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

Abstract

K289M is a variant of DNA polymerase β (pol β) 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 β, single-turnover kinetic experiments were performed using β,γ-CXY dGTP analogues with a wide range of leaving group monoacid dissociation constants (pKa4), including a corresponding set of novel β,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 β, 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 β.

DNA Polymerase β Cancer-Associated Variant I260M Exhibits Nonspecific Selectivity toward the β–γ Bridging Group of the Incoming dNTP

“…The steeper dihalogenated slopes for WT and I260M suggest that there is more sensitivity to charge stabilization during formation of TS in the presence of the dihalogenated analogues, which has been shown previously for WT pol β 25–27 . This indicates that the dihalogenated dTTP analogues are either exposed to or stimulating the formation of an altered binding pocket as compared to the other analogues.…”

“…This variant loses the dependence of log( k pol ) on p K a4 observed with WT enzyme during correct incorporation but not during misincorporation, which pointed to a fundamental change in the RDS of K289M pol β relative to WT, with a shift to a step prior to chemistry 27 . Moreover, it was found that several of the dNTP analogues tested improved the fidelity of the K289M cancer-associated variant.…”

Base Excision Repair in the Immune System: Small DNA Lesions With Big Consequences

“…In line with this, the Sweasy group has identified and characterized various functional POLB variants that have been implicated in the development of cancer (251)(252)(253)(254)(255). It was reported that POLB variants can be detected in up to 30% of human cancers (256).…”

Structures of LIG1 engaging with mutagenic mismatches inserted by polβ in base excision repair

“…Polβ has been reported to be mutated in 30% of a variety of human tumors such as lung, gastric, colorectal, and prostate cancer (37,38). Several of the cancer-associated polβ variants possess aberrant repair function in vitro such as a reduced fidelity stemming from impaired discrimination against incorrect nucleotide incorporation as reported for polβ cancer-associated mutant K289M (39,40). The expression of these variants in cells induces cellular transformation and genomic instability (41–46).…”