A pre-catalytic non-covalent step governs DNA polymerase β fidelity

Abstract

DNA polymerase beta (pol beta) selects the correct deoxyribonucleoside triphosphate for incorporation into the DNA polymer. Mistakes made by pol beta lead to mutations, some of which occur within specific sequence contexts to generate mutation hotspots. The adenomatous polyposis coli (APC) gene is mutated within specific sequence contexts in colorectal carcinomas but the underlying mechanism is not fully understood. In previous work, we demonstrated that a somatic colon cancer variant of pol beta, K289M, misincorporates deoxynucleotides at significantly increased frequencies over wild-type pol beta within a mutation hotspot that is present several times within the APC gene. Kinetic studies provide evidence that the rate-determining step of pol beta catalysis is phosphodiester bond formation and suggest that substrate selection is governed at this step. Remarkably, we show that, unlike WT, a pre-catalytic step in the K289M pol beta kinetic pathway becomes slower than phosphodiester bond formation with the APC DNA sequence but not with a different DNA substrate. Based on our studies, we propose that precatalytic conformational changes are of critical importance for DNA polymerase fidelity within specific DNA sequence contexts.