NMR studies of the conformations and interactions of substrates and ribonucleotide templates bound to the large fragment of DNA polymerase I

Ferrin, Lance J.; Mildvan, Albert S.

doi:10.1021/bi00366a023

Cited by 76 publications

(36 citation statements)

References 53 publications

(101 reference statements)

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“…Moreover, Mildvan also suggested the existence of what we call the prechemistry avenue: "an error prevention or verification step is needed, which follows the binding of the substrate and precedes the primer elongation step" (74). He further proposed that coordination of the enzymebound metal by the β-phosphate is the key step in activation of the chemical reaction (74,76,77).…”

Section: Prechemistry Avenuementioning

confidence: 99%

“…He further proposed that coordination of the enzymebound metal by the β-phosphate is the key step in activation of the chemical reaction (74,76,77). That competing pathways spur evolution of the active site geometry slowly in a substratedependent context (i.e., depending on correct vs incorrect base present) presents an intriguing mechanism for pol β's regulation of fidelity.…”

Section: Prechemistry Avenuementioning

confidence: 99%

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Regulation of DNA Repair Fidelity by Molecular Checkpoints: “Gates” in DNA Polymerase β's Substrate Selection

et al. 2006

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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 β (pol β), 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 β 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 β'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 ionpolymerase 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 † This work was supported by NSF Grant MCB-0316771, NIH Grants R01 GM55164 and R01 ES012692, and the donors of the American SUPPORTING INFORMATION AVAILABLEStudy of the R258A mutation in DNA polymerase β (Appendix A) and the prechemistry avenue (Appendix B). This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2007 August 13. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscriptfeatures that contribute to fidelity discrimination across various polymerases via such geometric, dynamic, and energetic selection criteria.DNA polymerases are essential for maintaining genomic order during DNA replication and repair (1) and thus for the long-term survival of a species. When...

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Section: Prechemistry Avenuementioning

confidence: 99%

Section: Prechemistry Avenuementioning

confidence: 99%

Regulation of DNA Repair Fidelity by Molecular Checkpoints: “Gates” in DNA Polymerase β's Substrate Selection

et al. 2006

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“…In the first reports on KF, it was suggested that the first conformational change may reflect reorientation of the triphosphate moiety of the incoming nucleotide to interact with side chains of the enzyme (28). This hypothesis was put forward to explain the observation made by Mildvan and colleagues that the orientation of the triphosphate and its interaction with divalent cation change in a KF ternary complex (38,39). Recently, it has been suggested that the conformational change defined kinetically may be related to changes in the orientation of the fingers subdomain observed crystallographically (12,40,41).…”

Section: Conformational Changes During 3d Pol -Catalyzed Nucleotide Imentioning

confidence: 99%

Poliovirus RNA-Dependent RNA Polymerase (3D^p^ol): Pre-Steady-State Kinetic Analysis of Ribonucleotide Incorporation in the Presence of Mg²⁺

2004

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We have solved the complete kinetic mechanism for correct nucleotide incorporation catalyzed by the RNA-dependent RNA polymerase from poliovirus, 3D pol . The phosphoryl-transfer step is flanked by two isomerization steps. The first conformational change may be related to reorientation of the triphosphate moiety of the bound nucleotide, and the second conformational change may be translocation of the enzyme into position for the next round of nucleotide incorporation. The observed rate constant for nucleotide incorporation by 3D pol (86 s -1 ) is dictated by the rate constants for both the first conformational change (300 s -1 ) and phosphoryl transfer (520 s -1 ). Changes in the stability of the "activated" ternary complex correlate best with changes in the observed rate constant for incorporation resulting from modification of the nucleotide. With the exception of UTP, the K d values for nucleotides are at least 10-fold lower than the cellular concentration of the corresponding nucleotide. Our data predict that transition mutations should occur at a frequency of 1/15000, transversion mutations should occur at a frequency of less than 1/150000, and incorporation of a 2′-deoxyribonucleotide with a correct base should occur at a frequency 1/7500. Together, these data support the conclusion that 3D pol is actually as faithful as an exonuclease-deficient, replicative DNA polymerase. We discuss the implications of this work on the development of RNA-dependent RNA polymerase inhibitors for use as antiviral agents.One positive aspect of the human immunodeficiency virus (HIV) 1 pandemic was the realization that, given the appropriate effort, antiviral agents targeting enzymes required for virus multiplication can be developed. The first HIV-encoded enzyme that emerged as a tractable target was the viral polymerase, reverse transcriptase (RT) (1-3). While most of the inhibitors of HIV RT currently in use are nucleoside analogues (1,4), many nonnucleoside analogue inhibitors of HIV RT have also been reported (1,4). In all cases, however, an understanding of the specificity and/or precise mechanism of action of these inhibitors has required evaluation of these compounds in the context of the complete kinetic mechanism for nucleotide incorporation catalyzed by this enzyme (1,4-7) and has benefited from structural information available for HIV RT in the absence and presence of substrates and/or inhibitors (8)(9)(10)(11)(12)(13)(14). Kinetic analysis of site-directed mutants of HIV RT has established some of the fundamental mechanisms employed by this enzyme to recognize its substrates, both nucleic acid and nucleotide, and to cooperate with other viral proteins during the process of converting the viral single-stranded RNA genome into double-stranded DNA (1,4,15,16). In addition, mechanistic studies of HIV RT have suggested alternative strategies to target regions of this enzyme distinct † This work was supported, in part, by a Howard Temin Award (CA75118) from the NCI, National Institutes of Health, and by a grant ...

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“…79 The importance of the -phosphate coordination in the assembly of polymerase active site for catalysis was also noted by Mildvan and co-workers. 80 Magnesium Ions Rearrangement. DNA polymerases across the different families have common two-metal ion coordination with few conserved acidic residues (e.g., two highly conserved aspartate residues and possibly either another aspartate or glutamate) in the enzyme active site.…”

Section: Orchestrated Order Of Events During Pol Closingmentioning

confidence: 99%

Conformational Transition Pathway of Polymerase β/DNA upon Binding Correct Incoming Substrate

Arora

Schlick

2005

J. Phys. Chem. B

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The closing conformational transition of wild-type polymerase bound to DNA template/primer before the chemical step (nucleotidyl transfer reaction) is simulated using the stochastic difference equation (in length version, "SDEL") algorithm that approximates long-time dynamics. The order of the events and the intermediate states during pol 's closing pathway are identified and compared to a separate study of pol using transition path sampling (TPS) (Radhakrishnan, R.; Schlick, T. Proc. Natl. Acad. Sci. USA 2004, 101, 5970-5975). Results highlight the cooperative and subtle conformational changes in the pol active site upon binding the correct substrate that may help explain DNA replication and repair fidelity. These changes involve key residues that differentiate the open from the closed conformation (Asp192, Arg258, Phe272), as well as residues contacting the DNA template/primer strand near the active site (Tyr271, Arg283, Thr292, Tyr296) and residues contacting the and γ phosphates of the incoming nucleotide (Ser180, Arg183, Gly189). This study compliments experimental observations by providing detailed atomistic views of the intermediates along the polymerase closing pathway and by suggesting additional key residues that regulate events prior to or during the chemical reaction. We also show general agreement between two sampling methods (the stochastic difference equation and transition path sampling) and identify methodological challenges involved in the former method relevant to large-scale biomolecular applications. Specifically, SDEL is very quick relative to TPS for obtaining an approximate path of medium resolution and providing qualitative information on the sequence of events; however, associated free energies are likely very costly to obtain because this will require both successful further refinement of the path segments close to the bottlenecks and large computational time.

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NMR studies of the conformations and interactions of substrates and ribonucleotide templates bound to the large fragment of DNA polymerase I

Cited by 76 publications

References 53 publications

Regulation of DNA Repair Fidelity by Molecular Checkpoints: “Gates” in DNA Polymerase β's Substrate Selection

Regulation of DNA Repair Fidelity by Molecular Checkpoints: “Gates” in DNA Polymerase β's Substrate Selection

Poliovirus RNA-Dependent RNA Polymerase (3D^p^ol): Pre-Steady-State Kinetic Analysis of Ribonucleotide Incorporation in the Presence of Mg²⁺

Conformational Transition Pathway of Polymerase β/DNA upon Binding Correct Incoming Substrate

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NMR studies of the conformations and interactions of substrates and ribonucleotide templates bound to the large fragment of DNA polymerase I

Cited by 76 publications

References 53 publications

Regulation of DNA Repair Fidelity by Molecular Checkpoints: “Gates” in DNA Polymerase β's Substrate Selection

Regulation of DNA Repair Fidelity by Molecular Checkpoints: “Gates” in DNA Polymerase β's Substrate Selection

Poliovirus RNA-Dependent RNA Polymerase (3Dpol): Pre-Steady-State Kinetic Analysis of Ribonucleotide Incorporation in the Presence of Mg2+

Conformational Transition Pathway of Polymerase β/DNA upon Binding Correct Incoming Substrate

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Poliovirus RNA-Dependent RNA Polymerase (3D^p^ol): Pre-Steady-State Kinetic Analysis of Ribonucleotide Incorporation in the Presence of Mg²⁺