Inactivation of DNA polymerase I (Klenow fragment) by adenosine 2',3'-epoxide 5'-triphosphate: evidence for the formation of a tight-binding inhibitor

Biochemistry. 1989 May 16;28(10):4374-82. doi: 10.1021/bi00436a038.


The suicidal inactivation of Escherichia coli DNA polymerase I by epoxy-ATP has been previously reported (Abboud et al., 1978). We have examined in detail the mechanism of this inactivation utilizing a synthetic DNA template-primer of defined sequence. Epoxy-ATP inactivates the large fragment of DNA polymerase I (the Klenow fragment) in a time- and concentration-dependent manner (KI = 21 microM; kinact = 0.021 s-1). Concomitant with inactivation is the incorporation of epoxy-AMP into the primer strand. The elongated DNA duplex directly inhibits the polymerase activity of the enzyme (no time dependence) and is resistant to degradation by the 3'----5' exonuclease and pyrophosphorylase activities of the enzyme. Inactivation of the enzyme results from slow (4 X 10(-4) s-1) dissociation of the intact epoxy-terminated template-primer from the enzyme and is thus characterized as a tight-binding inhibition. Surprisingly, while the polymerase activity of the enzyme is completely suppressed by epoxy-ATP, the 3'----5' exonuclease activity remains intact. The data presented demonstrate that even though the polymerase site is occupied with duplex DNA, the enzyme can bind a second DNA duplex and carry out exonucleolytic cleavage.

Publication types

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

MeSH terms

  • Adenosine Triphosphate / analogs & derivatives*
  • Adenosine Triphosphate / pharmacology
  • Binding Sites
  • DNA / metabolism
  • DNA Polymerase I / antagonists & inhibitors*
  • Escherichia coli / enzymology
  • Exodeoxyribonuclease V
  • Exodeoxyribonucleases / metabolism
  • Kinetics
  • Peptide Fragments / antagonists & inhibitors


  • Peptide Fragments
  • adenosine 2',3'-riboepoxide 5'-triphosphate
  • Adenosine Triphosphate
  • DNA
  • DNA Polymerase I
  • Exodeoxyribonucleases
  • Exodeoxyribonuclease V