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

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Abstract
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 (Kl = 21 .mu.M; 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'' .fwdarw. 5'' exonuclease and pyrophosphorylase activities of the enzyme. Inactivation of the enzyme results from slow (4 .times. 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'' .fwdarw. 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.