Identification of residues in the single-stranded DNA-binding site of the 8-kDa domain of rat DNA polymerase beta by UV cross-linking

J Biol Chem. 1993 Oct 25;268(30):22746-55.


Rat DNA polymerase beta (beta-pol) is a 39-kDa monomeric protein, organized in two structurally and functionally distinct domains. The 8-kDa NH2-terminal domain binds single-stranded (ss) DNA, whereas the 31-kDa COOH-terminal domain does not. To facilitate studies on ssDNA binding structure-function relationships of beta-pol, we overexpressed the 8-kDa domain in Escherichia coli, and purified the recombinant protein to homogeneity. Single-stranded nucleic acid binding of the recombinant 8-kDa domain was found to be similar to that previously reported for the 8-kDa fragment prepared by proteolysis of intact beta-pol (Kumar, A., Widen, S. G., Williams, K. R., Kedar, P. Karpel, R. L., and Wilson, S. H. (1990b) J. Biol. Chem. 265, 2124-2131; Casas-Finet, J. R., Kumar, A., Morris, G., Wilson, S. H., and Karpel, R. L. (1991) J. Biol. Chem. 266, 19618-19625). Residues in or near the DNA-binding pocket of the recombinant 8-kDa domain were examined by photochemical cross-linking to [32P] p(dT)16. Cross-linking was localized to a tryptic fragment spanning residues 28 through 35 and a V8 protease fragment spanning residues 27 through 58. Sequence analysis of the various [32P]p(dT)16-labeled proteins indicated that Ser30 and His34 were modified by cross-linking to p(dT)16. Therefore, these residues of the ssDNA-binding domain of beta-pol appear to be in close contact with this nucleic acid probe.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Autoradiography
  • Binding Sites
  • Chromatography, Ion Exchange
  • Cloning, Molecular
  • Cross-Linking Reagents
  • DNA Polymerase I / chemistry
  • DNA Polymerase I / isolation & purification
  • DNA Polymerase I / metabolism*
  • DNA, Single-Stranded / metabolism*
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli
  • Kinetics
  • Molecular Sequence Data
  • Molecular Weight
  • Osmolar Concentration
  • Peptide Fragments / chemistry
  • Peptide Fragments / isolation & purification
  • Peptide Fragments / metabolism
  • Phosphorus Radioisotopes
  • Poly T / isolation & purification
  • Poly T / metabolism
  • Rats
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Serine Endopeptidases
  • Trypsin
  • Ultraviolet Rays


  • Cross-Linking Reagents
  • DNA, Single-Stranded
  • Peptide Fragments
  • Phosphorus Radioisotopes
  • Recombinant Proteins
  • Poly T
  • DNA Polymerase I
  • Serine Endopeptidases
  • glutamyl endopeptidase
  • Trypsin