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

Journal Article · Tue Apr 04 00:00:00 EDT 1989 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00433a015· OSTI ID:5478618

Freeman-Wittig, M J; Welch, W Jr; Lewis, R A ^[1]

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1. Univ. of Nevada, Reno (USA)

Captan (N-((trichloromethyl)thio)-4-cyclohexene-1,2-dicarboximide) was shown to bind to DNA polymerase I from Escherichia coli. The ratio of ({sup 14}C)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. The interaction of captan with DNA pol I altered each of the three catalytic functions. The 3' {yields} 5' exonuclease and polymerase activities were inhibited, and the 5' {yields} 3' exonuclease activity was enhanced. In order to study the 5' {yields} 3' exonuclease activity more closely, ({sup 3}H)hpBR322 (DNA-({sup 3}H)RNA hybrid) was prepared from pBR322 plasmid DNA and used as a specific substrate for 5' {yields} 3' exonuclease activity. Collectively, the 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' {yields} 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. The final, irreversible step results in inhibited polymerase and 3' {yields} 5' exonuclease activities as well as enhanced 5' {yields} 3' exonuclease activity.

• OSTI ID: 5478618
• Journal Information: Biochemistry; (USA), Vol. 28:7; ISSN 0006-2960
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
DNA POLYMERASES
BIOCHEMICAL REACTION KINETICS
ENZYME ACTIVITY
IMIDES
CROSS-LINKING
CARBON 14 COMPOUNDS
DOSE-RESPONSE RELATIONSHIPS
ESCHERICHIA COLI
TRITIUM COMPOUNDS
BACTERIA
CHEMICAL REACTIONS
ENZYMES
HYDROGEN COMPOUNDS
KINETICS
LABELLED COMPOUNDS
MICROORGANISMS
NUCLEOTIDYLTRANSFERASES
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PHOSPHORUS-GROUP TRANSFERASES
POLYMERASES
POLYMERIZATION
REACTION KINETICS
TRANSFERASES
550201* - Biochemistry- Tracer Techniques