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

ABSTRACT: DNA polymerase ? (pol ?) selects the correct deoxyribonucleoside triphosphate for incorporation into the DNA polymer. Mistakes made by pol ? 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 ?, K289M, misincorporates deoxynucleotides at significantly increased frequencies over wild-type pol ? within a mutation hotspot that is present several times within the APC gene. Kinetic studies provide evidence that the rate-determining step of pol ? 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 ? 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.

SUBMITTER: Alnajjar KS

PROVIDER: S-EPMC7145665 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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

Alnajjar Khadijeh S KS Krylov Ivan S IS Negahbani Amirsoheil A Haratipour Pouya P Kashemirov Boris A BA Huang Ji J Mahmoud Mariam M McKenna Charles E CE Goodman Myron F MF Sweasy Joann B JB

Nucleic acids research 20191201 22

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, mi ...[more]

PMID: 31732732

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Project description:DNA polymerase beta (pol beta) from rat brain, overexpressed in Escherichia coli, was used as a model to study the factors responsible for substrate specificity [kpol, Kd(app) and kpol/Kd(app)] and fidelity during DNA synthesis. The roles of two active-site residues, Asn-279 and Tyr-271, were examined by construction of N279A, N279Q, Y271A, Y271F and Y271S mutants followed by structural analyses by NMR and CD and functional analyses by pre-steady-state kinetics. The results are summarized as follows. (i) None of the two-dimensional NMR spectra of the mutants was significantly perturbed relative to that for wild-type pol beta, suggesting that Tyr-271 and Asn-279 are not important for the global structure of the protein. (ii) CD analyses of guanidinium hydrochloride-induced denaturation showed that all mutants behaved similarly to the wild type in the free energy of denaturation, suggesting that Tyr-271 and Asn-279 are not critical for the conformational stability of pol beta. (iii) The Kd(app) for the correct dNTP was lower than that for the incorrect dNTP by a factor of 10-30 in the case of wild-type pol beta. Upon mutation to give N279A and N279Q, the Kd(app) for the correct dNTP increased by a factor of 15-25. As a consequence, the Kd(app) values for the correct and incorrect nucleotides were similar for N279A and N279Q, suggesting that the main function of the side chain of Asn-279 is in discrimination between the binding of correct and incorrect dNTPs. (iv) In the case of the Y271A mutant, the fidelity and the catalytic efficiency kpol/Kd(app) were little perturbed relative to the wild type. However, both the kpol and Kd(app) values for dNTP were 4-8 times lower in the case of the Y271A mutant than the corresponding values for wild-type pol beta. Since the chemical step may not be rate-limiting for wild-type pol beta, the effect on kpol could be quite significant if it is caused by a perturbation in the chemical step. (v) Pol beta displayed the greatest specificity towards the G:C base pair, which is incorporated during base excision repair of G:U and G:T mispairs. This specificity was slightly enhanced for the Y271F mutant.

Dataset Information

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

Publications

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

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