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

Khadijeh S Alnajjar,Amirsoheil Negahbani,Joann B Sweasy,Ivan S Krylov,Boris A Kashemirov,Pouya Haratipour,Myron F Goodman,Charles E Mckenna,Ji Huang,Mariam Mahmoud

doi:10.1093/nar/gkz1076

Khadijeh S Alnajjar, Amirsoheil Negahbani + Show 8 more

Open Access

https://doi.org/10.1093/nar/gkz1076

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Abstract

DNA polymerase β (pol β) selects the correct deoxyribonucleoside triphosphate for incorporation into the DNA polymer. Mistakes made by pol β 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 β, K289M, misincorporates deoxynucleotides at significantly increased frequencies over wild-type pol β within a mutation hotspot that is present several times within the APC gene. Kinetic studies provide evidence that the rate-determining step of pol β 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 β 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 pre-catalytic conformational changes are of critical importance for DNA polymerase fidelity within specific DNA sequence contexts.

Highlights

DNA polymerase ␤ functions during base excision repair (BER) to fill small DNA gaps that are created during the removal of damaged bases [1,2,3]
We show that the K289M mutation alters the enzyme such that it is unable to accommodate the adenomatous polyposis coli (APC) DNA substrate in a manner that promotes accurate DNA synthesis
Using a dNTP toolkit that monitors the dependence of the transition state (TS) on phosphodiester bond formation, we show that WT pol ␤ catalyzes the formation of a rate-limiting chemical TS in the presence of either the control or APC sequence contexts

Summary

Introduction

DNA polymerase ␤ (pol ␤) functions during base excision repair (BER) to fill small DNA gaps that are created during the removal of damaged bases [1,2,3]. It is imperative that pol ␤ selects the correct dNTP from a pool of structured dNTPs during the gap-filling step of BER, given its central role in this repair pathway. To provide mechanistic insights into substrate selection by pol ␤, we first characterized catalytically active mutator variants of this enzyme that we identified in genetic screens and in tumors (for examples see [7,8,9,10,11,12]). The majority of these pol ␤ mutations do not map to its active site. Our findings indicate that the pol ␤ mutator variants exhibit sequence context-specific misincorporation of nucleotides at hotpots that differ from wildtype pol ␤ (WT pol ␤) [13,14,15,16,17,18]

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