Crystal Structure Of DNA Polymerase Research Articles

Crystal structure of DNA polymerase I from Thermus phage G20c.

This study describes the structure of DNA polymerase I from Thermus phage G20c, termed PolI_G20c. This is the first structure of a DNA polymerase originating from a group of related thermophilic bacteriophages infecting Thermus thermophilus, including phages G20c, TSP4, P74-26, P23-45 and phiFA and the novel phage Tth15-6. Sequence and structural analysis of PolI_G20c revealed a 3'-5' exonuclease domain and a DNA polymerase domain, and activity screening confirmed that both domains were functional. No functional 5'-3' exonuclease domain was present. Structural analysis also revealed a novel specific structure motif, here termed SβαR, that was not previously identified in any polymerase belonging to the DNA polymerases I (or the DNA polymerase A family). The SβαR motif did not show any homology to the sequences or structures of known DNA polymerases. The exception was the sequence conservation of the residues in this motif in putative DNA polymerases encoded in the genomes of a group of thermophilic phages related to Thermus phage G20c. The structure of PolI_G20c was determined with the aid of another structure that was determined in parallel and was used as a model for molecular replacement. This other structure was of a 3'-5' exonuclease termed ExnV1. The cloned and expressed gene encoding ExnV1 was isolated from a thermophilic virus metagenome that was collected from several hot springs in Iceland. The structure of ExnV1, which contains the novel SβαR motif, was first determined to 2.19 Å resolution. With these data at hand, the structure of PolI_G20c was determined to 2.97 Å resolution. The structures of PolI_G20c and ExnV1 are most similar to those of the Klenow fragment of DNA polymerase I (PDB entry 2kzz) from Escherichia coli, DNA polymerase I from Geobacillus stearothermophilus (PDB entry 1knc) and Taq polymerase (PDB entry 1bgx) from Thermus aquaticus.

Crystal structures of DNA polymerase I capture novel intermediates in the DNA synthesis pathway.

High resolution crystal structures of DNA polymerase intermediates are needed to study the mechanism of DNA synthesis in cells. Here we report five crystal structures of DNA polymerase I that capture new conformations for the polymerase translocation and nucleotide pre-insertion steps in the DNA synthesis pathway. We suggest that these new structures, along with previously solved structures, highlight the dynamic nature of the finger subdomain in the enzyme active site.

Mapping Functional Substrate-Enzyme Interactions in the pol β Active Site through Chemical Biology: Structural Responses to Acidity Modification of Incoming dNTPs.

We report high-resolution crystal structures of DNA polymerase (pol) β in ternary complex with a panel of incoming dNTPs carrying acidity-modified 5'-triphosphate groups. These novel dNTP analogues have a variety of halomethylene substitutions replacing the bridging oxygen between Pβ and Pγ of the incoming dNTP, whereas other analogues have alkaline substitutions at the bridging oxygen. Use of these analogues allows the first systematic comparison of effects of 5'-triphosphate acidity modification on active site structures and the rate constant of DNA synthesis. These ternary complex structures with incoming dATP, dTTP, and dCTP analogues reveal the enzyme's active site is not grossly altered by the acidity modifications of the triphosphate group, yet with analogues of all three incoming dNTP bases, subtle structural differences are apparent in interactions around the nascent base pair and at the guanidinium groups of active site arginine residues. These results are important for understanding how acidity modification of the incoming dNTP's 5'-triphosphate can influence DNA polymerase activity and the significance of interactions at arginines 183 and 149 in the active site.

Mechanism of Error-Free DNA Replication Past Lucidin-Derived DNA Damage by Human DNA Polymerase κ.

DNA damage impinges on genetic information flow and has significant implications in human disease and aging. Lucidin-3-O-primeveroside (LuP) is an anthraquinone derivative present in madder root, which has been used as a coloring agent and food additive. LuP can be metabolically converted to genotoxic compound lucidin, which subsequently forms lucidin-specific N2-2'-deoxyguanosine (N2-dG) and N6-2'-deoxyadenosine (N6-dA) DNA adducts. Lucidin is mutagenic and carcinogenic in rodents but has low carcinogenic risks in humans. To understand the molecular mechanism of low carcinogenicity of lucidin in humans, we performed DNA replication assays using site-specifically modified oligodeoxynucleotides containing a structural analogue (LdG) of lucidin-N2-dG DNA adduct and determined the crystal structures of DNA polymerase (pol) κ in complex with LdG-bearing DNA and an incoming nucleotide. We examined four human pols (pol η, pol ι, pol κ, and Rev1) in their efficiency and accuracy during DNA replication with LdG; these pols are key players in translesion DNA synthesis. Our results demonstrate that pol κ efficiently and accurately replicates past the LdG adduct, whereas DNA replication by pol η, pol ι is compromised to different extents. Rev1 retains its ability to incorporate dCTP opposite the lesion albeit with decreased efficiency. Two ternary crystal structures of pol κ illustrate that the LdG adduct is accommodated by pol κ at the enzyme active site during insertion and postlesion-extension steps. The unique open active site of pol κ allows the adducted DNA to adopt a standard B-form for accurate DNA replication. Collectively, these biochemical and structural data provide mechanistic insights into the low carcinogenic risk of lucidin in humans.

The Closing Mechanism of DNA Polymerase I at Atomic Resolution

DNA polymerases must quickly and accurately distinguish between similar nucleic acids to form Watson-Crick base pairs and avoid DNA replication errors. Deoxynucleoside triphosphate (dNTP) binding to the DNA polymerase active site induces a large conformational change that is difficult to characterize experimentally on an atomic level. Here, we report an X-ray crystal structure of DNA polymerase I bound to DNA in the open conformation with a dNTP present in the active site. We use this structure to computationally simulate the open to closed transition of DNA polymerase in the presence of a Watson-Crick base pair. Our microsecond simulations allowed us to characterize the key steps involved in active site assembly, and propose the sequence of events involved in the prechemistry steps of DNA polymerase catalysis. They also reveal new features of the polymerase mechanism, such as a conserved histidine as a potential proton acceptor from the primer 3'-hydroxyl.

Molecular Dynamics Study of the Opening and Closing Mechanisms for DNA Polymerase I

The quick and accurate replication of genetic information (DNA) is critical to all life. DNA polymerase, a key enzyme for DNA replication in organisms, catalyzes the reaction that extends the primer strand of DNA during replication. Before catalysis, DNA polymerase has the daunting task of matching a template nucleic acid base with its corresponding Watson‐Crick base (dNTP) that must be added to the primer strand. The enzyme must have high specificity in order to distinguish one correct base for incorporation from three incorrect bases. Crystal structures of DNA polymerase have shown the enzyme in an “open” conformation in the absence of a dNTP in the active site and “closed” when a proper Watson‐Crick base pair is matched. Although crystal structures have given insight into these conformations of DNA polymerase in these three states, traditional biochemistry experimental techniques are unable to capture the details of the transition between these states and how the polymerase traverses the potential energy surface from open to closed and back again. In this study, we have utilized recent computational advances to simulate the opening and closing of the fingers domain starting from the of Bacillus stearothermophilus DNA polymerase Klenow fragment using dynamics on the microsecond time scale. We have fully characterized both processes, and determined the key events and movements critical to these large‐scale conformational changes. These simulations aid in the elucidation of the structural changes of DNA polymerase on an atomistic level not currently available with experimental measures.

Molecular dynamics study of the opening mechanism for DNA polymerase I.

During DNA replication, DNA polymerases follow an induced fit mechanism in order to rapidly distinguish between correct and incorrect dNTP substrates. The dynamics of this process are crucial to the overall effectiveness of catalysis. Although X-ray crystal structures of DNA polymerase I with substrate dNTPs have revealed key structural states along the catalytic pathway, solution fluorescence studies indicate that those key states are populated in the absence of substrate. Herein, we report the first atomistic simulations showing the conformational changes between the closed, open, and ajar conformations of DNA polymerase I in the binary (enzyme∶DNA) state to better understand its dynamics. We have applied long time-scale, unbiased molecular dynamics to investigate the opening process of the fingers domain in the absence of substrate for B. stearothermophilis DNA polymerase in silico. These simulations are biologically and/or physiologically relevant as they shed light on the transitions between states in this important enzyme. All closed and ajar simulations successfully transitioned into the fully open conformation, which is known to be the dominant binary enzyme-DNA conformation from solution and crystallographic studies. Furthermore, we have detailed the key stages in the opening process starting from the open and ajar crystal structures, including the observation of a previously unknown key intermediate structure. Four backbone dihedrals were identified as important during the opening process, and their movements provide insight into the recognition of dNTP substrate molecules by the polymerase binary state. In addition to revealing the opening mechanism, this study also demonstrates our ability to study biological events of DNA polymerase using current computational methods without biasing the dynamics.

Crystal Structure of DNA Polymerase β with DNA Containing the Base Lesion Spiroiminodihydantoin in a Templating Position

The first high-resolution crystal structure of spiroiminodihydantoin (dSp1) was obtained in the context of the DNA polymerase β active site and reveals two areas of significance. First, the structure verifies the recently determined S configuration at the spirocyclic carbon. Second, the distortion of the DNA duplex is similar to that of the single-oxidation product 8-oxoguanine. For both oxidized lesions, adaptation of the syn conformation results in similar backbone distortions in the DNA duplex. The resulting conformation positions the dSp1 A-ring as the base-pairing face whereas the B-ring of dSp1 protrudes into the major groove.

Perspective: pre-chemistry conformational changes in DNA polymerase mechanisms

In recent papers, there has been a lively exchange concerning theories for enzyme catalysis, especially the role of protein dynamics/pre-chemistry conformational changes in the catalytic cycle of enzymes. Of particular interest is the notion that substrate-induced conformational changes that assemble the polymerase active site prior to chemistry are required for DNA synthesis and impact fidelity (i.e., substrate specificity). High-resolution crystal structures of DNA polymerase β representing intermediates of substrate complexes prior to the chemical step are available. These structures indicate that conformational adjustments in both the protein and substrates must occur to achieve the requisite geometry of the reactive participants for catalysis. We discuss computational and kinetic methods to examine possible conformational change pathways that lead from the observed crystal structure intermediates to the final structures poised for chemistry. The results, as well as kinetic data from site-directed mutagenesis studies, are consistent with models requiring pre-chemistry conformational adjustments in order to achieve high fidelity DNA synthesis. Thus, substrate-induced conformational changes that assemble the polymerase active site prior to chemistry contribute to DNA synthesis even when they do not represent actual rate-determining steps for chemistry.

Crystal structure of DNA polymerase III β sliding clamp from Mycobacterium tuberculosis

The sliding clamp is a key component of DNA polymerase III (Pol III) required for genome replication. It is known to function with diverse DNA repair proteins and cell cycle-control proteins, making it a potential drug target. To extend our understanding of the structure/function relationship of the sliding clamp, we solved the crystal structure of the sliding clamp from Mycobacterium tuberculosis ( M. tuberculosis), a human pathogen that causes most cases of tuberculosis (TB). The sliding clamp from M. tuberculosis forms a ring-shaped head-to-tail dimer with three domains per subunit. Each domain contains two α helices in the inner ring that lie against two β sheets in the outer ring. Previous studies have indicated that many Escherichia coli clamp-binding proteins have a conserved LF sequence, which is critical for binding to the hydrophobic region of the sliding clamp. Here, we analyzed the binding affinities of the M. tuberculosis sliding clamp and peptides derived from the α and δ subunits of Pol III, which indicated that the LF motif also plays an important role in the binding of the α and δ subunits to the sliding clamp of M. tuberculosis.

Binuclear manganese(II) complexes in biological systems

The paper reports calculations on binuclear manganese(II) clusters related to those found in biological systems. B3LYP/6-311g(d,p) density functional theory (DFT) calculations are carried out specifically on a set of clusters that derive from the X-ray crystal structure of DNA polymerase β [Batra, V. K. et al., Structure 14, 757–766 (2006)]. Issues investigated are: (a) the relative energy of the lowest and highest spin states (antiferromagnetic (low spin/broken symmetry = LS/BS) or ferromagnetic); (b) the Mn(II)–Mn(II) distance; (c) the effect of water versus CH3OH (a sugar analogue) as a direct Mn ligand; and (d) the effect of the kind of oxygen bridge (mono-oxygen, single-oxygen from a carboxylate or phosphate, or a two-oxygen bridge from a carboxylate), on the geometry and energy. It was found generally that the antiferromagnetic state is lower in energy, that the Mn(II)–Mn(II) distance (which varies from 2.91 to 3.57 Å in the series studied) is longer for ferromagnetic states than for the corresponding low spin states, that the interchange of CH3OH and water does not affect the results, and that a single (carboxylate) oxygen as bridge leads to a lower energy than a corresponding two (carboxylate) oxygen bridge, as well as to a more compact structure.

Crystal structure of DNA polymerase from bacteriophage M2

Crystal structure of DNA polymerase from bacteriophage M2

The Hydrophobic Hinge Region of Rat DNA Polymerase β Is Critical for Substrate Binding Pocket Geometry

The hydrophobic hinge of DNA polymerase beta facilitates closing and stabilization of the enzyme once the nucleotide substrate has bound. Alteration of the hydrophobic nature of the hinge by the introduction of a hydrophilic glutamine residue in place of isoleucine 260 results in an inaccurate polymerase. The kinetic basis of infidelity is lack of discrimination during the binding of substrate. The I260Q polymerase beta variant has lower affinity than wild type enzyme for the correct substrate and much higher affinity for the incorrect substrate. Our results demonstrate that the hinge is important for formation of the substrate binding pocket. Our results are also consistent with the interpretation that DNA polymerase beta discriminates the correct from incorrect substrate during the binding step.

Crystal Structure of the Mycobacterium tuberculosis dUTPase: Insights into the Catalytic Mechanism

The structure of Mycobacterium tuberculosis dUTP nucleotidohydrolase (dUTPase) has been determined at 1.3Å resolution in complex with magnesium ion and the non-hydrolyzable substrate analog, α,β-imido dUTP. dUTPase is an enzyme essential for depleting potentially toxic concentrations of dUTP in the cell. Given the importance of its biological role, it has been proposed that inhibiting M.tuberculosis dUTPase might be an effective means to treat tuberculosis infection in humans. The crystal structure presented here offers some insight into the potential for designing a specific inhibitor of the M.tuberculosis dUTPase enzyme. The structure also offers new insights into the mechanism of dUTP hydrolysis by providing an accurate representation of the enzyme–substrate complex in which both the metal ion and dUTP analog are included. The structure suggests that inclusion of a magnesium ion is important for stabilizing the position of the α-phosphorus for an in-line nucleophilic attack. In the absence of magnesium, the α-phosphate of dUTP can have either of the two positions which differ by 4.5Å. A transiently ordered C-terminal loop further assists catalysis by shielding the general base, Asp83, from solvent thus elevating its pKa so that it might in turn activate a tightly bound water molecule for nucleophilic attack. The metal ion coordinates α, β, and γ phosphate groups with tridentate geometry identical with that observed in the crystal structure of DNA polymerase β complexed with magnesium and dNTP analog, revealing some common features in catalytic mechanism.

Structural design of a eukaryotic DNA repair polymerase: DNA polymerase β

DNA polymerase β, the smallest eukaryotic DNA polymerase, is designed to synthesize DNA in short DNA gaps during DNA repair. It is composed of two specialized domains that contribute essential enzymatic activities to base excision repair (BER). Its amino-terminal domain possesses a lyase activity necessary to remove the 5′-deoxyribose phosphate (dRP) intermediate generated during BER. Removal of the dRP moiety is often the rate-limiting step during BER. Failure to remove this group may initiate alternate BER pathways. The larger polymerase domain has nucleotidyl transferase activity. This domain has a modular organization with sub-domains that bind duplex DNA, catalytic metals, and the correct nucleoside triphosphate in a template-dependent manner. X-ray crystal structures of DNA polymerase β, with and without bound substrates, has inferred that domain, sub-domain, and substrate conformational changes occur upon ligand binding. Many of these conformational changes are distinct from those observed in structures of other DNA polymerases. This review will examine the structural aspects of DNA polymerase β that facilitate its role in BER.

DNA Polymerase β. 5. Dissecting the Functional Roles of the Two Metal Ions with Cr(III)dTTP1

DNA polymerases catalyze the synthesis of DNA with a fidelity ranging from 1000 to 100 000.2 Although the kinetic properties of DNA polymerases have been investigated extensively,3-5 the physical origin of this high fidelity is still poorly understood. The crystal structures of DNA polymerase â (Pol â) from rat brain show the presence of two Mg2+ ions in the active site6a,b,7 (Figure 1). Functionally, it has been established that DNA polymerases require divalent metal ions for catalysis.8,9 However, no kinetic evidence that two divalent metal ions are required has been obtained for DNA polymerases, nor have the roles of the two metal ions been dissected functionally. We here report the use of Cr(III)dNTP coupled with stopped-flow fluorescence to show the requirement of two metal ions, and to dissect their functional roles in the catalysis by Pol â from rat brain (overexpressed in Escherichia coli).5 Pol â can serve as a good model for nucleotidyl transfer reactions because of its small size (39 kDa) and simplicity (without exonuclease or proof-reading activities). Using a synthetic DNA primer/template containing the 2-aminopurine nucleotide analogue (2-AP) (Figure 2A), we recently reported two phases of fluorescence changes in the stopped-flow fluorescence assay of nucleotide incorporation catalyzed by Pol â (Figure 2B).10 The fast phase was attributed to a conformational change step because its rate dependence on [dTTP] is hyperbolic. The rate of the slow phase corresponds to product formation. With a dideoxynucleotide-terminated primer, we have successfully dissected the slow fluorescence change and the chemical step, and attributed the slow phase to a conformational change that is the rate-limiting step in the reaction.10 Cr(III)‚nucleotide complexes have been extensively used as analogues of Mg(II)‚nucleotide complexes to solve enzymological problems because of their exchange-inert properties.11-13 When the Pol â‚DNA complex was mixed with â,γ-bidentate Cr(ΙΙΙ)dTTP14 in the absence of Mg2+, only the fast phase was observed (Figure 2C). However, the slow phase was restored upon addition of Mg2+ (Figure 2D). Under the latter condition the substrates were turned over, and the rate of the slow phase was comparable to the rate of product (21-mer) formation measured by the rapid chemical quench assay. In the absence of any metal ions (dTTP alone) neither fluorescence changes nor catalysis occurred. These results clearly indicate that the fast conformational change is induced by the binding of Cr(III)dTTP and the slow conformational change by the binding of Mg2+. On the basis of the crystal structure shown in Figure 1, Cr3+ and Mg2+ should occupy the metal ion sites A and B, respectively.6c The results described above, taken together with our pre-steadystate kinetic analyses reported previously, strongly support the mechanism we proposed for Pol â:10