Binding of captan to DNA polymerase I from Escherichia coli and the concomitant effect on 5' .fwdarw. 3' exonuclease activity

Abstract

Captan (N-[(trichloromethyl)thio]-4-cyclohexene-1,2-dicarboximide) was shown to bind to DNA polymerase I from Escherichia coli. The ratio of [14C] captan bound to DNA pol I was 1:1 as measured by filter binding studies and sucrose gradient analysis. Preincubation of enzyme with polynucleotide prevented the binding of captan, but preincubation of enzyme with dGTP did not. Conversely, when the enzyme was preincubated with captan, neither polynucleotide nor dGTP binding was blocked. The modification of the enzyme by captan was described by an irreversible second-order rate process with a rate of 68 +/- 0.7 M-1 s-1. The interaction of captan with DNA pol I altered each of the three catalytic functions. The 3'----5' exonuclease and polymerase activities were inhibited, and the 5'----3' exonuclease activity was enhanced. In order to study the 5'----3' exonuclease activity more closely, [3H]hpBR322 (DNA-[3H]RNA hybrid) was prepared from pBR322 plasmid DNA and used as a specific substrate for 5'----3' exonuclease activity. When either DNA pol I or polynucleotide was preincubated with 100 microM captan, 5'----3' exonuclease activity exhibited a doubling of reaction rate as compared to the untreated sample. When 100 microM captan was added to the reaction in progress, 5'----3' exonuclease activity was enhanced to 150% of the control value. Collectively, these data support the hypothesis that captan acts on DNA pol I by irreversibly binding in the template-primer binding site associated with polymerase and 3'----5' exonuclease activities. It is also shown that the chemical reaction between DNA pol I and a single captan molecule proceeds through a Michaelis complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Cancer incidence among pesticide applicators exposed to captan in the Agricultural Health Study

“…Bacterial studies have shown that captan irreversibly binds DNA polymerase 1 of Escherichia coli and alters its catalytic functions [24]. In Salmonella typhimurium and E. coli, captan is mutagenic [25,26].…”

Exposure to the Fungicide Captan Induces DNA Base Alterations and Replicative Stress in Mammalian Cells

“…This suggests that in parallel to induction of the Fpg‐sensitive DNA lesions detected by the Fpg‐modified comet assay, captan also inhibits DNA replication, which is in good agreement with previous studies. Indeed, biochemical analyses have shown that captan could be a general inhibitor of both eukaryotic and prokaryotic DNA polymerases (Dillwith and Lewis ; Freeman‐Wittig et al ; Snyder ). These reports showed that the fungicide impairs initiation of polymerization, but not the elongation step of DNA synthesis, in an irreversible manner by competing with DNA regarding polymerase loading.…”