The primary structure of the 32-kDa subunit of human replication protein A

L F Erdile; M S Wold; T J Kelly

doi:10.1016/S0021-9258(19)39750-9

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The primary structure of the 32-kDa subunit of human replication protein A

Journal article

Open access

Peer reviewed

The primary structure of the 32-kDa subunit of human replication protein A

L F Erdile, M S Wold and T J Kelly

The Journal of biological chemistry, Vol.265(6), pp.3177-3182

02/25/1990

DOI: 10.1016/S0021-9258(19)39750-9

PMID: 2406247

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Abstract

Replication protein A (RP-A) is a complex of three polypeptides of molecular mass 70, 32, and 14 kDa, which is absolutely required for simian virus 40 DNA replication in vitro. We have isolated a cDNA coding for the 32-kDa subunit of RP-A. An oligonucleotide probe was constructed based upon a tryptic peptide sequence derived from whole RP-A, and clones were isolated from a lambda gt11 library containing HeLa cDNA inserts. The amino acid sequence predicted from the cDNA contains the peptide sequence obtained from whole RP-A along with two sequences obtained from tryptic peptides derived from sodium dodecyl sulfate-polyacrylamide gel-purified 32-kDa subunit. The coding sequence predicts a protein of 29,228 daltons, in good agreement with the electrophoretically determined molecular mass of the 32-kDa subunit. No significant homology was found with any of the sequences in the GenBank data base. The protein predicted from the cDNA has an N-terminal region rich in glycine and serine along with two acidic and two basic segments. Monoclonal antibodies have been raised against the 70- and 32-kDa subunits of RP-A. The cloned cDNA has been overexpressed in bacteria using an inducible T7 expression system. The protein made in bacteria is recognized by a monoclonal antibody that is specific for the 32-kDa subunit of RP-A. This monoclonal antibody against the 32-kDa subunit inhibits DNA replication in vitro.

DNA Replication

Amino Acid Sequence

Oligonucleotide Probes

Molecular Weight

Gene Library

Humans

Information Systems

Molecular Sequence Data

DNA-Binding Proteins - genetics

Sequence Homology, Nucleic Acid

Macromolecular Substances

DNA - genetics

Trypsin

Peptide Mapping

Escherichia coli - genetics

Base Sequence

Cloning, Molecular

Codon - genetics

Details

Title: Subtitle: The primary structure of the 32-kDa subunit of human replication protein A
Creators: L F Erdile - Department of Molecular Biology and Genetics, Johns Hopkins Medical School, Baltimore, Maryland 21205
M S Wold
T J Kelly
Resource Type: Journal article
Publication Details: The Journal of biological chemistry, Vol.265(6), pp.3177-3182
DOI: 10.1016/S0021-9258(19)39750-9
PMID: 2406247
NLM abbreviation: J Biol Chem
ISSN: 0021-9258
eISSN: 1083-351X
Publisher: United States
Grant note: GM42780 / NIGMS NIH HHS
Language: English
Date published: 02/25/1990
Academic Unit: Radiation Oncology; Biochemistry and Molecular Biology
Record Identifier: 9984024527602771

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