A Change in the Rate-Determining Step of Polymerization by the K289M DNA Polymerase ß Cancer-Associated Variant.

ABSTRACT

K289M is a variant of DNA polymerase ß (pol ß) that has previously been identified in colorectal cancer. The expression of this variant leads to a 16-fold increase in mutation frequency at a specific site in vivo and a reduction in fidelity in vitro in a sequence context-specific manner. Previous work shows that this reduction in fidelity results from a decreased level of discrimination against incorrect nucleotide incorporation at the level of polymerization. To probe the transition state of the K289M mutator variant of pol ß, single-turnover kinetic experiments were performed using ß,γ-CXY dGTP analogues with a wide range of leaving group monoacid dissociation constants (pKa4), including a corresponding set of novel ß,γ-CXY dCTP analogues. Surprisingly, we found that the values of the log of the catalytic rate constant (kpol) for correct insertion by K289M, in contrast to those of wild-type pol ß, do not decrease with increased leaving group pKa4 for analogues with pKa4 values of <11. This suggests that one of the relative rate constants differs for the K289M reaction in comparison to that of the wild type (WT). However, a plot of log(kpol) values for incorrect insertion by K289M versus pKa4 reveals a linear correlation with a negative slope, in this respect resembling kpol values for misincorporation by the WT enzyme. We also show that some of these analogues improve the fidelity of K289M. Taken together, our data show that Lys289 critically influences the catalytic pathway of pol ß.