PubMed Health⌕ Search

Biomedical subjects

William A Beard

Publications and source records attributed to William A Beard.

At least 19 recordsLinked to original sources
PubMed2007

Modifying the beta,gamma leaving-group bridging oxygen alters nucleotide incorporation efficiency, fidelity, and the catalytic mechanism of DNA polymerase beta.

DNA polymerase catalysis and fidelity studies typically compare incorporation of "right" versus "wrong" nucleotide bases where the leaving group is pyrophosphate. Here we use dGTP analogues replacing the beta,gamma-bridging O with CH2, CHF, CF2, or CCl2 to explore leaving-group effects on the nucleotidyl transfer mechanism and fidelity of DNA polymerase (pol) beta. T.G mismatches occur with fidelities similar to dGTP with the exception of the CH2 analogue, which is incorporated with 5-fold higher fidelity. All analogues are observed to bind opposite template C with Kds between 1 and 4 microM, and structural evidence suggests that the analogues bind in essentially the native conformation, making them suitable substrates for probing linear free energy relationships (LFERs) in transient-kinetics experiments. Importantly, Brnsted correlations of log(kpol) versus leaving-group pKa for both right and wrong base incorporation reveal similar sensitivities (betalg approximately -0.8) followed by departures from linearity, suggesting that a chemical step rather than enzyme conformational change is rate-limiting for either process. The location of the breaks relative to pKas of CF2, O, and the sterically bulky CCl2-bridging compounds suggests a modification-induced change in the mechanism by stabilization of leaving-group elimination. The results are addressed theoretically in terms of the energetics of successive primer 3'-O addition (bond forming) and pyrophosphate analogue elimination (bond breaking) reaction energy barriers.

Base Sequence↗
PubMed2006

Regulation of DNA repair fidelity by molecular checkpoints: "gates" in DNA polymerase beta's substrate selection.

With an increasing number of structural, kinetic, and modeling studies of diverse DNA polymerases in various contexts, a complex dynamical view of how atomic motions might define molecular "gates" or checkpoints that contribute to polymerase specificity and efficiency is emerging. Such atomic-level information can offer insights into rate-limiting conformational and chemical steps to help piece together mechanistic views of polymerases in action. With recent advances, modeling and dynamics simulations, subject to the well-appreciated limitations, can access transition states and transient intermediates along a reaction pathway, both conformational and chemical, and such information can help bridge the gap between experimentally determined equilibrium structures and mechanistic enzymology data. Focusing on DNA polymerase beta (pol beta), we present an emerging view of the geometric, energetic, and dynamic selection criteria governing insertion rate and fidelity mechanisms of DNA polymerases, as gleaned from various computational studies and based on the large body of existing kinetic and structural data. The landscape of nucleotide insertion for pol beta includes conformational changes, prechemistry, and chemistry "avenues", each with a unique deterministic or stochastic pathway that includes checkpoints for selective control of nucleotide insertion efficiency. For both correct and incorrect incoming nucleotides, pol beta's conformational rearrangements before chemistry include a cascade of slow and subtle side chain rearrangements, followed by active site adjustments to overcome higher chemical barriers, which include critical ion-polymerase geometries; this latter notion of a prechemistry avenue fits well with recent structural and NMR data. The chemical step involves an associative mechanism with several possibilities for the initial proton transfer and for the interaction among the active site residues and bridging water molecules. The conformational and chemical events and associated barriers define checkpoints that control enzymatic efficiency and fidelity. Understanding the nature of such active site rearrangements can facilitate interpretation of existing data and stimulate new experiments that aim to probe enzyme features that contribute to fidelity discrimination across various polymerases via such geometric, dynamic, and energetic selection criteria.

DNA Polymerase beta↗
PubMed2006

Magnesium-cationic dummy atom molecules enhance representation of DNA polymerase beta in molecular dynamics simulations: improved accuracy in studies of structural features and mutational effects.

Human DNA polymerase beta (pol beta) fills gaps in DNA as part of base excision DNA repair. Due to its small size it is a convenient model enzyme for other DNA polymerases. Its active site contains two Mg(2+) ions, of which one binds an incoming dNTP and one catalyzes its condensation with the DNA primer strand. Simulating such binuclear metalloenzymes accurately but computationally efficiently is a challenging task. Here, we present a magnesium-cationic dummy atom approach that can easily be implemented in molecular mechanical force fields such as the ENZYMIX or the AMBER force fields. All properties investigated here, namely, structure and energetics of both Michaelis complexes and transition state (TS) complexes were represented more accurately using the magnesium-cationic dummy atom model than using the traditional one-atom representation for Mg(2+) ions. The improved agreement between calculated free energies of binding of TS models to different pol beta variants and the experimentally determined activation free energies indicates that this model will be useful in studying mutational effects on catalytic efficiency and fidelity of DNA polymerases. The model should also have broad applicability to the modeling of other magnesium-containing proteins.

Binding Sites↗
PubMed2006

Structure of DNA polymerase beta with a benzo[c]phenanthrene diol epoxide-adducted template exhibits mutagenic features.

We have determined the crystal structure of the human base excision repair enzyme DNA polymerase beta (Pol beta) in complex with a 1-nt gapped DNA substrate containing a template N2-guanine adduct of the tumorigenic (-)-benzo[c]phenanthrene 4R,3S-diol 2S,1R-epoxide in the gap. Nucleotide insertion opposite this adduct favors incorrect purine nucleotides over the correct dCMP and hence can be mutagenic. The structure reveals that the phenanthrene ring system is stacked with the base pair immediately 3' to the modified guanine, thereby occluding the normal binding site for the correct incoming nucleoside triphosphate. The modified guanine base is displaced downstream and prevents the polymerase from achieving the catalytically competent closed conformation. The incoming nucleotide binding pocket is distorted, and the adducted deoxyguanosine is in a syn conformation, exposing its Hoogsteen edge, which can hydrogen-bond with dATP or dGTP. In a reconstituted base excision repair system, repair of a deaminated cytosine (i.e., uracil) opposite the adducted guanine was dramatically decreased at the Pol beta insertion step, but not blocked. The efficiency of gap-filling dCMP insertion opposite the adduct was diminished by >6 orders of magnitude compared with an unadducted templating guanine. In contrast, significant misinsertion of purine nucleotides (but not dTMP) opposite the adducted guanine was observed. Pol beta also misinserts a purine nucleotide opposite the adduct with ungapped DNA and exhibits limited bypass DNA synthesis. These results indicate that Pol beta-dependent base excision repair of uracil opposite, or replication through, this bulky DNA adduct can be mutagenic.

DNA Adducts↗
PubMed2006

Energy analysis of chemistry for correct insertion by DNA polymerase beta.

X-ray crystallographic structures of human DNA polymerase beta with nonhydrolyzable analogs containing all atoms in the active site required for catalysis provide a secure starting point for a theoretical analysis (quantum mechanics/molecular mechanics) of the mechanism of chemistry without biasing of modeling assumptions as required in previous studies. These structures provide the basis for a detailed quantum mechanics/molecular mechanics study of the path for the complete transfer of a monophosphate nucleoside donor to the sugar acceptor in the active site. The reaction is largely associative with the main energetic step preceded by proton transfer from the terminal primer deoxyribose O3' to Asp-256. The key residues that provide electrostatic stabilization of the transition state are identified and compared with those identified by mutational studies.

Binding Sites↗
PubMed2006

Determination of lysine pK values using [5-13C]lysine: application to the lyase domain of DNA Pol beta.

Determination of the protonation state of titratable protein residues is of critical importance for the interpretation of active site chemistry, as well as for understanding the role of electrostatic interactions in protein folding and stability. However, protein titration studies are limited by the fact that, at extreme pH values, increasing fractions of unfolded or partially unfolded structures may be present. This problem is particularly acute for lysine residues which have high pK values. In the present study, we point out that the use of the 13C resonance of lysine C-5 as a reporter for titration of the epsilon-amino group is preferable to the use of C-6 due to the 5-fold greater titration shift, so that reasonable results can be obtained using a two parameter fit of data obtained over a more limited pH range. A new synthetic procedure for [5-13C]lysine is described, and the pK value for Lys72 in the lyase domain of DNA polymerase beta has been determined using the [5-13C]lysine-labeled enzyme. The results agree well with recent studies of the Pol lambda lyase domain, demonstrating that the pK value for this residue is not optimized for Schiff base chemistry (Gao et al., Biochemistry 2006, 45, 1785-1794). We also have re-evaluated data for the pK of Lys73 in the TEM-1 beta-lactamase.

Binding Sites↗
PubMed2006

Magnesium-induced assembly of a complete DNA polymerase catalytic complex.

The molecular details of the nucleotidyl transferase reaction have remained speculative, as strategies to trap catalytic intermediates for structure determination utilize substrates lacking the primer terminus 3'-OH and catalytic Mg2+, resulting in an incomplete and distorted active site geometry. Since the geometric arrangement of these essential atoms will impact chemistry, structural insight into fidelity strategies has been hampered. Here, we present a crystal structure of a precatalytic complex of a DNA polymerase with bound substrates that include the primer 3'-OH and catalytic Mg2+. This catalytic intermediate was trapped with a nonhydrolyzable deoxynucleotide analog. Comparison with two new structures of DNA polymerase beta lacking the 3'-OH or catalytic Mg2+ is described. These structures provide direct evidence that both atoms are required to achieve a proper geometry necessary for an in-line nucleophilic attack of O3' on the alphaP of the incoming nucleotide.

Binding Sites↗
PubMed2006

Activities and mechanism of DNA polymerase beta.

DNA polymerase beta plays an essential role in the base excision repair pathway necessary to cleanse the genome of simple base lesions and abasic sites. Abasic sites arise in DNA from spontaneous base loss (depurination) and DNA-damage specific glycosylases that hydrolyze the N-glycosidic bond between the deoxyribose and the damaged base. DNA polymerase beta contributes two enzymatic activities: DNA synthesis and deoxyribose-phosphate removal through nucleotidyl transferase and lyase mechanisms, respectively. The active site for each of these activities resides on a distinct domain of the protein: 31-kDa polymerase domain and amino-terminal 8-kDa lyase domain. The simple organization of each domain and the ability to assay each activity have hastened our understanding of the faithful replication of DNA during repair synthesis and the flux of intermediates through single nucleotide base excision repair and its alternate pathways.

Animals↗
PubMed2005

A thymine isostere in the templating position disrupts assembly of the closed DNA polymerase beta ternary complex.

The high fidelity of the DNA polymerization process is critically important for the stability of the cellular genome. The role of template and incoming nucleotide base pairing in polymerase fidelity has recently been explored by the use of nucleotide isosteres, which preserve the steric but not the electronic properties of the corresponding bases. The DNA repair enzyme, DNA polymerase beta (Pol beta), is among the most discriminating, being inactive when the thymine isostere difluorotoluene (DFT) is present in the templating base position. To explore the physical basis for this inactivity, we have performed NMR studies on [methyl-13C]methionine-labeled Pol beta complexed with double-hairpin DNA, used to model the gapped nucleotide substrate, and having either a thymine or a DFT isostere at the templating base position. The six methionine residues distributed throughout the enzyme provide useful conformational probes of the lyase and polymerase domains and subdomains. Analysis of the proton shift of Met282 that results from formation of an abortive Pol beta-gapped DNA-dATP complex is consistent with an open to closed conformational change of the enzyme predicted from crystal structures. In contrast, the same resonance is nearly unshifted when a ternary complex is formed from dATP and gapped DNA in which a DFT isostere replaces thymine at the templating base position. Alternatively, the resonances of Met191 and Met155, located in the catalytic subdomain, show perturbations upon formation of the abortive ternary complex, which are qualitatively similar, but significantly weaker, than the changes observed when thymine is present at the templating base position. The changes in the Met155 and Met191 methyl resonances are in fact more similar to those observed in the binary Pol beta-dATP complex. These studies demonstrate that the block in catalysis is directly related to the absence of the set of conformational transitions that include the "open" to "closed" transition monitored by Met282.

Animals↗
PubMed2005

Mismatch-induced conformational distortions in polymerase beta support an induced-fit mechanism for fidelity.

Molecular dynamics simulations of DNA polymerase (pol) beta complexed with different incorrect incoming nucleotides (G x G, G x T, and T x T template base x incoming nucleotide combinations) at the template-primer terminus are analyzed to delineate structure-function relationships for aberrant base pairs in a polymerase active site. Comparisons, made to pol beta structure and motions in the presence of a correct base pair, are designed to gain atomically detailed insights into the process of nucleotide selection and discrimination. In the presence of an incorrect incoming nucleotide, alpha-helix N of the thumb subdomain believed to be required for pol beta's catalytic cycling moves toward the open conformation rather than the closed conformation as observed for the correct base pair (G x C) before the chemical reaction. Correspondingly, active-site residues in the microenvironment of the incoming base are in intermediate conformations for non-Watson-Crick pairs. The incorrect incoming nucleotide and the corresponding template residue assume distorted conformations and do not form Watson-Crick bonds. Furthermore, the coordination number and the arrangement of ligands observed around the catalytic and nucleotide binding magnesium ions are mismatch specific. Significantly, the crucial nucleotidyl transferase reaction distance (P(alpha)-O3') for the mismatches between the incoming nucleotide and the primer terminus is not ideally compatible with the chemical reaction of primer extension that follows these conformational changes. Moreover, the extent of active-site distortion can be related to experimentally determined rates of nucleotide misincorporation and to the overall energy barrier associated with polymerase activity. Together, our studies provide structure-function insights into the DNA polymerase-induced constraints (i.e., alpha-helix N conformation, DNA base pair bonding, conformation of protein residues in the vicinity of dNTP, and magnesium ions coordination) during nucleotide discrimination and pol beta-nucleotide interactions specific to each mispair and how they may regulate fidelity. They also lend further support to our recent hypothesis that additional conformational energy barriers are involved following nucleotide binding but prior to the chemical reaction.

Base Pair Mismatch↗
PubMed2005

Structural insight into the DNA polymerase beta deoxyribose phosphate lyase mechanism.

A large number of biochemical and genetic studies have demonstrated the involvement of DNA polymerase beta (Pol beta) in mammalian base excision repair (BER). Pol beta participates in BER sub-pathways by contributing gap filling DNA synthesis and lyase removal of the 5'-deoxyribose phosphate (dRP) group from the cleaved abasic site. To better understand the mechanism of the dRP lyase reaction at an atomic level, we determined a crystal structure of Pol beta complexed with 5'-phosphorylated abasic sugar analogs in nicked DNA. This DNA ligand represents a potential BER intermediate. The crystal structure reveals that the dRP group is bound in a non-catalytic binding site. The catalytic nucleophile in the dRP lyase reaction, Lys72, and all other potential secondary nucleophiles, are too far away to participate in nucleophilic attack on the C1' of the sugar. An approximate model of the dRP group in the expected catalytic binding site suggests that a rotation of 120 degrees about the dRP 3'-phosphate is required to position the epsilon-amino Lys72 close to the dRP C1'. This model also suggests that several other side chains are in position to facilitate the beta-elimination reaction. From results of mutational analysis of key residues in the dRP lyase active site, it appears that the substrate dRP can be stabilized in the observed non-catalytic binding conformation, hindering dRP lyase activity.

Binding Sites↗
PubMed2005

DNA polymerase lambda mediates a back-up base excision repair activity in extracts of mouse embryonic fibroblasts.

Mammalian DNA polymerase (pol) lambda is a member of the X-family of DNA polymerases and has striking enzymatic and structural similarities to mammalian DNA pol beta. Because pol beta provides two important enzymatic activities for base excision repair (BER), we examined whether pol lambda might also contribute to BER. We used extracts from mouse embryonic fibroblasts representing wild-type and null genotypes for pol beta and pol lambda. In combination with neutralizing antibodies against pol beta and pol lambda, our results show a BER deficiency in the pol lambda -/- cell extract compared with extract from isogenic wild-type cells. In addition, the pol lambda antibody strongly reduced in vitro BER in the pol beta -/- cell extract. These data indicate that pol lambda is able to contribute to BER in mouse fibroblast cell extract.

Animals↗
PubMed2005

Nucleotide-induced DNA polymerase active site motions accommodating a mutagenic DNA intermediate.

DNA polymerases occasionally insert the wrong nucleotide. For this error to become a mutation, the mispair must be extended. We report a structure of DNA polymerase beta (pol beta) with a DNA mismatch at the boundary of the polymerase active site. The structure of this complex indicates that the templating adenine of the mispair stacks with the primer terminus adenine while the templating (coding) cytosine is flipped out of the DNA helix. Soaking the crystals of the binary complex with dGTP resulted in crystals of a ternary substrate complex. In this case, the templating cytosine is observed within the DNA helix and forms Watson-Crick hydrogen bonds with the incoming dGTP. The adenine at the primer terminus has rotated into a syn-conformation to interact with the opposite adenine in a planar configuration. Yet, the 3'-hydroxyl on the primer terminus is out of position for efficient nucleotide insertion.

Crystallography, X-Ray↗
PubMed2004

DNA polymerase beta and flap endonuclease 1 enzymatic specificities sustain DNA synthesis for long patch base excision repair.

DNA polymerase beta (pol beta) and flap endonuclease 1 (FEN1) are key players in pol beta-mediated long-patch base excision repair (LP-BER). It was proposed that this type of LP-BER is accomplished through FEN1 removal of a 2- to 11-nucleotide flap created by pol beta strand displacement DNA synthesis. To understand how these enzymes might cooperate during LP-BER, we characterized purified human pol beta DNA synthesis by utilizing various BER intermediates, including single-nucleotide-gapped DNA, nicked DNA, and nicked DNA with various lengths of flaps all with a 5'-terminal tetrahydrofuran (THF) residue. We observed that nicked DNA and nicked-THF flap DNA were poor substrates for pol beta-mediated DNA synthesis; yet, DNA synthesis was strongly stimulated by purified human FEN1. FEN1 did not improve pol beta substrate binding. FEN1 cleavage activity was required for the stimulation, suggesting that FEN1 removed a barrier to pol beta DNA synthesis. In addition, FEN1 cleavage on both nicked and nicked-THF flap DNA resulted in a one-nucleotide gapped DNA molecule that was an ideal substrate for pol beta. This study demonstrates that pol beta cooperates with FEN1 to remove DNA damage via a "Hit and Run" mechanism, involving alternating short gap production by FEN1 and gap filling by pol beta, rather than through coordinated formation and removal of a strand-displaced flap.

DNA↗
PubMed2004

Dynamic characterization of a DNA repair enzyme: NMR studies of [methyl-13C]methionine-labeled DNA polymerase beta.

Crystallographic characterization of DNA polymerase beta (pol beta) has suggested that multiple-domain and subdomain motions occur during substrate binding and catalysis. NMR studies of [methyl-(13)C]methionine-labeled pol beta were conducted to characterize the structural and dynamic response to ligand binding. The enzyme contains seven methionine residues, one of which is at the amino terminus and is partially removed by the expression system. Three of the methyl resonances were readily assigned using site-directed mutants. Assignment of the resonances of Met155, Met158, and Met191 was more difficult due to the spatial proximity of these residues, so that assignments were based on NOESY-HSQC data and on the response to paramagnetic Co(2+) addition, as well as shift perturbations observed for the site-directed mutants. The response of the methyl resonances to substrate binding was evaluated by the serial addition of a template oligonucleotide, a downstream 5'-phosphorylated oligonucleotide, and a primer oligonucleotide to create a two-nucleotide-gapped DNA substrate. Addition of the single-stranded template DNA resulted in selective broadening of the methyl resonance of Met18 in the 8 kDa lyase domain, and this resonance then shifted and sharpened upon addition of a 5'-phosphate-terminated downstream complementary oligonucleotide. Conversion of the two-nucleotide-gapped DNA substrate to a single-nucleotide-gapped substrate by incorporation of ddCMP produced a small perturbation of the Met236 resonance, which makes contact with the primer strand in the crystal structure. The addition of a second equivalent of ddCTP to form the pol beta-DNA-ddCTP ternary complex resulted in significant shifts for the resonances corresponding to Met155, Met191, Met236, and Met282. The Met155 methyl resonance is severely broadened, while the Met191 and Met282 resonances exhibit significant but less extreme broadening. Since only Met236 makes contact with the substrate, the effects on Met155, Met236, and Met282 result from indirect conformational and dynamic perturbations. Previous crystallographic characterization of this abortive complex indicated that a polymerase subdomain or segment (alpha-helix N) repositions itself to form one face of the binding pocket for the nascent base pair. Met282 serves as a probe for motion in this segment. Addition of Mg(2+)-dATP to pol beta in the absence of DNA produced qualitatively similar but much smaller effects on Met191 and Met155, but did not strongly perturb Met282, leading to the conclusion that Mg(2+)-dATP alone is insufficient to produce the large conformational changes that are observed in the abortive complex involving the gapped DNA with a blocked primer and ddNTP. Thus, the NMR data indicate that the nucleotide-DNA interaction appears to be essential for conformational activation.

Animals↗
PubMed2004

Critical role of magnesium ions in DNA polymerase beta's closing and active site assembly.

To dissect the effects of the nucleotide-binding and catalytic metal ions on DNA polymerase mechanisms for DNA repair and synthesis, aside from the chemical reaction, we investigate their roles in the conformational transitions between closed and open states and assembly/disassembly of the active site of polymerase beta/DNA complexes before and after the chemical reaction of nucleotide incorporation. Using dynamics simulations, we find that closing before chemical reaction requires both divalent metal ions in the active site while opening after the chemical reaction is triggered by release of the catalytic metal ion. The critical closing is stabilized by the interaction of the incoming nucleotide with conserved catalytic residues (Asp190, Asp192, Asp256) and the two functional magnesium ions; without the catalytic ion, other protein residues (Arg180, Arg183, Gly189) coordinate the incomer's triphosphate group through the nucleotide-binding ion. Because we also note microionic heterogeneity near the active site, Mg(2+) and Na(+) ions can diffuse into the active site relatively rapidly, we suggest that the binding of the catalytic ion itself is not a rate-limiting conformational or overall step. However, geometric adjustments associated with functional ions and proper positioning in the active site, including subtle but systematic motions of protein side chains (e.g., Arg258), define slow or rate-limiting conformational steps that may guide fidelity mechanisms. These sequential rearrangements are likely sensitively affected when an incorrect nucleotide approaches the active site. Our suggestion that subtle and slow adjustments of the nucleotide-binding and catalytic magnesium ions help guide polymerase selection for the correct nucleotide extends descriptions of polymerase pathways and underscores the importance of the delicate conformational events both before and after the chemical reaction to polymerase efficiency and fidelity mechanisms.

Binding Sites↗