A kinetic study of rat recombinant DNA polymerase β: detection of a slow (hysteretic) transition in polymerase activity and inhibition by butylphenyl-deoxyguanosine triphosphate

Abstract

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.

Bleomycin-induced DNA repair synthesis in permeable human fibroblasts: mediation of long-patch and short-patch repair by distinct DNA polymerases

“…Studies using BuPdGTP support this interpretation. Whereas polymerase 7 is completely re- sistant to BuPdGTP (Khan et al, 1984), inhibition of aphidicolin-resistant repair synthesis by BuPdGTP is essentially identical with that reported for purified DNA polymerase ß (DiGiuseppe et al, 1989).…”

Multi-stage proofreading in DNA replication

“…Variations of both processivity and catalytic activity as a function of the recent history of the enzyme have been documented for E. coli DNA polymerase I (Bambara et al 1976;McClure & Chow, 1980;Detera & Wilson, 1982;Abbotts et al 1988), DNA polymerase a (Detera et al 1981;Grosse et al 1983) and DNA polymerase /? (DiGiuseppe et al 1989). Thus, while evidence for the original energy relay model is still lacking, there is considerable evidence that DNA polymerases exhibit both static and dynamic cooperativity, further implying that they can exist in multiple functional (and presumably, conformational) states.…”