The RPA32 subunit of human replication protein A contains a single-stranded DNA-binding domain

J Biol Chem. 1998 Feb 13;273(7):3932-6. doi: 10.1074/jbc.273.7.3932.

Abstract

Replication protein A (RPA) is a conserved nuclear single-stranded DNA (ssDNA)-binding protein. Human RPA (hRPA) comprises three subunits of approximately 70, 32, and 14 kDa (hRPA70, hRPA32 and hRPA14). RPA is known to bind ssDNA through two ssDNA-binding domains in the RPA70 subunit. Here, we demonstrate that the complex of hRPA32 and hRPA14 has an ssDNA-binding domain. Limited proteolysis of the hRPA14.32 complex defined a core dimer composed of the central region of hRPA32 (amino acids 43-171) and RPA14. The core dimer bound ssDNA with an affinity of approximately 10-50 microM, which is at least 100-fold more avid than the DNA-binding affinity of the intact dimer. Analysis of the predicted secondary structure of hRPA32 suggests that amino acids 63-150 of hRPA32 form an ssDNA-binding domain similar in structure to each of those in hRPA70. The complex of hRPA14 and hRPA32-(43-171) in turn formed a trimeric complex with the C-terminal region of hRPA70 (amino acids 436-616). The ssDNA-binding affinity of this trimeric complex was 3 to 5-fold higher than hRPA14.32-(43-171) alone, suggesting a role for the C terminus of hRPA70 in ssDNA binding.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Binding Sites
  • DNA, Single-Stranded / metabolism*
  • DNA-Binding Proteins / analysis
  • DNA-Binding Proteins / chemistry*
  • Escherichia coli / genetics
  • Humans
  • Molecular Sequence Data
  • Peptide Fragments / analysis
  • Protein Conformation
  • Protein Structure, Secondary
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Replication Protein A
  • Sequence Alignment
  • Sequence Analysis
  • Serine Endopeptidases / metabolism
  • Trypsin / metabolism

Substances

  • DNA, Single-Stranded
  • DNA-Binding Proteins
  • Peptide Fragments
  • RPA1 protein, human
  • Recombinant Proteins
  • Replication Protein A
  • Serine Endopeptidases
  • glutamyl endopeptidase
  • Trypsin