Modifying the β,γ Leaving-Group Bridging Oxygen Alters Nucleotide Incorporation Efficiency, Fidelity, and the Catalytic Mechanism of DNA Polymerase β

Sucato, Christopher A.; Upton, Thomas G.; Kashemirov, B. A.; Batra, V.K.; Martínek, Václav; Xiang, Yun; Beard, William A.; Pedersen, Lars C.; Wilson, Samuel H.; McKenna, Charles E.; Florián, Jan; Warshel, A.; Goodman, Myron F.

doi:10.1021/bi061517b

Cited by 96 publications

(255 citation statements)

References 51 publications

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“…Because single nucleotide insertion appears to be limited by a chemical step rather than conformational adjustments (16,49), the increased rate of insertion exhibited by the R258A mutant must be due to a change in the thermodynamic equilibrium with non-productive complexes rather than a kinetic change in the rate-limiting step. Scheme 1 illustrates structural adjustments that occur in the enzyme and/or substrates of the ternary substrate complex.…”

Section: Discussionmentioning

confidence: 99%

Substrate-induced DNA Polymerase β Activation

Beard

Shock

Batra

et al. 2014

Journal of Biological Chemistry

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Background: Highly conserved residues of DNA polymerase ␤ alter their interactions as the enzyme transitions from open and closed conformations. Results: Site-directed mutagenesis coupled with kinetic/structural characterization of key mutants defines the role(s) in enzyme activation. Conclusion: These residues both hasten and deter correct and incorrect nucleotide insertion. Significance: Conformational equilibrium(s) of the precatalytic ternary substrate complex influences the observed rate of nucleotide insertion.

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Section: Discussionmentioning

confidence: 99%

Substrate-induced DNA Polymerase β Activation

Beard

Shock

Batra

et al. 2014

Journal of Biological Chemistry

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“…Specifically, recent experimental studies (Sucato et al 2007(Sucato et al , 2008) presented a new probe for leaving group effects during the nucleotide transfer reaction catalyzed by Pol b, in which the b,c-bridging oxygens of the dGTP were replaced by a series of substituted methylene groups (X=CYZ, where Y and Z=H, halogen or another substituent, Fig. 45).…”

Section: Some Points On the Interpretation Of Lfer In Polymerasesmentioning

confidence: 99%

Why nature really chose phosphate

Kamerlin

Sharma

Prasad

et al. 2013

Quart. Rev. Biophys.

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315

429

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Phosphoryl transfer plays key roles in signaling, energy transduction, protein synthesis, and maintaining the integrity of the genetic material. On the surface, it would appear to be a simple nucleophile displacement reaction. However, this simplicity is deceptive, as, even in aqueous solution, the low-lying d-orbitals on the phosphorus atom allow for eight distinct mechanistic possibilities, before even introducing the complexities of the enzyme catalyzed reactions. To further complicate matters, while powerful, traditional experimental techniques such as the use of linear free-energy relationships (LFER) or measuring isotope effects cannot make unique distinctions between different potential mechanisms. A quarter of a century has passed since Westheimer wrote his seminal review, ‘Why Nature Chose Phosphate’ (Science 235 (1987), 1173), and a lot has changed in the field since then. The present review revisits this biologically crucial issue, exploring both relevant enzymatic systems as well as the corresponding chemistry in aqueous solution, and demonstrating that the only way key questions in this field are likely to be resolved is through careful theoretical studies (which of course should be able to reproduce all relevant experimental data). Finally, we demonstrate that the reason that naturereallychose phosphate is due to interplay between two counteracting effects: on the one hand, phosphates are negatively charged and the resulting charge-charge repulsion with the attacking nucleophile contributes to the very high barrier for hydrolysis, making phosphate esters among the most inert compounds known. However, biology is not only about reducing the barrier to unfavorable chemical reactions. That is, the same charge-charge repulsion that makes phosphate ester hydrolysis so unfavorable also makes it possible to regulate, by exploiting the electrostatics. This means that phosphate ester hydrolysis can not only be turned on, but also be turned off, by fine tuning the electrostatic environment and the present review demonstrates numerous examples where this is the case. Without this capacity for regulation, it would be impossible to have for instance a signaling or metabolic cascade, where the action of each participant is determined by the fine-tuned activity of the previous piece in the production line. This makes phosphate esters the ideal compounds to facilitate life as we know it.

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“…Earlier structures of DNA pol β with incoming nucleoside triphosphate analogs with CF 2 substituted for O α;β or O β;γ indicated that this substitution is well tolerated (35,36). Recently, even O β;γ has been substituted by CXY group (X;Y ¼ H, F, Cl, Br, and /or CH 3 ) (37, 38).…”

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confidence: 99%

Synthesis and biological evaluation of fluorinated deoxynucleotide analogs based on bis-(difluoromethylene)triphosphoric acid

Prakash

Zibinsky

Upton

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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It is difficult to overestimate the importance of nucleoside triphosphates in cellular chemistry: They are the building blocks for DNA and RNA and important sources of energy. Modifications of biologically important organic molecules with fluorine are of great interest to chemists and biologists because the size and electronegativity of the fluorine atom can be used to make defined structural alterations to biologically important molecules. Although the concept of nonhydrolyzable nucleotides has been around for some time, the progress in the area of modified triphosphates was limited by the lack of synthetic methods allowing to access bisCF 2 -substituted nucleotide analogs-one of the most interesting classes of nonhydrolyzable nucleotides. These compounds have "correct" polarity and the smallest possible steric perturbation compared to natural nucleotides. No other known nucleotides have these advantages, making bisCF 2 -substituted analogs unique. Herein, we report a concise route for the preparation of hitherto unknown highly acidic and polybasic bis(difluoromethylene)triphosphoric acid 1 using a phosphorous(III)/phosphorous(V) interconversion approach. The analog 1 compared to triphosphoric acid is enzymatically nonhydrolyzable due to substitution of two bridging oxygen atoms with CF 2 groups, maintaining minimal perturbations in steric bulkiness and overall polarity of the triphosphate polyanion. The fluorinated triphosphoric acid 1 was used for the preparation of the corresponding fluorinated deoxynucleotides (dNTPs). One of these dNTP analogs (dT) was demonstrated to fit into DNA polymerase beta (DNA pol β) binding pocket by obtaining a 2.5 Å resolution crystal structure of a ternary complex with the enzyme. Unexpected dominating effect of triphosphate∕Mg 2þ interaction over Watson-Crick hydrogen bonding was found and discussed.DNA polymerase beta | nonhydrolyzable nucleotides | fluorinated triphosphate | pentabasic acid | isopolarity and bioisotericity

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Modifying the β,γ Leaving-Group Bridging Oxygen Alters Nucleotide Incorporation Efficiency, Fidelity, and the Catalytic Mechanism of DNA Polymerase β

Cited by 96 publications

References 51 publications

Substrate-induced DNA Polymerase β Activation

Substrate-induced DNA Polymerase β Activation

Why nature really chose phosphate

Synthesis and biological evaluation of fluorinated deoxynucleotide analogs based on bis-(difluoromethylene)triphosphoric acid

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