Insight into the Catalytic Mechanism of DNA Polymerase β:  Structures of Intermediate Complexes<sup>,</sup>

Arndt, Joseph W.; Gong, Weimin; Zhong, Xi; Showalter, Alexander K.; Liu, Jia; Dunlap, Christopher A.; Lin, Zheng; Paxson, C.; Tsai, Ming‐Daw; Chan, Michael K.

doi:10.1021/bi002176j

Cited by 126 publications

(191 citation statements)

References 24 publications

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“…It is generally assumed that a conformational change in the polymerase is ratelimiting for each nucleotide addition step. Such a change would have to be different for each base-pairing, but recent fluorescent studies of DNA polymerase-␤ have failed to detect a ratelimiting conformational change (41). Furthermore, Fersht (42) has presented arguments that a rate-limiting conformational change would not enhance specificity without altering the transition state.…”

Section: Discussionmentioning

confidence: 99%

Amino Acid Substitutions at Conserved Tyrosine 52 Alter Fidelity and Bypass Efficiency of Human DNA Polymerase η

Glick

Chau

Vigna

et al. 2003

Journal of Biological Chemistry

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DNA polymerase (Pol ) is a member of a new class of DNA polymerases that is able to copy DNA containing damaged nucleotides. These polymerases are highly error-prone during copying of unaltered DNA templates. We analyzed the relationship between bypass efficiency and fidelity of DNA synthesis by introducing substitutions for Tyr-52, a highly conserved amino acid, within the human DNA polymerase (hPol ) finger domain. Most substitutions for Tyr-52 caused reduction in bypass of UV-associated damage, measured by the ability to rescue the viability of UV-sensitive yeast cells at a high UV dose. For most mutants, the reduction in bypass ability paralleled the reduction in polymerization activity. Interestingly, the hPol Y52E mutant exhibited a greater reduction in bypass efficiency than polymerization activity. The reduction in bypass efficiency was accompanied by an up to 11-fold increase in the incorporation of complementary nucleotides relative to noncomplementary nucleotides. The fidelity of DNA synthesis, measured by copying a gapped M13 DNA template in vitro, was also enhanced as much as 15-fold; the enhancement resulted from a decrease in transitions, which were relatively frequent, and a large decrease in transversions. Our demonstration that an amino acid substitution within the active site enhances the fidelity of DNA synthesis by hPol , one of the most inaccurate of DNA polymerases, supports the hypothesis that even error-prone DNA polymerases function in base selection.Accurate DNA replication is required for the maintenance of a species. The accuracy of cellular DNA replication results from the exceptionally high fidelity of DNA synthesis by replicative DNA polymerases and multiple mechanisms for the repair of DNA damage. As a result, the mutation rate in human cells has been estimated to be as low as 1 ϫ 10 Ϫ10 mutations/nucleotide/ cell division (1). Replicational accuracy is maintained despite the large number of lesions caused by both exogenous and endogenous DNA damaging agents. For example, evidence suggests that spontaneous depurination of DNA can result in 10,000 abasic sites per cell per day (2), and an even larger number of lesions are produced as a result of DNA damage by reactive oxygen species (3). Many of the lesions are likely to escape DNA repair mechanisms and therefore are present in DNA templates during copying by replicative DNA polymerases. Bulky lesions can block the progression of DNA replication, forcing the cell to employ additional mechanisms to complete replication in the presence of unrepaired lesions (4, 5).Recently, a new family of DNA polymerases, referred to as the Y-family, has been identified and categorized as translesion synthesis DNA polymerases. The Y-family of DNA polymerases is highly conserved through evolution. Thus far, four Y-family DNA polymerases have been identified in human cells: DNA polymerase (hPol ) 1 (6, 7), DNA polymerase (hPol ) (8), DNA polymerase (hPol ) (9), and the DNA-dependent dCMP transferase Rev1 (10). In contrast to replicative polymerases, ...

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Section: Discussionmentioning

confidence: 99%

Amino Acid Substitutions at Conserved Tyrosine 52 Alter Fidelity and Bypass Efficiency of Human DNA Polymerase η

Glick

Chau

Vigna

et al. 2003

Journal of Biological Chemistry

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“…DNA polymerase b-activity and carcinogenesis (Lang et al 2004 ;Wang et al 1992). Understanding the precise mechanism of phosphate diester hydrolysis in solution is key to being able to fully understand the precise catalytic mechanism of diesterases such as DNA polymerase, which, at this point, remains unclear (Arndt et al 2001 ;Florián et al 2003aFlorián et al , 2005.…”

Section: The Mechanism(s) Of Phosphate Diester Hydrolysismentioning

confidence: 99%

Why nature really chose phosphate

Kamerlin

Sharma

Prasad

et al. 2013

Quart. Rev. Biophys.

339

448

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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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“…Thus, the question of whether a conformational change or chemical step may be rate-limiting for T7 pol needs to be better understood. Indeed, recent kinetic data for pol ␤ indicates that the chemistry step may be rate-limiting for incorporation of a correct nucleotide (30,36).…”

Section: Computational Issues and Approachesmentioning

confidence: 99%

“…Two Mg 2ϩ ions, which are clearly distinguished in the crystal structure of the T7 pol ternary complex (26), play important roles in catalysis and fidelity (27,28). The binding of dNTP substrate to the polymerase-DNA complex is usually accompanied by a transition from an open to closed protein conformation (24,29,30). The fidelity contributions from binding and conformational transitions have been studied by all-atom computer simulations (31-33).…”

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confidence: 99%

Computer simulations of protein functions: Searching for the molecular origin of the replication fidelity of DNA polymerases

Florián

Goodman

Warshel

2005

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

102

143

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The use of computers to simulate the functions of complex biological macromolecules is essential to achieve a microscopic description of biological processes and to model and interpret experimental data. Here we apply theoretical computational approaches to investigate the fidelity of T7 DNA polymerase, divided into discrete steps that include contributions from substrate binding, pKa shifts, and rate constants for the PO bondbreaking and bond-making processes. We begin by defining the discrimination between right and wrong nucleotides in terms of the free energy landscape for the dNMP incorporation reaction. We then use the linear response approximation and the empirical valence bond methods to obtain converging results for the contribution of the binding and chemical steps to the overall fidelity. These approaches are successful in reproducing general trends in the observed polymerase incorporation fidelity. The calculations demonstrate the potential for further integration of theoretical and experimental studies to analyze high-and low-fidelity DNA polymerases.T he emergence of force field analysis along with the introduction of a convenient Cartesian representation (1) has led to general computer programs aimed at describing conformational and vibrational states of arbitrary molecules and the energy refinement of protein structures (2). The ability to represent protein structures by computer programs suggested that similar methods could be used in studies of the actual biological function of proteins. At that time, however, it was not clear how to extend force field approaches to studies of bond-breaking and bond-making reactions, nor was it obvious how to devise general approaches for computer modeling of biological processes. Perhaps one could have adopted methods similar to those used in studying the reactions of small molecules. However, advances in the rigorous modeling of chemical processes in small molecules seemed insufficient to apply to large biological macromolecules, including proteins and enzymes. It was found, however, that relatively simple intuitive approaches could be used effectively to simulate a wide range of protein functions (3).An important advance was the development of methods that used the reaction in solution as a reference system for the investigation of the same reaction in the protein. Using solution reference systems eliminated the need to evaluate the potential surface of the reacting molecule with high absolute accuracy, thus paving the way for efficient computer simulations of redox centers (4), ligand binding and pK a calculations (5), signal transduction (6), mechanochemistry (7), proton pumping (8) and enzyme function in general (9). Different methods have been developed for modeling diverse functions, ranging from semiclassical surface hopping for fast light-induced processes (e.g., vision and photosynthesis) (3) to hybrid quantum mechanical͞molecular mechanics approaches to investigate enzymatic reactions (9). To demonstrate such approaches, we focus here on the fidelit...

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Insight into the Catalytic Mechanism of DNA Polymerase β: Structures of Intermediate Complexes^,

Cited by 126 publications

References 24 publications

Amino Acid Substitutions at Conserved Tyrosine 52 Alter Fidelity and Bypass Efficiency of Human DNA Polymerase η

Amino Acid Substitutions at Conserved Tyrosine 52 Alter Fidelity and Bypass Efficiency of Human DNA Polymerase η

Why nature really chose phosphate

Computer simulations of protein functions: Searching for the molecular origin of the replication fidelity of DNA polymerases

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Insight into the Catalytic Mechanism of DNA Polymerase β: Structures of Intermediate Complexes,

Cited by 126 publications

References 24 publications

Amino Acid Substitutions at Conserved Tyrosine 52 Alter Fidelity and Bypass Efficiency of Human DNA Polymerase η

Amino Acid Substitutions at Conserved Tyrosine 52 Alter Fidelity and Bypass Efficiency of Human DNA Polymerase η

Why nature really chose phosphate

Computer simulations of protein functions: Searching for the molecular origin of the replication fidelity of DNA polymerases

Contact Info

Product

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Insight into the Catalytic Mechanism of DNA Polymerase β: Structures of Intermediate Complexes^,