Photoaffinity labeling of DNA template-primer binding site in Escherichia coli DNA polymerase I. Identification of involved amino acids

J Biol Chem. 1994 Aug 26;269(34):21828-34.


We have used two self-annealing template-primers (TPs) to covalently cross-link the Klenow fragment of Escherichia coli DNA polymerase I in its polymerase mode. The specificity of cross-linking is demonstrated by the observation that other template-primers, but not the template or primer alone, readily compete with self-annealing TPs. The enzyme-TP covalent complex is catalytically active and can incorporate one nucleotide on the primer terminus of the immobilized template-primer. Using a peptide mapping approach, we have identified a 17-amino acid tryptic peptide spanning residues 759-775 as a major constituent of the TP binding domain. Amino acid sequence analysis further revealed that Ile-765, Tyr-766 in the O-helix and Ser-769, Phe-771 in the O1-helix of the three-dimensional crystal structure of the Klenow fragment constitute the attachment site for TP.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Affinity Labels
  • Amino Acid Sequence
  • Base Sequence
  • Binding, Competitive
  • Cations, Divalent / pharmacology
  • Cross-Linking Reagents
  • DNA Polymerase I / metabolism*
  • DNA Primers / metabolism*
  • DNA-Binding Proteins / metabolism*
  • Dose-Response Relationship, Radiation
  • Escherichia coli / enzymology*
  • Exodeoxyribonuclease V
  • Exodeoxyribonucleases / metabolism
  • Molecular Sequence Data
  • Osmolar Concentration
  • Peptide Fragments / chemistry
  • Sequence Analysis
  • Trypsin / metabolism
  • Ultraviolet Rays


  • Affinity Labels
  • Cations, Divalent
  • Cross-Linking Reagents
  • DNA Primers
  • DNA-Binding Proteins
  • Peptide Fragments
  • DNA Polymerase I
  • Exodeoxyribonucleases
  • Exodeoxyribonuclease V
  • Trypsin