Dpo4 is hindered in extending a G.T mismatch by a reverse wobble

The ability or inability of a DNA polymerase to extend a mispair directly affects the establishment of genomic mutations. We report here kinetic analyses of the ability of Dpo4, a Y-family polymerase from Sulfolobus solfataricus, to extend from all mispairs opposite a template G or T. Dpo4 is equally inefficient at extending these mispairs, which include, surprisingly, a G.T mispair expected to conform closely to Watson-Crick geometry. To elucidate the basis of this, we solved the structure of Dpo4 bound to G.T-mispaired primer template in the presence of an incoming nucleotide. As a control, we also determined the structure of Dpo4 bound to a matched A-T base pair at the primer terminus. The structures offer a basis for the low efficiency of Dpo4 in extending a G.T mispair: a reverse wobble that deflects the primer 3'-OH away from the incoming nucleotide.     

Further characterization of the interaction between the Epstein-Barr virus DNA polymerase catalytic subunit and its accessory subunit with regard to the 3'-to-5' exonucleolytic activity and stability of initiation complex at primer terminus.

The Epstein-Barr virus (EBV) DNA polymerase catalytic subunit, BALF5 gene product, possesses an intrinsic 3'-to 5' proofreading exonuclease activity in addition to 5'-to-3' DNA polymerase activity (T. Tsurumi, A. Kobayashi, K. Tamai, T. Daikoku, R. Kurachi, and Y. Nishiyama, J. Virol. 67:4651-4658, 1993). The exonuclease hydrolyzed both double-and single-stranded DNA substrates with 3'-to-5' directionality, releasing deoxyribonucleoside 5'-monophosphates. The double-strand exonucleolytic activity catalyzed by the BALF5 polymerase catalytic subunit was very sensitive to high ionic strength, whereas the single-strand exonucleolytic activity was moderately resistant. The addition of the BMRF1 polymerase accessory subunit to the reaction enhanced the double-strand exonucleolytic activity in the presence of high concentrations of ammonium sulfate (fourfold stimulation at 75 mM ammonium sulfate). Optimal stimulation was obtained when the molar ratio of BMRF1 protein to BALF5 protein was 2 and higher, identical to the values required for reconstituting the optimum DNA polymerizing activity (T. Tsurumi, T. Daikoku, R. Kurachi, and Y. Nishiyama, J. Virol. 67:7648-7653, 1993). Furthermore, product size analyses revealed that the polymerase catalytic subunit alone excised a few nucleotides from the 3' termini of the primer hybridized to template DNA and that the addition of the BMFR1 polymerase accessory subunit stimulated the nucleotide excision several times. In contrast, the hydrolysis of single-stranded DNA by the BALF5 protein was not affected by the addition of the BMRF1 polymerase accessory subunit at all. These observations suggest that the BMRF1 polymerase accessory subunit forms a complex with the BALF5 polymerase catalytic subunit to stabilize the interaction of the holoenzyme complex with the 3'-OH end of the primer on the template DNA during exonucleolysis. On the other hand, challenger DNA experiments revealed that the BALF5 polymerase catalytic subunit alone stably binds to the primer terminus in a stationary state, whereas the reconstituted polymerase holoenzyme is unstable. The instability of the initiation complex of the EBV DNA polymerase would allow the rapid removal of the EBV DNA polymerase holoenzyme from the lagging strand after it has replicated up to the previous Okazaki fragment. This feature of the EBV DNA polymerase holoenzyme in a stationary state is in marked contrast to the moving holoenzyme complex tightly bound to the primer end during polymerization and exonucleolysis.     

Crystal structures of a template-independent DNA polymerase: murine terminal deoxynucleotidyltransferase

The crystal structure of the catalytic core of murine terminal deoxynucleotidyltransferase (TdT) at 2.35 A resolution reveals a typical DNA polymerase beta-like fold locked in a closed form. In addition, the structures of two different binary complexes, one with an oligonucleotide primer and the other with an incoming ddATP-Co(2+) complex, show that the substrates and the two divalent ions in the catalytic site are positioned in TdT in a manner similar to that described for the human DNA polymerase beta ternary complex, suggesting a common two metal ions mechanism of nucleotidyl transfer in these two proteins. The inability of TdT to accommodate a template strand can be explained by steric hindrance at the catalytic site caused by a long lariat-like loop, which is absent in DNA polymerase beta. However, displacement of this discriminating loop would be sufficient to unmask a number of evolutionarily conserved residues, which could then interact with a template DNA strand. The present structure can be used to model the recently discovered human polymerase mu, with which it shares 43% sequence identity.     

Exonuclease associated with bacteriophage T5-Induced DNA polymerase.

T-5-induced DNA polymerase has been shown to possess a 3' leads to 5'-exonucleolytic activity. The exonuclease acts on both native and denatured DNA, but the apparent rate of degradation of denatured DNA is about five times faster than that for native DNA. The enzyme appears to act only on 3'-OH ends and produces mainly 5'-dNMP's. Like polymerase activity, exonuclease activity shows a pH optimum around 8.6. Mg2+, dithiothreitol, and N-ethylmaleimide had identical effects on both the activities. Nicked DNA was almost totally protected from exonuclease action under synthetic conditions, i.e., in the presence of 4dNTP's. Denatured DNA was partly degraded in the early phase of incubation with 4dNTP's, presumably due to unhybridized tails at the 3'-OH primer ends. However, the exonuclease activity was operative in both cases under synthetic conditions, as evidenced by template-dependent conversion of [3H]dTTP to [3H]dTMP.     

Effect of oxidatively damaged DNA on the active site preorganization during nucleotide incorporation in a high fidelity polymerase from Bacillus stearothermophilus

We study the effect of the oxidative lesion 8-oxoguanine (8oxoG) on the preorganization of the active site for DNA replication in the closed (active) state of the Bacillus fragment (BF), a Klenow analog from Bacillus stearothermophilus. Our molecular dynamics and free energy simulations of explicitly solvated model ternary complexes of BF bound to correct dCTP/incorrect dATP opposite guanine (G) and 8oxoG bases in DNA suggest that the lesion introduces structural and energetic changes at the catalytic site to favor dATP insertion. Despite the formation of a stable Watson-Crick pairing in the 8oxoG:dCTP system, the catalytic geometry is severely distorted to possibly slow down catalysis. Indeed, our calculated free energy landscapes associated with active site preorganization suggest additional barriers to assemble an efficient catalytic site, which need to be overcome during dCTP incorporation opposite 8oxoG relative to that opposite undamaged G. In contrast, the catalytic geometry for the Hoogsteen pairing in the 8oxoG:dATP system is highly organized and poised for efficient nucleotide incorporation via the "two-metal-ion" catalyzed phosphoryl transfer mechanism. However, the free energy calculations suggest that the catalytic geometry during dATP incorporation opposite 8oxoG is considerably less plastic than that during dCTP incorporation opposite G despite a very similar, well organized catalytic site for both systems. A correlation analysis of the dynamics trajectories suggests the presence of significant coupling between motions of the polymerase fingers and the primary distance for nucleophilic attack (i.e., between the terminal primer O3' and the dNTP P(alpha.) atoms) during correct dCTP incorporation opposite undamaged G. This coupling is shown to be disrupted during nucleotide incorporation by the polymerase with oxidatively damaged DNA/dNTP substrates. We also suggest that the lesion affects DNA interactions with key polymerase residues, thereby affecting the enzymes ability to discriminate against non-complementary DNA/dNTP substrates. Taken together, our results provide a unified structural, energetic, and dynamic platform to rationalize experimentally observed relative nucleotide incorporation rates for correct dCTP/incorrect dATP insertion opposite an undamaged/oxidatively damaged template G by BF.     

Insight into the catalytic mechanism of DNA polymerase beta: structures of intermediate complexes

The catalytic reaction mediated by DNA polymerases is known to require two Mg(II) ions, one associated with dNTP binding and the other involved in metal ion catalysis of the chemical step. Here we report a functional intermediate structure of a DNA polymerase with only one metal ion bound, the DNA polymerase beta-DNA template-primer-chromium(III).2'-deoxythymidine 5'-beta,gamma-methylenetriphosphate [Cr(III).dTMPPCP] complex, at 2.6 A resolution. The complex is distinct from the structures of other polymerase-DNA-ddNTP complexes in that the 3'-terminus of the primer has a free hydroxyl group. Hence, this structure represents a fully functional intermediate state. Support for this contention is provided by the observation of turnover in biochemical assays of crystallized protein as well as from the determination that soaking Pol beta crystals with Mn(II) ions leads to formation of the product complex, Pol beta-DNA-Cr(III).PCP, whose structure is also reported. An important feature of both structures is that the fingers subdomain is closed, similar to structures of other ternary complexes in which both metal ion sites are occupied. These results suggest that closing of the fingers subdomain is induced specifically by binding of the metal-dNTP complex prior to binding of the catalytic Mg(2+) ion. This has led us to reevaluate our previous evidence regarding the existence of a rate-limiting conformational change in Pol beta's reaction pathway. The results of stopped-flow studies suggest that there is no detectable rate-limiting conformational change step.     

Novel 3'-ribonuclease and 3'-phosphatase activities of the bacterial non-homologous end-joining protein, DNA ligase D

Pseudomonas aeruginosa DNA ligase D (PaeLigD) exemplifies a family of bacterial DNA end-joining proteins that consist of a ligase domain fused to a polymerase domain and a putative nuclease module. The LigD polymerase preferentially adds single ribonucleotides at blunt DNA ends and, as we show here, is also capable of adding up to 4 ribonucleotides to a DNA primer-template. We report that PaeLigD has an intrinsic ability to resect the short tract of 3'-ribonucleotides of a primer-template substrate to the point at which the primer strand has a single 3'-ribonucleotide remaining. The failure to digest beyond this point reflects a requirement for a 2'-OH group on the penultimate nucleoside of the primer strand. Replacing the 2'-OH by a 2'-F, 2'-NH2, 2'-OCH3, or 2'-H abolishes the resection reaction. The ribonucleotide resection activity resides within a 187-amino acid N-terminal nuclease domain and is the result of at least two component steps: (i) the 3'-terminal nucleoside is first removed to yield a primer strand with a ribonucleoside 3'-PO4 terminus, and (ii) the 3'-PO4 is hydrolyzed to a 3'-OH. The 3'-ribonuclease and 3'-phosphatase activities are both dependent on a divalent cation, specifically manganese. PaeLigD preferentially remodels the 3'-ends of a duplex primer-template substrate rather than a single strand of identical composition, and it prefers DNA primer strands containing a short 3'-ribonucleotide tract to an all-RNA primer. The nuclease domain of PaeLigD and its bacterial homologs has no apparent structural or mechanistic similarity to previously characterized nucleases. Thus, we surmise that it exemplifies a novel phosphoesterase family, defined in part by conserved residues Asp-50, Arg-52, and His-84, which are essential for the 3'-ribonuclease and 3'-phosphatase reactions.     

Catalytic mechanism of human DNA polymerase lambda with Mg2+ and Mn2+ from ab initio quantum mechanical/molecular mechanical studies

DNA polymerases play a crucial role in the cell cycle due to their involvement in genome replication and repair. Understanding the reaction mechanism by which these polymerases carry out their function can provide insights into these processes. Recently, the crystal structures of human DNA polymerase lambda (Pollambda) have been reported both for pre- and post-catalytic complexes [García-Díaz et al., DNA Repair 3 (2007), 1333]. Here we employ the pre-catalytic complex as a starting structure for the determination of the catalytic mechanism of Pollambda using ab initio quantum mechanical/molecular mechanical methods. The reaction path has been calculated using Mg(2+) and Mn(2+) as the catalytic metals. In both cases the reaction proceeds through a two-step mechanism where the 3'-OH of the primer sugar ring is deprotonated by one of the conserved Asp residues (D490) in the active site before the incorporation of the nucleotide to the nascent DNA chain. A significant charge transfer is observed between both metals and some residues in the active site as the reaction proceeds. The optimized reactant and product structures agree with the reported crystal structures. In addition, the calculated reaction barriers for both metals are close to experimentally estimated barriers. Energy decomposition analysis to explain individual residue contributions suggests that several amino acids surrounding the active site are important for catalysis. Some of these residues, including R420, R488 and E529, have been implicated in catalysis by previous mutagenesis experiments on the homologous residues on Polbeta. Furthermore, Pollambda residues R420 and E529 found to be important from the energy decomposition analysis, are homologous to residues R183 and E295 in Polbeta, both of which are linked to cancer. In addition, residues R386, E391, K422 and K472 appear to have an important role in catalysis and could be a potential target for mutagenesis experiments. There is partial conservation of these residues across the Pol X family of DNA polymerases.     

Biochemical and structural insights into substrate binding and catalytic mechanism of mammalian poly(A) polymerase

Polyadenylation of messenger RNA precursors is an essential process in eukaryotes. Poly(A) polymerase (PAP), a member of the nucleotidyltransferase family that includes DNA polymerase beta, incorporates ATP at the 3' end of mRNAs in a template-independent manner. Although the structures of mammalian and yeast PAPs are known, their mechanism of ATP selection has remained elusive. In a recent bovine PAP structure complexed with an analog of ATP and Mn2+, strictly conserved residues interact selectively with the adenine base, but the nucleotide was found in a "non-productive" conformation. Here we report a second bovine crystal structure, obtained in the presence of Mg2+, where 3'-dATP adopts a "productive" conformation similar to that seen in yeast PAP or DNA polymerase beta. Mutational analysis and activity assays with ATP analogs suggest a role in catalysis for one of the two adenine-binding sites revealed by our structural data. The other site might function to prevent futile hydrolysis of ATP. In order to investigate the role of metals in catalysis we performed steady state kinetics experiments under distributive polymerization conditions. These tests suggest a sequential random mechanism in vitro in the presence of ATP and RNA, without preference for a particular order of binding of the two substrates. In vivo, however, where polyadenylation is processive and the primer does not dissociate from the enzyme, an ordered mechanism with the primer as the leading substrate is more likely.     

Site-specific modification of functional groups in genomic hepatitis delta virus (HDV) ribozyme.

Human hepatitis delta (HDV) ribozyme is one of small ribozymes, such as hammerhead and hairpin ribozymes, etc. Its secondary structure shows pseudoknot structure composed of four stems (I to IV) and three single-stranded regions (SSrA, -B and -C). The 3D structure of 3'-cleaved product of genomic HDV ribozyme provided extensive information about tertiary hydrogen bonding interactions between nucleotide bases, phosphate oxygens and 2'OHs including new stem structure P1.1. To analyze the role of these hydrogen bond networks in the catalytic reaction, site-specific atomic-level modifications (such as deoxynucleotides, deoxyribosyl-2-aminopurine, deoxyribosylpurine, 7-deaza-ribonucleotide and inosine) were incorporated in the smallest trans-acting HDV ribozyme (47-mer). Kinetic analysis of these ribozyme variants demonstrated the importance of the two W-C base pairs of P1.1 for cleavage; in addition, the results suggest that all hydrogen bond interactions detected in the crystal structure involving 2'-OH and N7 atoms are present in the active ribozyme structure. In most of the variants, the relative reduction in kobs caused by substitution of the 2'-OH group correlated with the number of hydrogen bonds affected by the substitution. However G74 and C75 may have more than one hydrogen bond involving the 2'-OH in both the trans- and cis-acting HDV ribozyme. Moreover, in variants in which N7 was deleted, kobs was reduced 5- to 15-fold, it may suggest that N7 assists in coordinating Mg2+ ions or water molecules which bind with weak affinity in the active structure.     

Cleavage specificity of Saccharomyces cerevisiae flap endonuclease 1 suggests a double-flap structure as the cellular substrate

Flap endonuclease 1 (FEN1) is a structure-specific nuclease that cleaves substrates containing unannealed 5'-flaps during Okazaki fragment processing. Cleavage removes the flap at or near the point of annealing. The preferred substrate for archaeal FEN1 or the 5'-nuclease domains of bacterial DNA polymerases is a double-flap structure containing a 3'-tail on the upstream primer adjacent to the 5'-flap. We report that FEN1 in Saccharomyces cerevisiae (Rad27p) exhibits a similar specificity. Cleavage was most efficient when the upstream primer contained a 1-nucleotide 3'-tail as compared with the fully annealed upstream primer traditionally tested. The site of cleavage was exclusively at a position one nucleotide into the annealed region, allowing human DNA ligase I to seal all resulting nicks. In contrast, a portion of the products from traditional flap substrates is not ligated. The 3'-OH of the upstream primer is not critical for double-flap recognition, because Rad27p is tolerant of modifications. However, the positioning of the 3'-nucleotide defines the site of cleavage. We have tested substrates having complementary tails that equilibrate to many structures by branch migration. FEN1 only cleaved those containing a 1-nucleotide 3'-tail. Equilibrating substrates containing 12-ribonucleotides at the end of the 5'-flap simulates the situation in vivo. Rad27p cleaves this substrate in the expected 1-nucleotide 3'-tail configuration. Overall, these results suggest that the double-flap substrate is formed and cleaved during eukaryotic DNA replication in vivo.     

Conformational changes during normal and error-prone incorporation of nucleotides by a Y-family DNA polymerase detected by 2-aminopurine fluorescence

Y-family polymerases are specialized to carry out DNA synthesis past sites of DNA damage. Their active sites make fewer contacts to their substrates, consistent with the remarkably low fidelity of these DNA polymerases when copying undamaged DNA. We have used DNA containing the fluorescent reporter 2-aminopurine (2-AP) to study the reaction pathway of the Y-family polymerase Dbh. We detected 3 rapid noncovalent steps between binding of a correctly paired dNTP and the rate-limiting step for dNTP incorporation. These early steps resemble those seen with high-fidelity DNA polymerases, such as Klenow fragment, and include a step that may be related to the unstacking of the 5' neighbor of the templating base that is seen in polymerase ternary complex crystal structures. A significant difference between Dbh and high-fidelity polymerases is that Dbh generates no fluorescence changes subsequent to dNTP binding if the primer lacks a 3'OH, suggesting that the looser active site of Y-family polymerases may enforce reliance on the correct substrate structure in order to assemble the catalytic center. Dbh, like other bypass polymerases of the DinB subgroup, generates single-base deletion errors at an extremely high frequency by skipping over a template base that is part of a repetitive sequence. Using 2-AP as a reporter to study the base-skipping process, we determined that Dbh uses a mechanism in which the templating base slips back to pair with the primer terminus while the base that was originally paired with the primer terminus becomes unpaired.     

Y265H mutator mutant of DNA polymerase beta. Proper teometric alignment is critical for fidelity

DNA polymerases have the unique ability to select a specific deoxynucleoside triphosphate from a pool of similarly structured substrates. One of these enzymes, DNA polymerase beta, offers a simple system to relate polymerase structure to the fidelity of DNA synthesis. In this study, a mutator DNA polymerase beta, Y265H, was identified using an in vivo genetic screen. Purified Y265H produced errors at a 40-fold higher frequency than the wild-type protein in a forward mutation assay. At 37 degrees C, transient kinetic analysis demonstrated that the alteration caused a 111-fold decrease in the maximum rate of polymerization and a 117-fold loss in fidelity for G misincorporation opposite template A. Our data suggest that the maximum rate of polymerization was reduced, because Y265H was dramatically impaired in its ability to perform nucleotidyl transfer in the presence of the correct nucleotide substrate. In contrast, at 20 degrees C, the mutant protein had a fidelity similar to wild-type enzyme. Both proteins at 20 degrees C demonstrate a rapid change in protein conformation, followed by a slow chemical step. These data suggest that proper geometric alignment of template, 3'-OH of the primer, magnesium ions, dNTP substrates, and the active site residues of DNA polymerase beta are important factors in polymerase fidelity and provide the first evidence that Tyr-265 is important for this alignment to occur properly in DNA polymerase beta.     

Crystal structure of human ISG20, an interferon-induced antiviral ribonuclease

ISG20 is an interferon-induced antiviral exoribonuclease that acts on single-stranded RNA and also has minor activity towards single-stranded DNA. It belongs to the DEDDh group of RNases of the DEDD exonuclease superfamily. We have solved the crystal structure of human ISG20 complexed with two Mn2+ ions and uridine 5'-monophosphate (UMP) at 1.9 A resolution. Its structure, including that of the active site, is very similar to those of the corresponding domains of two DEDDh-group DNases, the epsilon subunit of Escherichia coli DNA polymerase III and E. coli exonuclease I, strongly suggesting that its catalytic mechanism is identical to that of the two DNases. However, ISG20 also has distinctive residues, Met14 and Arg53, to accommodate hydrogen bonds with the 2'-OH group of the UMP ribose, and these residues may be responsible for the preference of ISG20 for RNA substrates.     

Role of the catalytic metal during polymerization by DNA polymerase lambda

The incorporation of dNMPs into DNA by polymerases involves a phosphoryl transfer reaction hypothesized to require two divalent metal ions. Here we investigate this hypothesis using as a model human DNA polymerase lambda (Pol lambda), an enzyme suggested to be activated in vivo by manganese. We report the crystal structures of four complexes of human Pol lambda. In a 1.9 A structure of Pol lambda containing a 3'-OH and the non-hydrolyzable analog dUpnpp, a non-catalytic Na+ ion occupies the site for metal A and the ribose of the primer-terminal nucleotide is found in a conformation that positions the acceptor 3'-OH out of line with the alpha-phosphate and the bridging oxygen of the pyrophosphate leaving group. Soaking this crystal in MnCl2 yielded a 2.0 A structure with Mn2+ occupying the site for metal A. In the presence of Mn2+, the conformation of the ribose is C3'-endo and the 3'-oxygen is in line with the leaving oxygen, at a distance from the phosphorus atom of the alpha-phosphate (3.69 A) consistent with and supporting a catalytic mechanism involving two divalent metal ions. Finally, soaking with MnCl2 converted a pre-catalytic Pol lambda/Na+ complex with unreacted dCTP in the active site into a product complex via catalysis in the crystal. These data provide pre- and post-transition state information and outline in a single crystal the pathway for the phosphoryl transfer reaction carried out by DNA polymerases.