The primary structure of the 32-kDa subunit of human replication protein A.
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.
Department of Molecular Biology and Genetics, Johns Hopkins Medical School, Baltimore, Maryland 21205.
L F
LF
Erdile
M S
MS
Wold
T J
TJ
Kelly
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D004247:DNA / Q000235:genetics; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D006801:Humans; D007256:Information Systems; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015345:Oligonucleotide Probes; D010449:Peptide Mapping; D012689:Sequence Homology, Nucleic Acid; D014357:Trypsin
D003062:Codon; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D015345:Oligonucleotide Probes; D004247:DNA; D014357:Trypsin