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Erdile, LF, Wold, MS and Kelly, TJ (1990) The primary structure of the 32-kDa subunit of human replication protein A. J. Biol. Chem. 265:3177-82

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.

PubMed

Amino Acid Sequence; Base Sequence; Cloning, Molecular; Codon/genetics; DNA/genetics; DNA Replication; DNA-Binding Proteins/genetics; Escherichia coli/genetics; Gene Library; Humans; Information Systems; Macromolecular Substances; Molecular Sequence Data; Molecular Weight; Oligonucleotide Probes; Peptide Mapping; Sequence Homology, Nucleic Acid; Trypsin