Nucleic Acids Research, 1989, Vol. 17, No. 8 3079-3089
© 1989
ENZYMOLOGY |
A kinetic study of rat recombinant DNA polymerase ß: detection of a slow (hysteretic) transition in polymerase activity and inhibition by butylphenyl-deoxyguanosine triphosphate
Department of Pathology, Washington University School of Medicine St Louis, MO 63110, USA 1Department of Pharmacology, University of Massachusetts Medical School Worcester. MA 01605, USA
*To whom correspondence should be addressed
Received December 6, 1988. Revised February 22, 1989. Accepted February 22, 1989.
We have identified and characterized a distinct non-linearity in the time course of the reaction of mammalian DNA polymerase ß 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 ß-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 ß-polymerase. When poly(dC) oligo(dG) is used as template primer,inhibition of the initial velocity is competitive with dGTP with a K1 of 1.25µM. On activated DNA, however,ß-polymerase displays sensitivity to BuPdGTP which overlaps with that previously reported for DNA polymerase 
; 100µM 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 ß-polymerase.