Binding of captan to DNA polymerase I from Escherichia coli and the concomitant effect on 5'----3' exonuclease activity.
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)
Freeman-Wittig
M J
MJ
Department of Biochemistry, University of Nevada, Reno 89557.
Welch
W
W
Lewis
R A
RA
D002215:Captan / Q000378:metabolism* / Q000494:pharmacology; D004256:DNA Polymerase I / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D010957:Plasmids; D011485:Protein Binding
D004256:DNA Polymerase I; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V; D002215:Captan