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

作者：Alnajjar Khadijeh S; Garcia Barboza Beatriz; Negahbani Amirsoheil; Nalzhjiri Maryam; Kashemirov Boris; McKenna Charles; Goodman Myron F; Sweasy Joann B^*

来源：Biochemistry, 2017, 56(15): 2096-2105.

DOI：10.1021/acs.biochem.6b01230

K289M is a variant of DNA polymerase beta (pol beta) 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 beta, single-turnover kinetic experiments were performed using beta,gamma-CXY dGTP analogues with a wide range of leaving group monoacid dissociation constants (pK(a4)), including a corresponding set of novel beta,gamma-CXY dCTP analogues. Surprisingly, we found that the values of the log of the catalytic rate constant (k(pol)) for correct insertion by K289M, in contrast to those of wild-type pol beta, do not decrease with increased leaving group pK(a4) for analogues with pK(a4) 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(k(pol)) values for incorrect insertion by K289M versus pK(a4) reveals a linear correlation with a negative slope, in this respect resembling k(pol) 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 beta.

• 出版日期2017-4-18