Photoaffinity labeling of the Klenow fragment with 8-azido-dATP

J Biol Chem. 1990 Mar 25;265(9):4821-7.

Abstract

The photoaffinity compound 8-azido-dATP was used as a probe for the deoxyribonucleoside triphosphate-binding site of the large fragment of DNA polymerase I. Azido-dATP specifically modified a saturable binding site within the Klenow fragment, and each of the four natural deoxyribonucleoside triphosphate substrates competed with labeling at this site in proportion to its binding constant, as previously defined by equilibrium dialysis. Analysis of tryptic peptides after azido-dATP modification revealed five major cross-linking products, which apparently arose from five distinct photoadducts formed near Tyr-766.

MeSH terms

  • Adenosine Triphosphate / analogs & derivatives*
  • Adenosine Triphosphate / metabolism
  • Affinity Labels / metabolism*
  • Amino Acid Sequence
  • Azides / metabolism*
  • Binding Sites
  • Binding, Competitive
  • DNA Polymerase I / genetics
  • DNA Polymerase I / metabolism*
  • DNA-Directed DNA Polymerase / genetics
  • DNA-Directed DNA Polymerase / metabolism*
  • Escherichia coli / enzymology*
  • Escherichia coli / genetics
  • Kinetics
  • Models, Molecular
  • Molecular Sequence Data
  • Peptide Fragments / genetics
  • Peptide Fragments / metabolism*
  • Plasmids
  • Protein Conformation
  • Trypsin

Substances

  • Affinity Labels
  • Azides
  • Peptide Fragments
  • 8-azidoadenosine 5'-triphosphate
  • Adenosine Triphosphate
  • DNA Polymerase I
  • DNA-Directed DNA Polymerase
  • Trypsin