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Identification of critical residues for the tight binding of both correct and incorrect nucleotides to human DNA polymerase ?.

ABSTRACT: DNA polymerase ? (Pol ?) is a novel X-family DNA polymerase that shares 34% sequence identity with DNA polymerase ?. Pre-steady-state kinetic studies have shown that the Pol ?-DNA complex binds both correct and incorrect nucleotides 130-fold tighter, on average, than the DNA polymerase ?-DNA complex, although the base substitution fidelity of both polymerases is 10(-)(4) to 10(-5). To better understand Pol ?'s tight nucleotide binding affinity, we created single-substitution and double-substitution mutants of Pol ? to disrupt the interactions between active-site residues and an incoming nucleotide or a template base. Single-turnover kinetic assays showed that Pol ? binds to an incoming nucleotide via cooperative interactions with active-site residues (R386, R420, K422, Y505, F506, A510, and R514). Disrupting protein interactions with an incoming correct or incorrect nucleotide impacted binding to each of the common structural moieties in the following order: triphosphate?base>ribose. In addition, the loss of Watson-Crick hydrogen bonding between the nucleotide and the template base led to a moderate increase in K(d). The fidelity of Pol ? was maintained predominantly by a single residue, R517, which has minor groove interactions with the DNA template.

SUBMITTER: Brown JA

PROVIDER: S-EPMC2957559 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

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Identification of critical residues for the tight binding of both correct and incorrect nucleotides to human DNA polymerase λ.

Brown Jessica A JA Pack Lindsey R LR Sherrer Shanen M SM Kshetry Ajay K AK Newmister Sean A SA Fowler Jason D JD Taylor John-Stephen JS Suo Zucai Z

Journal of molecular biology 20100921 4

DNA polymerase λ (Pol λ) is a novel X-family DNA polymerase that shares 34% sequence identity with DNA polymerase β. Pre-steady-state kinetic studies have shown that the Pol λ-DNA complex binds both correct and incorrect nucleotides 130-fold tighter, on average, than the DNA polymerase β-DNA complex, although the base substitution fidelity of both polymerases is 10(-)(4) to 10(-5). To better understand Pol λ's tight nucleotide binding affinity, we created single-substitution and double-substitut ...[more]

PMID: 20851705

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Project description:RNA polymerase (RNAP) is the primary machine responsible for transcription. Its ability to distinguish between correct (cognate) and incorrect (noncognate) nucleoside triphosphates (NTPs) is important for fidelity control in transcription. In this work, we investigated the substrate selection mechanism of T7 RNAP from the perspective of energetics. The dissociation free energies were determined for matched and unmatched base pairs in the preinsertion complex using the umbrella sampling method. A clear hydrogen-bond-rupture peak is observed in the potential of mean force curve for a matched base pair, whereas no such peaks are present in the position of mean force profiles for unmatched ones. The free-energy barrier could prevent correct substrates from being separated from the active site. Therefore, when NTPs diffuse into the active site, correct ones will stay for chemistry once they establish effective base pairing contacts with the template nucleotide, whereas incorrect ones will be withdrawn from the active site and rejected back to solution. This result provides an important energy evidence for the substrate selection mechanism of RNAP. Then we elucidated energetics and molecular details for correct NTP binding to the active site of the insertion complex. Our observations reveal that strong interactions act on the triphosphate of NTP to constrain its movement, whereas relatively weak interactions serve to position the base in the correct conformation. Triple interactions, hydrophobic contacts from residues M635 and Y639, base stacking from the 3' RNA terminal nucleotide, and base pairing from the template nucleotide act together to position the NTP base in a catalytically competent conformation. At last, we observed that incorrect NTPs cannot be as well-stabilized as the correct one in the active site when they are misincorporated in the insertion site. It is expected that our work can be helpful for comprehensively understanding details of this basic step in genetic transcription.

Project description:Incorporating the cognate instead of non-cognate substrates is crucial for DNA polymerase function. Here we analyze molecular dynamics simulations of DNA polymerase μ (pol μ) bound to different non-cognate incoming nucleotides including A:dCTP, A:dGTP, A(syn):dGTP, A:dATP, A(syn):dATP, T:dCTP, and T:dGTP to study the structure-function relationships involved with aberrant base pairs in the conformational pathway; while a pol μ complex with the A:dTTP base pair is available, no solved non-cognate structures are available. We observe distinct differences of the non-cognate systems compared to the cognate system. Specifically, the motions of active-site residue His329 and Asp330 distort the active site, and Trp436, Gln440, Glu443 and Arg444 tend to tighten the nucleotide-binding pocket when non-cognate nucleotides are bound; the latter effect may further lead to an altered electrostatic potential within the active site. That most of these "gate-keeper" residues are located farther apart from the upstream primer in pol μ, compared to other X family members, also suggests an interesting relation to pol μ's ability to incorporate nucleotides when the upstream primer is not paired. By examining the correlated motions within pol μ complexes, we also observe different patterns of correlations between non-cognate systems and the cognate system, especially decreased interactions between the incoming nucleotides and the nucleotide-binding pocket. Altered correlated motions in non-cognate systems agree with our recently proposed hybrid conformational selection/induced-fit models. Taken together, our studies propose the following order for difficulty of non-cognate system insertions by pol μ: T:dGTP<A(syn):dATP<T:dCTP<A:dGTP<A(syn):dGTP<A:dCTP<A:dATP. This sequence agrees with available kinetic data for non-cognate nucleotide insertions, with the exception of A:dGTP, which may be more sensitive to the template sequence. The structures and conformational aspects predicted here are experimentally testable.

Dataset Information

Identification of critical residues for the tight binding of both correct and incorrect nucleotides to human DNA polymerase ?.

Publications

Identification of critical residues for the tight binding of both correct and incorrect nucleotides to human DNA polymerase λ.

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