A kinetic study of rat recombinant DNA polymerase beta: detection of a slow (hysteretic) transition in polymerase activity and inhibition by butylphenyl-deoxyguanosine triphosphate.
We have identified and characterized a distinct non-linearity in the time course of the reaction of mammalian DNA polymerase beta with synthetic polynucleotides. Nucleotide incorporation is biphasic; an initial burst of activity decays exponentially to a lower steady-state velocity. This slow transition in polymerase activity is not due to substrate depletion, abortive complex formation, or enzyme inactivation. The data are consistent with description of the beta-polymerase as a hysteretic enzyme, a finding which provides a potential explanation for the non-hyperbolic kinetics which have been reported previously for this polymerase. We have also found, in contrast to some previous data, that the nucleotide analogue, N2-(p-n-butylphenyl)-2'-deoxyguanosine-5'-triphosphate (BuPdGTP), is an inhibitor of the beta-polymerase. When poly(dC).oligo(dG) is used as template.primer, inhibition of the initial velocity is competitive with dGTP with a Ki of 1.25 microM. On activated DNA, however, beta-polymerase displays sensitivity to BuPdGTP which overlaps with that previously reported for DNA polymerase delta; 100 microM BuPdGTP is required to inhibit the initial velocity of a dGTP-deficient, truncated assay. Finally, we demonstrate that, in addition to its inhibition of initial velocity, BuPdGTP also modulates both the rate constant of the slow transition in polymerase activity, and the steady-state velocity of the beta-polymerase.
DiGiuseppe
J A
JA
Department of Pathology, Washington University School of Medicine, St Louis, MO 63110.
Wright
G E
GE
Dresler
S L
SL
D000818:Animals; D004256:DNA Polymerase I / Q000037:antagonists & inhibitors / Q000378:metabolism*; D003848:Deoxyguanine Nucleotides / Q000494:pharmacology; D066298:In Vitro Techniques; D007700:Kinetics; D011089:Polydeoxyribonucleotides / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins; D013698:Templates, Genetic
D003848:Deoxyguanine Nucleotides; D011089:Polydeoxyribonucleotides; D011994:Recombinant Proteins; C016681:poly(dG).poly(dC); C040364:N(2)-(4-n-butylphenyl)-2'-deoxyguanosine 5'-triphosphate; D004256:DNA Polymerase I