Quantum Chemical Investigation of Enzymatic Activity in DNA Polymerase β. A Mechanistic Study

Abashkin, Yuri G.; Erickson, and John W.; Burt, Stanley K.

doi:10.1021/jp003629x

Cited by 43 publications

(82 citation statements)

References 24 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The related chemical reaction distance (P R -O3′) between the matched or mismatched incoming nucleotide and the primer terminus is larger than expected for phosphoryl transfer by a dissociative mechanism (54,65,66). The base of the incorrect nucleotide and its template partner stack against each other rather than forming Watson-Crick hydrogen bonds.…”

Section: Discussionmentioning

confidence: 94%

Mismatch-Induced Conformational Distortions in Polymerase β Support an Induced-Fit Mechanism for Fidelity

et al. 2005

View full text Add to dashboard Cite

Molecular dynamics simulations of DNA polymerase (pol) complexed with different incorrect incoming nucleotides (G‚G, G‚T, and T‚T template base‚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 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, R-helix N of the thumb subdomain believed to be required for pol 's catalytic cycling moves toward the open conformation rather than the closed conformation as observed for the correct base pair (G‚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 R -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., R-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 -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.

show abstract

Section: Discussionmentioning

confidence: 94%

Mismatch-Induced Conformational Distortions in Polymerase β Support an Induced-Fit Mechanism for Fidelity

et al. 2005

View full text Add to dashboard Cite

show abstract

“…This configuration for H3T suggests a direct proton transfer pathway from the 3Ј hydroxyl primer terminus to Asp-256. Proton transfer to an ␣-phosphate oxygen suggested by a cluster model calculation (12) appears unlikely because such a proton transfer pathway will require large structural rearrangement of the active site, which is hindered by the surrounding residues in the initial structure. A water-mediated proton transfer pathway is also possible if water molecules are able to penetrate and interact with H3T.…”

Section: Resultsmentioning

confidence: 99%

“…DNA pol ␤ has been characterized by x-ray crystallography in various liganded states (3)(4)(5)(6)(7)(8), and the dynamics were characterized by NMR (9, 10) and time-resolved fluorescence (11). A number of theoretical studies have been carried out with pol ␤ in an effort to understand details of catalysis (12)(13)(14)(15). Additionally, computer modeling of conformational changes deduced from x-ray crystallography before and after ligand binding and catalysis have provided insight into substrate discrimination (16)(17)(18)(19)(20)(21)(22)(23).…”

mentioning

confidence: 99%

Energy analysis of chemistry for correct insertion by DNA polymerase β

Peng

Pedersen

Batra

et al. 2006

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

197

View full text Add to dashboard Cite

X-ray crystallographic structures of human DNA polymerase ␤ with nonhydrolyzable analogs containing all atoms in the active site required for catalysis provide a secure starting point for a theoretical analysis (quantum mechanics͞molecular 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 mechanics͞molecular 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.quantum mechanics ͉ molecular mechanics ͉ reaction mechanism D NA polymerases play a central role in the replication and repair of DNA. High-fidelity proofreading-deficient polymerases typically produce one error per million nucleotides synthesized (1). DNA polymerase ␤ (pol ␤) is the simplest eukaryotic DNA polymerase and catalyzes the templatedirected nucleotidyl transfer reaction during the repair of ''simple'' base lesions with moderate fidelity, producing approximately one error per 3,000 nt synthesized during base excision DNA repair (2). DNA pol ␤ has been characterized by x-ray crystallography in various liganded states (3-8), and the dynamics were characterized by NMR (9, 10) and time-resolved fluorescence (11). A number of theoretical studies have been carried out with pol ␤ in an effort to understand details of catalysis (12-15). Additionally, computer modeling of conformational changes deduced from x-ray crystallography before and after ligand binding and catalysis have provided insight into substrate discrimination (16-23). These structural and computational studies can be interpreted in the wealth of kinetic and site-directed mutagenesis studies available for pol ␤ (2).A recent structural study of the precatalytic complex including pol ␤, DNA template, primer and dNTP analogue, 2Ј-deoxyuridine-5Ј-(␣,␤)-imido triphosphate, provides a ternary complex structure including a hydroxyl group (O3Ј) on the primer terminus with two bound Mg ions (24). Both Mg ions are found to be octahedrally coordinated, with each ion bound firmly to a nonbridging oxygen on the dNTP ␣-phosphate and each with a distinct bound water molecule. The primer O3Ј atom, often missing in other x-ray crystal structures and necessary for the nucleophilic attack, is securely bound in the inner coordination sphere of the catalytic Mg ion. As all of the vital components for the reaction are in place in this experimental structure, we have a secure initial reaction system that minimizes the chance of introducing modeling bias at the outset of a theoretical study.All families of DNA polymerases are believed to catalyze the nucleotidyl transfer reaction through a universal two-metal-ion mechanis...

show abstract

“…As an alternative, we carried out a series of managed quantum mechanics calculations using a cluster model of the active site to obtain estimates of the energy differences/ barrier between the two states. Cluster models have been used in previous theoretical studies of DNA polymerases (15,16). Our cluster model was constructed from the crystal structure, and during the geometry optimization and potential energy scans, the model closely resembles the active-site structure.…”

Section: Resultsmentioning

confidence: 99%

Incorrect nucleotide insertion at the active site of a G:A mismatch catalyzed by DNA polymerase β

Peng

Batra

Pedersen

et al. 2008

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

100

View full text Add to dashboard Cite

Based on a recent ternary complex crystal structure of human DNA polymerase ␤ with a G:A mismatch in the active site, we carried out a theoretical investigation of the catalytic mechanism of incorrect nucleotide incorporation using molecular dynamics simulation, quantum mechanics, combined quantum mechanics, and molecular mechanics methods. A two-stage mechanism is proposed with a nonreactive active-site structural rearrangement prechemistry step occurring before the nucleotidyl transfer reaction. The free energy required for formation of the prechemistry state is found to be the major factor contributing to the decrease in the rate of incorrect nucleotide incorporation compared with correct insertion and therefore to fidelity enhancement. Hence, the transition state and reaction barrier for phosphodiester bond formation after the prechemistry state are similar to that for correct insertion reaction. Key residues that provide electrostatic stabilization of the transition state are identified.combined quantum mechanics and molecular mechanics ͉ incorrect nucleotide incorporation ͉ nucleotidyl transfer ͉ two-stage mechanism I nsertion fidelity of DNA polymerases plays a central role in the replication and repair of DNA (1). DNA polymerase ␤ (pol ␤) is the simplest eukaryotic DNA polymerase. It catalyzes the template-directed nucleotidyl transfer reaction during the repair of ''simple'' base lesions with moderate fidelity, producing approximately one error per 3,000 nucleotides synthesized during base excision DNA repair (2). There is great interest in how pol ␤ achieves such fidelity without an intrinsic proofreading exonuclease. This interest is stimulated by a wealth of kinetic and site-directed mutagenesis studies available for pol ␤ (2, 3).A recent structural study of the precatalytic complex [pol ␤, DNA template, primer and a nonreactive deoxynucleoside triphosphate (dNTP) analogue, 2Ј-deoxy-adenosine-5Ј-(␣,␤)-methylene triphosphate (dAMPCPP)] provided for the first time a ternary complex structure with an active-site G:A mismatch (4). The two Mn 2ϩ ions in the active site were found to be octahedrally coordinated. The primer O3Ј group was found to be replaced by a water molecule, relative to its position in an earlier structure of a ternary complex with a matched (i.e., WatsonCrick) base pair (5).All families of DNA polymerases are believed to catalyze the nucleotidyl transfer reaction through a universal two-metal-ion mechanism (6). Reaction pathways for correct nucleotide incorporation have been extensively investigated by using various models. However, the reaction pathway for incorrect insertion is still unknown. Earlier studies had found that the rate of catalytic reaction for the correct base pair is a crucial factor in achieving high fidelity in DNA synthesis (7). The incorrect nucleotide insertion reaction catalyzed by DNA polymerases exhibiting modest to high fidelity is much slower than for the correct insertion (8, 9).A variety of computational techniques have been used in this study, including cl...

show abstract

Quantum Chemical Investigation of Enzymatic Activity in DNA Polymerase β. A Mechanistic Study

Cited by 43 publications

References 24 publications

Mismatch-Induced Conformational Distortions in Polymerase β Support an Induced-Fit Mechanism for Fidelity

Mismatch-Induced Conformational Distortions in Polymerase β Support an Induced-Fit Mechanism for Fidelity

Energy analysis of chemistry for correct insertion by DNA polymerase β

Incorrect nucleotide insertion at the active site of a G:A mismatch catalyzed by DNA polymerase β

Contact Info

Product

Resources

About