Characterization of a cDNA encoding the 70-kDa single-stranded DNA-binding subunit of human replication protein A and the role of the protein in DNA replication.
Replication protein A (RP-A) is a three-subunit single-stranded DNA-binding protein that has been isolated from human cells. RP-A is essential for SV40 DNA replication and may also be important in genetic recombination. The sequence of a cDNA encoding the 70-kDa subunit of human RP-A is reported. The 616-amino acid predicted open reading frame of the human protein is 31% identical with the 621-amino acid open reading frame of the 70-kDa subunit of RP-A from the yeast Saccharomyces cerevisiae. Both proteins share a highly conserved putative metal binding domain of the 4-cysteine type. The human cDNA directs production in Escherichia coli of a 70-kDa protein that reacts with a monoclonal antibody directed against the 70-kDa subunit of human RP-A. The recombinant 70-kDa subunit, purified from bacteria, exhibits single-stranded DNA binding activity comparable to that of the complete RP-A complex. The 70-kDa subunit is able to substitute for the complete human RP-A complex in stimulating the activity of DNA polymerase alpha-primase on a poly(dA).oligo(dT) template. However, the 70-kDa subunit alone cannot substitute for the complete RP-A complex in SV40 DNA replication in vitro, suggesting an important functional role for the other subunits.
Erdile
L F
LF
Department of Molecular Biology and Genetics, Johns Hopkins Medical School, Baltimore, Maryland 21205.
Heyer
W D
WD
Kolodner
R
R
Kelly
T J
TJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D004247:DNA / Q000235:genetics*; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000502:physiology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005800:Genes, Fungal; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids; D051716:Replication Protein A; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid
D004268:DNA-Binding Proteins; C497167:RPA1 protein, human; D051716:Replication Protein A; D004247:DNA