William A Beard

National Institute of Environmental Health Sciences, Durham, North Carolina, United States

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Publications (128)704.69 Total impact

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    ABSTRACT: Mammalian DNA polymerase (pol) β is the founding member of a large group of DNA polymerases now termed the X-family. DNA polymerase β has been kinetically, structurally, and biologically well characterized and can serve as a phylogenetic reference. Accordingly, we have performed a phylogenetic analysis to understand the relationship between pol β and other members of the X-family of DNA polymerases. The bacterial X-family DNA polymerases, Saccharomyces cerevisiae pol IV, and four mammalian X-family polymerases appear to be directly related. These enzymes originated from an ancient common ancestor characterized in two Bacillus species. Understanding distinct functions for each of the X-family polymerases, evolving from a common bacterial ancestor is of significant interest in light of the specialized roles of these enzymes in DNA metabolism.
    DNA repair. 08/2014; 22C:77-88.
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    ABSTRACT: Cytosine methylation and demethylation in tracks of CpG dinucleotides is an epigenetic mechanism for control of gene expression. The initial step in the demethylation process can be deamination of 5-methylcytosine producing the TpG alteration and T:G mispair, and this step is followed by thymine DNA glycosylase (TDG) initiated base excision repair (BER). A further consideration is that guanine in the CpG dinucleotide may become oxidized to 7,8-dihydro-8-oxoguanine (8-oxoG), and this could affect the demethylation process involving TDG-initiated BER. However, little is known about the enzymology of BER of tandemly altered CpG dinucleotides; e.g., Tp8-oxoG. Here, we investigated interactions between this altered dinucleotide and purified BER enzymes, the DNA glycosylases TDG and 8-oxoG DNA glycosylase 1 (OGG1), apurinic/apyrimidinic (AP) endonuclease 1, DNA polymerase β and DNA ligases. The overall TDG-initiated BER of the Tp8-oxoG dinucleotide is significantly reduced. Specifically, TDG and DNA ligase activities are reduced by a 3[prime]-flanking 8-oxoG. In contrast, OGG1-initiated BER pathway is blocked due to the 5[prime]-flanking T:G mispair; this reduces OGG1, AP endonuclease 1, and DNA polymerase β activities. Further, in TDG-initiated BER, TDG remains bound to its product AP site blocking OGG1 access to the adjacent 8-oxoG. These results reveal BER enzyme specificities enabling suppression of OGG1-initiated BER and coordination of TDG-initiated BER at this tandem alteration in the CpG dinucleotide.
    Journal of Biological Chemistry 04/2014; · 4.65 Impact Factor
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    ABSTRACT: DNA polymerase (pol) β is a multi-domain enzyme with two enzymatic activities that plays a central role in the overlapping base excision repair and single strand break repair pathways. The high frequency of pol β variants identified in tumor-derived tissues suggests a possible role in the progression of cancer, making it of interest to determine the functional consequences of these variants. Pol β containing a proline substitution for leucine 22 in the lyase domain (LD), identified in gastric tumors, has been reported to exhibit severe impairment of both lyase and polymerase activities. NMR spectroscopic evaluations of both pol β and the isolated LD containing the L22P mutation demonstrate destabilization sufficient to result in LD-selective unfolding with minimal structural perturbations to the polymerase domain. Unexpectedly, addition of single-stranded or hairpin DNA resulted in partial refolding of the mutated lyase domain, both in isolation and for the full-length enzyme. Further, formation of an abortive ternary complex using Ca2+ and a complementary dNTP indicates that the fraction of pol β(L22P) containing the folded LD undergoes conformational activation similar to that of the wild-type enzyme. Kinetic characterization of the polymerase activity of L22P pol β indicates that the L22P mutation compromises DNA binding, but nearly wild-type catalytic rates can be observed at elevated substrate concentrations. The organic osmolyte trimethylamine N-oxide (TMAO) is similarly able to induce folding and kinetic activation of both polymerase and lyase activities of the mutant. Kinetic data indicate synergy between the TMAO cosolvent and substrate binding. NMR data indicate that the effect of the DNA results primarily from interaction with the folded LD(L22P), while the effect of the TMAO results primarily from destabilization of the unfolded LD(L22P). These studies illustrate that substrate-induced catalytic activation of pol β provides an optimal enzyme conformation even in the presence of a strongly destabilizing point mutation. Accordingly, it remains to be determined whether this mutation alters the threshold of cellular repair activity needed for routine genome maintenance or whether the 'inactive' variant interferes with DNA repair.
    Biochemistry 03/2014; · 3.38 Impact Factor
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    ABSTRACT: Kinetics studies of dNTP analogs having pyrophosphate-mimicking β,γ-pCXYp leaving groups with variable X,Y-substitution reveal striking differences in the chemical transition-state energy for DNA polymerase β that depend on all aspects of base pairing configurations, including whether the incoming dNTP is a purine or pyrimidine and if base pairings are right (T•A, G•C) or wrong (T•G, G•T). Brønsted plots of the catalytic rate constant (log(kpol)) vs pKa4 for the leaving group exhibit Linear Free Energy Relationships (LFERs) with negative slopes ranging from -0.6 to -2.0, consistent with chemical rate-determining transition-states in which the active site adjusts to charge stabilization demand during chemistry depending on base-pair configuration. The Brønsted slopes and also intercepts differ dramatically, and provide the first direct evidence that dNTP base recognition by the enzyme-primer-template complex triggers a conformational change in the catalytic region of the active site, that significantly modifies the rate-determining chemical step.
    Biochemistry 03/2014; · 3.38 Impact Factor
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    ABSTRACT: To interpret recent structures of the human DNA repair enzyme DNA polymerase β (pol β) differing in the number of Mg(2+) ions, we apply transition path sampling (TPS) to assess the effect of differing ion placement on the transition from the open one-metal to the closed two-metal state. We find that the closing pathway depends on the initial ion position, both in terms of the individual transition states and associated energies. The energy barrier of the conformational pathway varies from 25 to 58 kJ/mol, compared to the conformational energy barrier of 42 kJ/mol for the wild-type pol β reported previously. Moreover, we find a preferred ion route located in the center of the enzyme, parallel to the DNA. Within this route, the conformational pathway is similar to that of the overall open to closed transition of pol β but outside it, especially when ion starts near active site residues Arg258 and Asp190, the conformational pathway diverges significantly. We hypothesize that our findings should apply generally to pol β, since R283 is relatively far from the active site. Our hypothesis suggests further experimental and computational work. Our studies also underscore the common feature that less active mutants have less stable closed states than their open states, in marked contrast to the wild-type enzyme, where the closed state is significantly more stable than the open form.
    Journal of the American Chemical Society 02/2014; · 10.68 Impact Factor
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    Cell cycle (Georgetown, Tex.) 01/2014; 13(5). · 5.24 Impact Factor
  • Chemical Reviews 12/2013; · 41.30 Impact Factor
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    ABSTRACT: Human DNA polymerase (pol) β is essential for base excision repair. We previously reported a triple somatic mutant of pol β (p.P261L/T292A/I298T) found in an early onset prostate tumor. This mutation abolishes polymerase activity, and the wild-type allele was not present in the tumor, indicating a complete deficiency in pol β function. The effect on polymerase activity is unexpected because the point mutations that comprise the triple mutant are not part of the active site. Herein, we demonstrate the mechanism of this loss-of-function. In order to understand the effect of the individual point mutations we biochemically analyzed all single and double mutants that comprise the triple mutant. We found that the p.I298T mutation is responsible for a marked instability of the triple mutant protein at 37˚C. At room temperature the triple mutant's low efficiency is also due to a decrease in the apparent binding affinity for the dNTP substrate, which is due to the p.T292A mutation. Furthermore, the triple mutant displays lower fidelity for transversions in vitro, due to the p.T292A mutation. We conclude that distinct mutations of the triple pol β mutant are responsible for the loss of activity, lower fidelity, and instability observed in vitro.
    International Journal of Oncology 07/2013; · 2.66 Impact Factor
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    ABSTRACT: DNA polymerase (pol) β is a model polymerase involved in gap-filling DNA synthesis utilizing two metals to facilitate nucleotidyl transfer. Previous structural studies have trapped catalytic intermediates by utilizing substrate analogs (dideoxy-terminated primer or nonhydrolysable incoming nucleotide). To identify additional intermediates during catalysis, we now employ natural substrates (correct and incorrect nucleotides) and follow product formation in real time with 15 different crystal structures. We are able to observe molecular adjustments at the active site that hasten correct nucleotide insertion and deter incorrect insertion not appreciated previously. A third metal binding site is transiently formed during correct, but not incorrect, nucleotide insertion. Additionally, long incubations indicate that pyrophosphate more easily dissociates after incorrect, compared to correct, nucleotide insertion. This appears to be coupled to subdomain repositioning that is required for catalytic activation/deactivation. The structures provide insights into a fundamental chemical reaction that impacts polymerase fidelity and genome stability.
    Cell 07/2013; 154(1):157-68. · 31.96 Impact Factor
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    ABSTRACT: DNA polymerase β (pol β) is a bifunctional enzyme widely studied for its roles in base excision DNA repair where one key function is gap-filling DNA synthesis. In spite of significant progress in recent years, the atomic level mechanism of the DNA synthesis reaction has remained poorly understood. Based on crystal structures of pol β in complex with its substrates and theoretical considerations of amino acids and metals in the active site, we have proposed that a nearby carboxylate group of Asp256 enables the reaction by accepting a proton from the primer O3´group, thus activating O3´as the nucleophile in the reaction path. Here, we tested this proposal by altering the side chain of Asp256 to Glu and then exploring the impact of this conservative change on the reaction. The D256E enzyme is more than 1,000-fold less active than the wild-type enzyme, and the crystal structures are subtly different in the active sites of the D256E and wild-type enzymes. Theoretical analysis of DNA synthesis by the D256E enzyme shows that the O3´proton still transfers to the nearby carboxylate of residue 256. However, the electrostatic stabilization and location of the O3´ proton transfer during the reaction path are dramatically altered compared with wild-type. Surprisingly, this is due to repositioning of the Arg254 side chain in the Glu256 enzyme active site, such that Arg254 is not in position to stabilize the proton transfer from O3´. The theoretical results with the wild-type enzyme indicate early charge reorganization associated with the O3´ proton transfer, and this does not occur in the D256E enzyme. The charge reorganization is mediated by the catalytic magnesium ion in the active site.
    Journal of the American Chemical Society 05/2013; · 10.68 Impact Factor
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    ABSTRACT: To aid in the characterization of the relationship of structure and function for human immunodeficiency virus type-1 reverse transcriptase (HIV-1 RT), this investigation utilized DNAs containing benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE)-modified primers and templates as a probe of the architecture of this complex. BPDE lesions that differed in their stereochemistry around the C10 position were covalently linked to N (6)-adenine and positioned in either the primer or template strand of a duplex template-primer. HIV-1 RT exhibited a stereoisomer-specific and strand-specific difference in replication when the BPDE-lesion was placed in the template versus the primer strand. When the C10 R-BPDE adduct was positioned in the primer strand in duplex DNA, 5 nucleotides from the 3΄ end of the primer terminus, HIV-1 RT could not fully replicate the template, producing truncated products; this block to further synthesis did not affect rates of dissociation or DNA binding affinity. Additionally, when the adducts were in the same relative position, but located in the template strand, similar truncated products were observed with both the C10 R and C10 S BPDE adducts. These data suggest that the presence of covalently-linked intercalative DNA adducts distant from the active site can lead to termination of DNA synthesis catalyzed by HIV-1 RT.
    PLoS ONE 01/2013; 8(9):e72131. · 3.53 Impact Factor
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    ABSTRACT: Human 8-oxoguanine DNA glycosylase (OGG1) excises the mutagenic oxidative DNA lesion 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Kinetic characterization of OGG1 is undertaken to measure the rates of 8-oxoG excision and product release. When the OGG1 concentration is lower than substrate DNA, time courses of product formation are biphasic; a rapid exponential phase (i.e. burst) of product formation is followed by a linear steady-state phase. The initial burst of product formation corresponds to the concentration of enzyme properly engaged on the substrate, and the burst amplitude depends on the concentration of enzyme. The first-order rate constant of the burst corresponds to the intrinsic rate of 8-oxoG excision and the slower steady-state rate measures the rate of product release (product DNA dissociation rate constant, koff). Here, we describe steady-state, pre-steady-state, and single-turnover approaches to isolate and measure specific steps during OGG1 catalytic cycling. A fluorescent labeled lesion-containing oligonucleotide and purified OGG1 are used to facilitate precise kinetic measurements. Since low enzyme concentrations are used to make steady-state measurements, manual mixing of reagents and quenching of the reaction can be performed to ascertain the steady-state rate (koff). Additionally, extrapolation of the steady-state rate to a point on the ordinate at zero time indicates that a burst of product formation occurred during the first turnover (i.e. y-intercept is positive). The first-order rate constant of the exponential burst phase can be measured using a rapid mixing and quenching technique that examines the amount of product formed at short time intervals (<1 sec) before the steady-state phase and corresponds to the rate of 8-oxoG excision (i.e. chemistry). The chemical step can also be measured using a single-turnover approach where catalytic cycling is prevented by saturating substrate DNA with enzyme (E>S). These approaches can measure elementary rate constants that influence the efficiency of removal of a DNA lesion.
    Journal of Visualized Experiments 01/2013;
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    ABSTRACT: A major base lesion resulting from oxidative stress is 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxoG) that has ambiguous coding potential. Error-free DNA synthesis involves 8-oxoG adopting an anti-conformation to base pair with cytosine whereas mutagenic bypass involves 8-oxoG adopting a syn-conformation to base pair with adenine. Left unrepaired the syn-8-oxoG/dAMP base pair results in a G-C to T-A transversion. During base excision repair of this mispair, DNA polymerase (pol) β is confronted with gap filling opposite 8-oxoG. To determine how pol β discriminates between anti- and syn-8-oxoG, we introduced a point mutation (R283K) to alter insertion specificity. Kinetic studies demonstrate that this substitution results in an increased fidelity opposite 8-oxoG. Structural studies with R283K pol β show that the binary DNA complex has 8-oxoG in equilibrium between anti- and syn-forms. Ternary complexes with incoming dCTP resemble the wild-type enzyme, with templating anti-8-oxoG base pairing with incoming cytosine. In contrast to wild-type pol β, the ternary complex of the R283K mutant with an incoming dATP-analogue and templating 8-oxoG resembles a G-A mismatched structure with 8-oxoG adopting an anti-conformation. These results demonstrate that the incoming nucleotide is unable to induce a syn-8-oxoG conformation without minor groove DNA polymerase interactions that influence templating (anti-/syn-equilibrium) of 8-oxoG while modulating fidelity.
    Nucleic Acids Research 12/2012; · 8.81 Impact Factor
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    ABSTRACT: Although the primary function of DNA polymerase (pol) β is associated with gap-filling DNA synthesis as part of the DNA base excision repair pathway, translesion synthesis activity has also been described. To further understand the potential role of pol β-catalyzed translesion DNA synthesis (TLS) and the structure-function relationships of specific residues in pol β, wild-type and selected mutants of pol β were used in TLS assays with DNA substrates containing bulky polycyclic aromatic hydrocarbon-adducted oligonucleotides. Stereo-specific (+) and (-)-anti-trans-(C10S and C10R) benzo[a]pyrene-7,8- dihydrodiol-9-10-epoxide (BPDE) adducts were covalently attached to both the N(6)-adenine and N(2)-guanine in the major and minor grooves, respectively. For all substrates tested, the presence of the BPDE adducts greatly decreased the efficiency of nucleotide incorporation opposite the lesion and the stereochemistry of the adducts also further modulated the efficiency of the insertion step, such that lesions pointing toward the oncoming polymerase were considerably more blocking than those oriented away from the polymerase. In the absence of a downstream DNA strand, the extension step beyond the adduct was extremely inefficient, relative to a dinucleotide gap-filling reaction, such that in the presence of the downstream DNA, dinucleotide incorporation was strongly favored. In general, analyses of the TLS activities of four pol β mutants revealed similar overall properties but wild-type pol β exhibited more than 50-fold greater extension and bypass of the C10S-dA adducts as compared to a low fidelity mutant R283K expected to interact with the templating base. Replication bypass investigations were further extended to include analyses of HIV-1 reverse transcriptase, and these studies revealed patterns of inhibition very similar to that observed for pol β.
    Chemical Research in Toxicology 11/2012; · 3.67 Impact Factor
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    ABSTRACT: In recent papers, there has been a lively exchange concerning theories for enzyme catalysis, especially the role of protein dynamics/pre-chemistry conformational changes in the catalytic cycle of enzymes. Of particular interest is the notion that substrate-induced conformational changes that assemble the polymerase active site prior to chemistry are required for DNA synthesis and impact fidelity (i.e., substrate specificity). High-resolution crystal structures of DNA polymerase β representing intermediates of substrate complexes prior to the chemical step are available. These structures indicate that conformational adjustments in both the protein and substrates must occur to achieve the requisite geometry of the reactive participants for catalysis. We discuss computational and kinetic methods to examine possible conformational change pathways that lead from the observed crystal structure intermediates to the final structures poised for chemistry. The results, as well as kinetic data from site-directed mutagenesis studies, are consistent with models requiring pre-chemistry conformational adjustments in order to achieve high fidelity DNA synthesis. Thus, substrate-induced conformational changes that assemble the polymerase active site prior to chemistry contribute to DNA synthesis even when they do not represent actual rate-determining steps for chemistry.
    Theoretical Chemistry Accounts 11/2012; 131:1287. · 2.14 Impact Factor
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    ABSTRACT: Recently, we synthesized the first individual β,γ-CHX-dGTP diastereomers [(R)- or (S)-CHX, where X is F or Cl] and determined their structures in ternary complexes with DNA polymerase β (pol β). We now report stereospecificity by pol β on the mixed β,γ-CHX diastereomer pairs using nuclear magnetic resonance and on the separate diastereomers using transient kinetics. For both the F and Cl diastereomers, the R isomer is favored over the S isomer for G·C correct incorporation, with stereospecificities [(k(pol)/K(d))(R)/(k(pol)/K(d))(S)] of 3.8 and 6.3, respectively, and also for G·T misincorporation, with stereospecificities of 11 and 7.8, respectively. Stereopreference for the (R)-CHF-dGTP diastereomer was abolished for k(pol) but not K(d) with mutant pol β (R183A). These compounds constitute a new class of stereochemical probes for active site interactions involving halogen atoms. As Arg183 is unique in family X pols, the design of CXY deoxyribonucleotide analogues to enhance interaction is a possible strategy for inhibiting BER selectively in cancer cells.
    Biochemistry 10/2012; · 3.38 Impact Factor
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    ABSTRACT: Human 8-oxoguanine DNA glycosylase (OGG1) is a key enzyme involved in removing 7,8-dihydro-8-oxoguanine (8-oxoG), a highly mutagenic DNA lesion generated by oxidative stress. The removal of 8-oxoG by OGG1 is affected by the local DNA sequence, and this feature most likely contributes to observed mutational hotspots in genomic DNA. To elucidate the influence of local DNA sequence on 8-oxoG excision activity of OGG1, we conducted steady-state, pre-steady-state, and single-turnover kinetic evaluation of OGG1 in alternate DNA sequence contexts. The sequence context effect was studied for a mutational hotspot at a CpG dinucleotide. Altering either the global DNA sequence or the 5(')-flanking unmodified base pair failed to influence the excision of 8-oxoG. Methylation of the cytosine 5(') to 8-oxoG also did not affect 8-oxoG excision. In contrast, a 5(')-neighboring mismatch strongly decreased the rate of 8-oxoG base removal. Substituting the 5(')-C in the CpG dinucleotide with T, A, or tetrahydrofuran (i.e., T:G, A:G and tetrahydrofuran:G mispairs) resulted in a 10-, 13-, and 4-fold decrease in the rate constant for 8-oxoG excision, respectively. A greater loss in activity was observed when T:C or A:C was positioned 5(') of 8-oxoG (59- and 108-fold, respectively). These results indicate that neighboring structural abnormalities 5(') to 8-oxoG deter its repair thereby enhancing its mutagenic potential.
    Journal of Biological Chemistry 09/2012; · 4.65 Impact Factor
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    ABSTRACT: DNA polymerase and substrate conformational changes are essential for high-fidelity DNA synthesis. Structures of DNA polymerase (pol) β in complex with DNA show the enzyme in an "open" conformation. Subsequent to binding the nucleotide, the polymerase "closes" around the nascent base pair with two metals positioned for chemistry. However, structures of substrate/active site intermediates prior to closure are lacking. By destabilizing the closed complex, we determined unique ternary complex structures of pol β with correct and incorrect incoming nucleotides bound to the open conformation. These structures reveal that Watson-Crick hydrogen bonding is assessed upon initial complex formation. Importantly, nucleotide-bound states representing intermediate metal coordination states occur with active site assembly. The correct, but not incorrect, nucleotide maintains Watson-Crick hydrogen bonds during interconversion of these states. These structures indicate that the triphosphate of the incoming nucleotide undergoes rearrangement prior to closure, providing an opportunity to deter misinsertion and increase fidelity.
    Structure 09/2012; · 5.99 Impact Factor
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    ABSTRACT: The influence of water: crystallization of (R/S)-α,β-CHF-dATP with the preorganized pol β-DNA complex shows that (S)-α,β-CHF-dATP is preferentially bound to the active site with the C=F fluorine proximal to a structural water bound to Asp276.
    ChemBioChem 03/2012; 13(4):528-30. · 3.74 Impact Factor

Publication Stats

3k Citations
704.69 Total Impact Points

Institutions

  • 1995–2014
    • National Institute of Environmental Health Sciences
      • • Laboratory of Structural Biology (LSB)
      • • Laboratory of Molecular Genetics (LMG)
      Durham, North Carolina, United States
  • 1993–2014
    • National Institutes of Health
      • • Structural Biophysics Laboratory
      • • Chemical Biology Laboratory
      Maryland, United States
  • 2012
    • CUNY Graduate Center
      New York City, New York, United States
  • 2007–2012
    • University of Southern California
      • Department of Chemistry
      Los Angeles, CA, United States
  • 1999–2011
    • Research Triangle Park Laboratories, Inc.
      Raleigh, North Carolina, United States
  • 2004–2007
    • New York University
      • Department of Chemistry
      New York City, NY, United States
  • 2002–2004
    • Howard Hughes Medical Institute
      Maryland, United States
  • 1994–1998
    • University of Texas Medical Branch at Galveston
      • Department of Biochemistry and Molecular Biology
      Galveston, Texas, United States
  • 1996
    • Novosibirsk Institute of Organic Chemistry
      Novo-Nikolaevsk, Novosibirsk, Russia