Dataset Information

DNTP-dependent conformational transitions in the fingers subdomain of Klentaq1 DNA polymerase: insights into the role of the "nucleotide-binding" state.

ABSTRACT: Conformational selection plays a key role in the polymerase cycle.Klentaq1 exists in conformational equilibrium between three states (open, closed, and “nucleotide-binding”) whose level of occupancy is determined by the bound substrate.The “nucleotide-binding” state plays a pivotal role in the reaction pathway.Direct evidence is provided for the role of a conformationally distinct “nucleotide-binding” state during dNTP incorporation. DNA polymerases are responsible for the accurate replication of DNA. Kinetic, single-molecule, and x-ray studies show that multiple conformational states are important for DNA polymerase fidelity. Using high precision FRET measurements, we show that Klentaq1 (the Klenow fragment of Thermus aquaticus DNA polymerase 1) is in equilibrium between three structurally distinct states. In the absence of nucleotide, the enzyme is mostly open, whereas in the presence of DNA and a correctly base-pairing dNTP, it re-equilibrates to a closed state. In the presence of a dNTP alone, with DNA and an incorrect dNTP, or in elevated MgCl2 concentrations, an intermediate state termed the "nucleotide-binding" state predominates. Photon distribution and hidden Markov modeling revealed fast dynamic and slow conformational processes occurring between all three states in a complex energy landscape suggesting a mechanism in which dNTP delivery is mediated by the nucleotide-binding state. After nucleotide binding, correct dNTPs are transported to the closed state, whereas incorrect dNTPs are delivered to the open state.

SUBMITTER: Rothwell PJ

PROVIDER: S-EPMC3650393 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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dNTP-dependent conformational transitions in the fingers subdomain of Klentaq1 DNA polymerase: insights into the role of the "nucleotide-binding" state.

Rothwell Paul J PJ Allen William J WJ Sisamakis Evangelos E Kalinin Stanislav S Felekyan Suren S Widengren Jerker J Waksman Gabriel G Seidel Claus A M CA

The Journal of biological chemistry 20130322 19

<h4>Background</h4>Conformational selection plays a key role in the polymerase cycle.<h4>Results</h4>Klentaq1 exists in conformational equilibrium between three states (open, closed, and “nucleotide-binding”) whose level of occupancy is determined by the bound substrate.<h4>Conclusion</h4>The “nucleotide-binding” state plays a pivotal role in the reaction pathway.<h4>Significance</h4>Direct evidence is provided for the role of a conformationally distinct “nucleotide-binding” state during dNTP in ...[more]

PMID: 23525110

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Project description:HIV-1 reverse transcriptase shares the key features of high fidelity polymerases, such as a closed architecture of the active site, but displays a level of fidelity that is intermediate to that of high fidelity, replicative polymerases and low fidelity translesion synthesis (TLS) polymerases. The beta3-beta4 loop of the HIV-1 RT fingers subdomain makes transient contacts with the dNTP and template base. To investigate the role of active site architecture in HIV-1 RT fidelity, we truncated the beta3-beta4 loop, eliminating contact between Lys65 and the gamma-phosphate of dNTP. The mutant, in a manner reminiscent of TLS polymerases, was only able to incorporate a nucleotide that was capable of base-pairing with the template nucleotide, but not a nucleotide shape-analog incapable of Watson-Crick hydrogen bonding. Unexpectedly, however, the deletion mutant differed from the TLS polymerases in that it displayed an increased fidelity. The increased fidelity was associated with reduced dNTP binding affinity as measured using the dead end complex formation. In an effort to delineate the specific amino acid residue in the deleted segment responsible for this phenotype, we examined the K65 residue. Two substitution mutants, K65R and K65A were studied. The K65A mutant behaved similarly to the deletion mutant displaying dependence on Watson-Crick hydrogen bonding, increased fidelity and reduced dNTP-binding, while the K65R was more akin to wild-type enzyme. These results underscore the key role of the K65 residue in the phenotype observed in the deletion mutant. Based on the well-known electrostatic interaction between K65 and the gamma-phosphate moiety of incoming dNTP substrate in the ternary complex structure of HIV-1 RT, we conclude that non-discriminatory interactions between beta3-beta4 loop and the dNTP in wild-type HIV-1 RT help lower dNTP selectivity. Our results show that the fidelity of dNTP insertion is influenced by protein interactions with the triphosphate moiety.

Dataset Information

DNTP-dependent conformational transitions in the fingers subdomain of Klentaq1 DNA polymerase: insights into the role of the "nucleotide-binding" state.

Publications

dNTP-dependent conformational transitions in the fingers subdomain of Klentaq1 DNA polymerase: insights into the role of the "nucleotide-binding" state.

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