Structural Comparison of DNA Polymerase Architecture Suggests a Nucleotide Gateway to the Polymerase Active Site

Wu, Sangwook; Beard, William A.; Pedersen, Lee G.; Wilson, Samuel H.

doi:10.1021/cr3005179

Cited by 40 publications

(58 citation statements)

References 58 publications

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“…Early studies by Petruska et al (6) compared matched and mismatched DNA duplex stabilities, using bluntended duplexes. However, most polymerase-mediated DNA synthesis occurs on templates extending well beyond the primer terminus, and DNA polymerases make contacts with the basepaired duplex substrate not only at the primer terminus, but also with the unpaired template strand multiple bases downstream of the primer (13)(14)(15). Thus, it is important to take into consideration the influence of this overhanging template strand on stability and fidelity.…”

Section: Resultsmentioning

confidence: 99%

Kinetic selection vs. free energy of DNA base pairing in control of polymerase fidelity

Oertell

Harcourt

Mohsen

et al. 2016

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

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What is the free energy source enabling high-fidelity DNA polymerases (pols) to favor incorporation of correct over incorrect base pairs by 10 3 -to 10 4 -fold, corresponding to free energy differences of ΔΔG inc ∼ 5.5-7 kcal/mol? Standard ΔΔG°values (∼0.3 kcal/mol) calculated from melting temperature measurements comparing matched vs. mismatched base pairs at duplex DNA termini are far too low to explain pol accuracy. Earlier analyses suggested that pol active-site steric constraints can amplify DNA free energy differences at the transition state (kinetic selection). A recent paper [Olson et al. (2013) J Am Chem Soc 135:1205-1208] used Vent pol to catalyze incorporations in the presence of inorganic pyrophosphate intended to equilibrate forward (polymerization) and backward (pyrophosphorolysis) reactions. A steady-state leveling off of incorporation profiles at long reaction times was interpreted as reaching equilibrium between polymerization and pyrophosphorolysis, yielding apparent ΔG°= −RT ln K eq , indicating ΔΔG°of 3.5-7 kcal/mol, sufficient to account for pol accuracy without need of kinetic selection. Here we perform experiments to measure and account for pyrophosphorolysis explicitly. We show that forward and reverse reactions attain steady states far from equilibrium for wrong incorporations such as G opposite T. Therefore, ΔΔG°i nc values obtained from such steady-state evaluations of K eq are not dependent on DNA properties alone, but depend largely on constraints imposed on right and wrong substrates in the polymerase active site. -fold, corresponding to free energy differences ΔΔG inc ∼ 5.5-7 kcal/mol (1). Kinetic studies have identified a variety of "checkpoints" favoring the selection of R over W. Kinetic checkpoints are triggered by substrate binding; the ternary pol-DNA-dNTP complex is stabilized with dRTP and destabilized with dWTP. The associated conformational changes drive the reaction forward toward incorporation with dRTP and backward toward substrate release with dWTP favoring incorporation of R over W (reviewed in refs. 1-4). The rate-determining steps can be different for different pols and may also differ for R and W for a single pol (3,4).A fundamental issue is to identify possible sources of free energy that might be large enough to account for high pol incorporation fidelity. Seemingly an obvious source might be the differences in stability of matched and mismatched base pairs in the DNA itself, which involve both H-bonding and base-stacking interactions (5). In an early experiment, used here as an example, equilibrium constants (K eq ) for double-stranded DNA (dsDNA) containing R and W base pairs at blunt-end termini were obtained by measuring melting temperatures in aqueous solution and used to infer standard free energy differences ΔΔG°∼ 0.3 kcal/mol (6). Because these were far too small to account for fidelity, it was then proposed that steric constraints imposed by the pol active site could "amplify" the small free energy differences between R and W base pairing to attain ΔΔG°...

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Section: Resultsmentioning

confidence: 99%

Kinetic selection vs. free energy of DNA base pairing in control of polymerase fidelity

Oertell

Harcourt

Mohsen

et al. 2016

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

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“…Because many DNA polymerases undergo subdomain motions that accompany substrate binding, the active site also transitions from an open, solvent-exposed state to a closed, less solventaccessible state (11). In this context, it should be noted that these calculations are performed in an environment of the closed polymerase active site and are thus sensitive to the internal chemical equilibrium at the enzyme active site.…”

Section: Discussionmentioning

confidence: 99%

“…DNA polymerase (pol) β has served as a model eukaryotic DNA polymerase for computational, structural, mechanistic, kinetic, and biological studies (10)(11)(12). Based on the structure of DNA bound to the exonuclease site of Escherichia coli DNA polymerase I, a general two-metal ion mechanism for the nucleotidyl transferase (nuclease and DNA synthesis) reaction was proposed 25 y ago (13).…”

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confidence: 99%

Requirement for transient metal ions revealed through computational analysis for DNA polymerase going in reverse

Perera

Freudenthal

Beard

et al. 2015

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

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DNA polymerases facilitate faithful insertion of nucleotides, a central reaction occurring during DNA replication and repair. DNA synthesis (forward reaction) is "balanced," as dictated by the chemical equilibrium by the reverse reaction of pyrophosphorolysis. Two closely spaced divalent metal ions (catalytic and nucleotide-binding metals) provide the scaffold for these reactions. The catalytic metal lowers the pK a of O3′ of the growing primer terminus, and the nucleotide-binding metal facilitates substrate binding. Recent time-lapse crystallographic studies of DNA polymerases have identified an additional metal ion (product metal) associated with pyrophosphate formation, leading to the suggestion of its possible involvement in the reverse reaction. Here, we establish a rationale for a role of the product metal using quantum mechanical/molecular mechanical calculations of the reverse reaction in the confines of the DNA polymerase β active site. Additionally, site-directed mutagenesis identifies essential residues and metal-binding sites necessary for pyrophosphorolysis. The results indicate that the catalytic metal site must be occupied by a magnesium ion for pyrophosphorolysis to occur. Critically, the product metal site is occupied by a magnesium ion early in the pyrophosphorolysis reaction path but must be removed later. The proposed dynamic nature of the active site metal ions is consistent with crystallographic structures. The transition barrier for pyrophosphorolysis was estimated to be significantly higher than that for the forward reaction, consistent with kinetic activity measurements of the respective reactions. These observations provide a framework to understand how ions and active site changes could modulate the internal chemical equilibrium of a reaction that is central to genome stability.NA polymerases are responsible for high-fidelity DNA synthesis during replication and repair of the genome (1). Although there are at least 17 human DNA polymerases, they all use a general nucleotidyl transferase DNA synthesis reaction. This reaction requires deoxynucleoside triphosphates (dNTPs), divalent metal ions, and DNA substrate with a primer 3′-OH annealed to a coding template strand. An inline nucleophilic attack of the primer 3′-oxyanion on P α of the incoming dNTP results in products with DNA extended by one nucleotide [i.e., deoxynucleoside monophosphate (dNMP)] and pyrophosphate (PP i ). If the enzyme does not release PP i , pyrophosphorolysis (reverse reaction) can generate dNTP and a DNA strand that is one nucleotide shorter (Fig. 1) (2).Although the forward DNA synthesis reaction is preferred, the pyrophosphorolysis reaction can be biologically significant. Because DNA polymerases are an attractive chemotherapeutic target, chainterminating nucleoside drugs are often used in a strategy of blocking DNA synthesis (3-5). However, drug resistance to chain-terminating agents is influenced by the ability of a stalled DNA polymerase to remove chain-terminating nucleotides through pyrophosphorolysis (6-8)...

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“…DNA-dependent DNA polymerases are key enzymes in DNA replication and are classified into six main families named A, B, C, D, X and Y (Garcia-Diaz & Bebenek, 2007). DNA polymerases (EC 2.7.7.7) play a pivotal role in DNA replication and repair, and those from thermophilic organisms have extensive biotechnological applications in polymerase chain reaction (PCR) methods (Wu et al, 2014). These include the amplification of specific genes or regions of interest in the genome, DNA sequencing and site-directed mutagenesis, as well as diagnostic and forensic work.…”

Section: Introductionmentioning

confidence: 99%

“…Despite the structural differences between the polymerase enzymes from different classes, the arrangement of the catalytic carboxyl groups in the palm domain and the bound metal ions are strongly conserved with respect to the bound DNA molecule (Wu et al, 2014). Of the two bound metal ions, which are denoted A and B, metal ion A is thought to activate the 3 0 -hydroxyl group of the primer strand by lowering its pK a .…”

Section: Introductionmentioning

confidence: 99%

Structure of the family B DNA polymerase from the hyperthermophilic archaeonPyrobaculum calidifontis

Guo¹,

Zhang²,

Coker³

et al. 2017

Acta Cryst Sect D Struct Biol

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The family B DNA polymerase from Pyrobaculum calidifontis (Pc-polymerase) consists of 783 amino acids and is magnesium-ion dependent. It has an optimal pH of 8.5, an optimal temperature of 75 C and a half-life of 4.5 h at 95 C, giving it greater thermostability than the widely used Taq DNA polymerase. The enzyme is also capable of PCR-amplifying larger DNA fragments of up to 7.5 kb in length. It was shown to have functional, error-correcting 3 0 -5 0 exonuclease activity, as do the related high-fidelity DNA polymerases from Pyrococcus furiosus, Thermococcus kodakarensis KOD1 and Thermococcus gorgonarius, which have extensive commercial applications. Pc-polymerase has a quite low sequence identity of approximately 37% to these enzymes, which, in contrast, have very high sequence identity to each other, suggesting that the P. calidifontis enzyme is distinct. Here, the structure determination of Pc-polymerase is reported, which has been refined to an R factor of 24.47% and an R free of 28.81% at 2.80 Å resolution. The domains of the enzyme are arranged in a circular fashion to form a disc with a narrow central channel. One face of the disc has a number of connected crevices in it, which allow the protein to bind duplex and single-stranded DNA. The central channel is thought to allow incoming nucleoside triphosphates to access the active site. The enzyme has a number of unique structural features which distinguish it from other archaeal DNA polymerases and may account for its high processivity. A model of the complex with the primer-template duplex of DNA indicates that the largest conformational change that occurs upon DNA binding is the movement of the thumb domain, which rotates by 7.6 and moves by 10.0 Å . The surface potential of the enzyme is dominated by acidic groups in the central region of the molecule, where catalytic magnesium ions bind at the polymerase and exonuclease active sites. The outer regions are richer in basic amino acids that presumably interact with the sugar-phosphate backbone of DNA. The large number of salt bridges may contribute to the high thermal stability of this enzyme.

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Structural Comparison of DNA Polymerase Architecture Suggests a Nucleotide Gateway to the Polymerase Active Site

Cited by 40 publications

References 58 publications

Kinetic selection vs. free energy of DNA base pairing in control of polymerase fidelity

Kinetic selection vs. free energy of DNA base pairing in control of polymerase fidelity

Requirement for transient metal ions revealed through computational analysis for DNA polymerase going in reverse

Structure of the family B DNA polymerase from the hyperthermophilic archaeonPyrobaculum calidifontis

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