Inactivation of DNA Polymerase I (Klenow Fragment) by Adenosine 2',3'-Epoxide 5'-Triphosphate: Evidence for the Formation of a Tight-Binding Inhibitor

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 (K_I=21 μM; k_inact=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 × 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.