DNA Polymerase Fidelity:  Kinetics, Structure, and Checkpoints

Joyce, Catherine M.; Benkovic, Stephen J.

doi:10.1021/bi048422z

Cited by 300 publications

(508 citation statements)

References 46 publications

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“…But since this step is not likely to be rate limiting, it is sufficient just to calculate the proton transfer reaction energy, ΔG PT , which in turn requires calculating the pK a shift of the proton donor. The pK a shift of the ribose 3′OH group in the protein is calculated by using the FEP method and the following relationship 53 (4) ΔG PT is then calculated as (5) The pK a of the ribose 3′OH in water is taken as 13. 54 Here pK a,3′OH = 13 + ΔpK a and pH = 7 are used in the calculations.…”

Section: Methodsmentioning

confidence: 99%

“…36) and DNA polymerases in general. [4][5][6] Experimental evidence indicates that the open to closed subdomain transition is too rapid to be rate determining. 37-40 Instead, we think it is considerably more likely that fidelity is determined by the binding energy plus the activation barrier of the chemical step, and that the barrier for conformational changes between the open and closed forms is not likely to determine the fidelity in Pol β.…”

Section: Introductionmentioning

confidence: 99%

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Exploring the role of large conformational changes in the fidelity of DNA polymerase β

et al. 2007

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The relationships between the conformational landscape, nucleotide insertion catalysis and fidelity of DNA polymerase β are explored by means of computational simulations. The simulations indicate that the transition states for incorporation of right (R) and wrong (W) nucleotides reside in substantially different protein conformations. The protein conformational changes that reproduce the experimentally observed fidelity are significantly larger than the small rearrangements that usually accompany motions from the reactant state to the transition state in common enzymatic reactions. Once substrate binding has occurred, different constraints imposed on the transition states for insertion of R and W nucleotides render it highly unlikely that both transition states can occur in the same closed structure, because the predicted fidelity would then be many orders of magnitude too large. Since the conformational changes reduce the transition state energy of W incorporation drastically they decrease fidelity rather than increase it. Overall, a better agreement with experimental data is attained when the R is incorporated through a transition state in a closed conformation and W is incorporated through a transition state in one or perhaps several partially open conformations. The generation of free energy surfaces for R and W also allow us to analyze proposals about the relationship between induced fit and fidelity.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Exploring the role of large conformational changes in the fidelity of DNA polymerase β

et al. 2007

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“…21). We therefore predicted that only the addition of complementary dNTP, and not the three non-complementary dNTPs, would yield the cluster of longer dwell time events at ∼24 pA amplitude (Fig.…”

mentioning

confidence: 91%

Sequence-specific detection of individual DNA polymerase complexes in real time using a nanopore

et al. 2007

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Nanoscale pores have potential to be used as biosensors and are an established tool for analysing the structure and composition of single DNA or RNA molecules 1-3 . Recently, nanopores have been used to measure the binding of enzymes to their DNA substrates 4,5 . In this technique, a polynucleotide bound to an enzyme is drawn into the nanopore by an applied voltage. The force exerted on the charged backbone of the polynucleotide by the electric field is used to examine the enzyme-polynucleotide interactions. Here we show that a nanopore sensor can accurately identify DNA templates bound in the catalytic site of individual DNA polymerase molecules. Discrimination among unbound DNA, binary DNA/polymerase complexes, and ternary DNA/polymerase/ deoxynucleotide triphosphate complexes was achieved in real time using finite state machine logic. This technique is applicable to numerous enzymes that bind or modify DNA or RNA including exonucleases, kinases and other polymerases.We describe a nanopore device that monitors ionic current through a single protein pore inserted in a lipid bilayer (Fig. 1a). The limiting aperture of the pore is just sufficient to accommodate single-stranded DNA (ssDNA) 6,7 , and the adjacent pore vestibule can accommodate doublestranded (duplex) DNA (dsDNA) 7-9 . In the absence of DNA, the open channel current (I o ) through the α-haemolysin pore is 60 pA at 180 mV applied potential in 0.3 M KCl. DNA capture in the nanopore results in a decrease in the current (I). The DNA resides in the pore for a time (t D ) until it leaves, moving to the trans compartment (Fig. 1b). These two parameters, I and t D , together with current noise, are typically used to report results from nanopore experiments 6,10-19 .We used a nanopore instrument to probe the interaction of the Klenow fragment (KF) of Escherichia coli DNA polymerase I with its DNA substrate. This substrate is a duplex DNA formed by base-pairing of a short ssDNA primer with a longer template DNA. The KF catalyses DNA replication by the sequential addition of nucleotides to the primer strand, dictated by Watson-Crick complementarity to the template strand 20 . In contrast with earlier studies examining Exonuclease I/DNA complexes 4 and EcoRI/DNA complexes 5 , our nanopore Capture and translocation of a model DNA template (14 bp hairpin with a 36-nucleotide 5′ overhang and 2′-3′ dideoxycytidine terminus) resulted in a cluster of events with a median duration of 1 ms and an average blockade amplitude I = 20 pA (Fig. 2a). When the KF (2 μM) was subsequently added to the cis compartment under conditions where catalytic activity had been demonstrated in separate experiments (see Supplementary Information, Fig. S1), a second population of events emerged with a 3-ms median dwell time and a higher blockade current (I = 23 pA, Fig. 2b). This class of events is enzyme-concentration-dependent (see Supplementary Information, Fig. S2 and Table S1), consistent with nanopore capture of a DNA/ KF binary complex.Addition of a deoxynucleotide triphosphat...

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“…Here, despite significant experimental milestones in studies of the fidelity of DNA polymerases (e.g. Echols & Goodman, 1991 ;Goodman, 2002 ;Goodman et al 1993 ;Johnson, 1993 ;Joyce & Benkovic, 2004 ;Kunkel & Bebenek, 2000 ;Showalter et al 2006), we still do not have a clear quantitative picture of the energetics of this progress. The progress made in using structural information to explore the question of fidelity will be reviewed in Section 4.3.5.…”

Section: Dna Polymerases and Replication Fidelity 431 Introductionmentioning

confidence: 97%

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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DNA Polymerase Fidelity: Kinetics, Structure, and Checkpoints

Cited by 300 publications

References 46 publications

Exploring the role of large conformational changes in the fidelity of DNA polymerase β

Exploring the role of large conformational changes in the fidelity of DNA polymerase β

Sequence-specific detection of individual DNA polymerase complexes in real time using a nanopore

Why nature really chose phosphate

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