molecular and cellular biology

Details

Title: Subtitle: Recombinant replication protein A : expression, complex formation, and functional characterization
Creators: Leigh A Henricksen - Univ. Iowa, dep. biochemistry, Iowa City IA 42242, United States
Christopher B Umbricht - Johns Hopkins University School of Medicine
M. S WOLD - Univ. Iowa, dep. biochemistry, Iowa City IA 42242, United States
Resource Type: Journal article
Publication Details: The Journal of biological chemistry, Vol.269(15), pp.11121-11132
DOI: 10.1016/S0021-9258(19)78100-9
PMID: 8157639
NLM abbreviation: J Biol Chem
ISSN: 0021-9258
eISSN: 1083-351X
Publisher: American Society for Biochemistry and Molecular Biology; Bethesda, MD
Language: English
Date published: 1994
Academic Unit: Radiation Oncology; Biochemistry and Molecular Biology
Record Identifier: 9984024559902771

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