Dataset Information

Prechemistry nucleotide selection checkpoints in the reaction pathway of DNA polymerase I and roles of glu710 and tyr766.

ABSTRACT: The accuracy of high-fidelity DNA polymerases such as DNA polymerase I (Klenow fragment) is governed by conformational changes early in the reaction pathway that serve as fidelity checkpoints, identifying inappropriate template-nucleotide pairings. The fingers-closing transition (detected by a fluorescence resonance energy transfer-based assay) is the unique outcome of binding a correct incoming nucleotide, both complementary to the templating base and with a deoxyribose (rather than ribose) sugar structure. Complexes with mispaired dNTPs or complementary rNTPs are arrested at an earlier stage, corresponding to a partially closed fingers conformation, in which weak binding of DNA and nucleotide promote dissociation and resampling of the substrate pool. A 2-aminopurine fluorescence probe on the DNA template provides further information about the steps preceding fingers closing. A characteristic 2-aminopurine signal is observed on binding a complementary nucleotide, regardless of whether the sugar is deoxyribose or ribose. However, mispaired dNTPs show entirely different behavior. Thus, a fidelity checkpoint ahead of fingers closing is responsible for distinguishing complementary from noncomplementary nucleotides and routing them toward different outcomes. The E710A mutator polymerase has a defect in the early fidelity checkpoint such that some complementary dNTPs are treated as if they were mispaired. In the Y766A mutant, the early checkpoint functions normally, but some correctly paired dNTPs do not efficiently undergo fingers closing. Thus, both mutator alleles cause a blurring of the distinction between correct and incorrect base pairs and result in a larger fraction of errors passing through the prechemistry fidelity checkpoints.

SUBMITTER: Bermek O

PROVIDER: S-EPMC3770053 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

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Prechemistry nucleotide selection checkpoints in the reaction pathway of DNA polymerase I and roles of glu710 and tyr766.

Bermek Oya O Grindley Nigel D F ND Joyce Catherine M CM

Biochemistry 20130827 36

The accuracy of high-fidelity DNA polymerases such as DNA polymerase I (Klenow fragment) is governed by conformational changes early in the reaction pathway that serve as fidelity checkpoints, identifying inappropriate template-nucleotide pairings. The fingers-closing transition (detected by a fluorescence resonance energy transfer-based assay) is the unique outcome of binding a correct incoming nucleotide, both complementary to the templating base and with a deoxyribose (rather than ribose) sug ...[more]

PMID: 23937394

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Project description:With an increasing number of structural, kinetic, and modeling studies of diverse DNA polymerases in various contexts, a complex dynamical view of how atomic motions might define molecular "gates" or checkpoints that contribute to polymerase specificity and efficiency is emerging. Such atomic-level information can offer insights into rate-limiting conformational and chemical steps to help piece together mechanistic views of polymerases in action. With recent advances, modeling and dynamics simulations, subject to the well-appreciated limitations, can access transition states and transient intermediates along a reaction pathway, both conformational and chemical, and such information can help bridge the gap between experimentally determined equilibrium structures and mechanistic enzymology data. Focusing on DNA polymerase beta (pol beta), we present an emerging view of the geometric, energetic, and dynamic selection criteria governing insertion rate and fidelity mechanisms of DNA polymerases, as gleaned from various computational studies and based on the large body of existing kinetic and structural data. The landscape of nucleotide insertion for pol beta includes conformational changes, prechemistry, and chemistry "avenues", each with a unique deterministic or stochastic pathway that includes checkpoints for selective control of nucleotide insertion efficiency. For both correct and incorrect incoming nucleotides, pol beta's conformational rearrangements before chemistry include a cascade of slow and subtle side chain rearrangements, followed by active site adjustments to overcome higher chemical barriers, which include critical ion-polymerase geometries; this latter notion of a prechemistry avenue fits well with recent structural and NMR data. The chemical step involves an associative mechanism with several possibilities for the initial proton transfer and for the interaction among the active site residues and bridging water molecules. The conformational and chemical events and associated barriers define checkpoints that control enzymatic efficiency and fidelity. Understanding the nature of such active site rearrangements can facilitate interpretation of existing data and stimulate new experiments that aim to probe enzyme features that contribute to fidelity discrimination across various polymerases via such geometric, dynamic, and energetic selection criteria.

Dataset Information

Prechemistry nucleotide selection checkpoints in the reaction pathway of DNA polymerase I and roles of glu710 and tyr766.

Publications

Prechemistry nucleotide selection checkpoints in the reaction pathway of DNA polymerase I and roles of glu710 and tyr766.

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