Improved PCR performance and fidelity of double mutant Neq A523R/N540R DNA polymerase

We previously reported that Neq A523R DNA polymerase is more efficient in PCR than wild-type Neq DNA polymerase, and amplifies products more rapidly. Neq A523R DNA polymerase also amplifies templates more rapidly than Pfu DNA polymerase, but has a lower fidelity than Pfu DNA polymerase. To improve product yield and the fidelity of amplification simultaneously, we constructed and characterized the double mutant Neq A523R/N540R. The yield of PCR products was greater for Neq A523R/N540R DNA polymerase than wild-type and other mutant DNA polymerases, and the Neq double mutant catalyzed amplification of a 12-kb PCR product from a lambda template with an extension time of 3 min. The PCR error rate of Neq A523R/N540R DNA polymerase (6.3 × 10⁻⁵) was roughly similar to that of Pfu DNA polymerase (4.8 × 10⁻⁵), but much lower than those of wild-type Neq DNA polymerase (57.2 × 10⁻⁵), Neq A523R DNA polymerase (13.1 × 10⁻⁵), and Neq N540R DNA polymerase (37.7 × 10⁻⁵). These results indicated that A523R and N540R mutations of Neq DNA polymerase had synergistic effects on its fidelity.     

Structural factors that determine selectivity of a high fidelity DNA polymerase for deoxy-, dideoxy-, and ribonucleotides

In addition to discriminating against base pair mismatches, DNA polymerases exhibit a high degree of selectivity for deoxyribonucleotides over ribo- or dideoxynucleotides. It has been proposed that a single active site residue (steric gate) blocks productive binding of nucleotides containing 2'-hydroxyls. Although this steric gate plays a role in sugar moiety discrimination, its interactions do not account fully for the observed behavior of mutants. Here we present 10 high resolution crystal structures and enzyme kinetic analyses of Bacillus DNA polymerase I large fragment variants complexed with deoxy-, ribo-, and dideoxynucleotides and a DNA substrate. Taken together, these data present a more nuanced and general mechanism for nucleotide discrimination in which ensembles of intermediate conformations in the active site trap non-cognate substrates. It is known that the active site O-helix transitions from an open state in the absence of nucleotide substrates to a ternary complex closed state in which the reactive groups are aligned for catalysis. Substrate misalignment in the closed state plays a fundamental part in preventing non-cognate nucleotide misincorpation. The structures presented here show that additional O-helix conformations intermediate between the open and closed state extremes create an ensemble of binding sites that trap and misalign non-cognate nucleotides. Water-mediated interactions, absent in the fully closed state, play an important role in formation of these binding sites and can be remodeled to accommodate different non-cognate substrates. This mechanism may extend also to base pair discrimination.     

Genetic mapping of the Saccharomyces cerevisiae DNA polymerase I gene and characterization of a pol1 temperature-sensitive mutant altered in DNA primase-polymerase complex stability

Summary The cloned DNA polymerase I gene has been used to map the POL1 locus on the left arm of chromosome XIV, between MET4 and TOP2. Temperature-sensitive mutants in POL1 have been obtained by in vitro mutagenesis of the cloned gene and in vivo replacement of the wild-type allele with the mutated copy. Physiological and biochemical characterization of one temperature-sensitive mutant (pol1-1) shows that cells shifted to the non-permissive temperature can complete one round of cell division and DNA replication before they arrest. Analysis of DNA polymerase I in crude extracts and in partially purified preparations indicates that the pol1-1 mutation results in a conformational change and affects the stability of the DNA primase-polymerase complex.     

Molecular dynamics studies on HIV-1 protease: a comparison of the flap motions between wild type protease and the M46I/G51D double mutant

The emergence of drug-resistant mutants of HIV-1 is a tragic effect associated with conventional long-treatment therapies against acquired immunodeficiency syndrome. These mutations frequently involve the aspartic protease encoded by the virus; knowledge of the molecular mechanisms underlying the conformational changes of HIV-1 protease mutants may be useful in developing more effective and longer lasting treatment regimes. The flap regions of the protease are the target of a particular type of mutations occurring far from the active site. These mutations modify the affinity for both substrate and ligands, thus conferring resistance. In this work, molecular dynamics simulations were performed on a native wild type HIV-1 protease and on the drug-resistant M46I/G51D double mutant. The simulation was carried out for a time of 3.5 ns using the GROMOS96 force field, with implementation of the SPC216 explicit solvation model. The results show that the flaps may exist in an ensemble of conformations between a “closed” and an “open” conformation. The behaviour of the flap tips during simulations is different between the native enzyme and the mutant. The mutation pattern leads to stabilization of the flaps in a semi-open configuration.     

Efficient and high fidelity incorporation of dye-terminators by a novel archaeal DNA polymerase mutant

We examined the molecular basis of ddNTP selectivity in archaeal family B DNA polymerases by randomly mutagenizing the gene encoding Thermococcus sp. JDF-3 DNA polymerase and screening mutant libraries for improved ddNTP incorporation. We identified two mutations, P410L and A485T, that improved ddNTP uptake, suggesting the contribution of P410 and A485 to ddNTP/dNTP selectivity in archaeal DNA polymerases. The importance of A485 was identified previously in mutagenesis studies employing Pfu (A486) and Vent (A488) DNA polymerases, while the contribution of P410 to ddNTP/dNTP selectivity has not been reported. We demonstrate that a combination of mutations (P410L/A485T) has an additive effect in improving ddNTP incorporation by a total of 250-fold. To assess the usefulness of the JDF-3 P410L/A485T in fluorescent-sequencing applications, we compared the archaeal mutant to Taq F667Y with respect to fidelity and kinetic parameters for DNA and dye-ddNTPs. Although the Taq F667Y and JDF-3 P410L/A485T mutants exhibit similar K(m) and V(max) values for dye-ddNTPs in single-base extension assays, the archaeal mutant exhibits higher fidelity due to a reduced tendency to form certain (ddG:dT, ddT:dC) mispairs. DNA polymerases exhibiting higher insertion fidelity are expected to provide greater accuracy in SNP frequency determinations by single-base extension and in multiplex minisequencing assays.     

New structural snapshots provide molecular insights into the mechanism of high fidelity DNA synthesis

Time-lapse X-ray crystallography allows visualization of intermediate structures during the DNA polymerase catalytic cycle. Employing time-lapse crystallography with human DNA polymerase β has recently allowed us to capture and solve novel intermediate structures that are not stable enough to be analyzed by traditional crystallography. The structures of these intermediates reveals exciting surprises about active site metal ions and enzyme conformational changes as the reaction proceeds from the ground state to product release. In this perspective, we provide an overview of recent advances in understanding the DNA polymerase nucleotidyl transferase reaction and highlight both the significance and mysteries of enzyme efficiency and specificity that remain to be solved.     

Mutability of DNA polymerase I: implications for the creation of mutant DNA polymerases

DNA polymerases of the Family A catalyze the addition of deoxynucleotides to a primer with high efficiency, processivity, and selectivity-properties that are critical to their function both in nature and in the laboratory. These polymerases tolerate many amino acid substitutions, even in regions that are evolutionarily conserved. This tolerance can be exploited to create DNA polymerases with novel properties and altered substrate specificities, using rational design and molecular evolution. These efforts have focused mainly on the Family A DNA polymerises -Taq, E. coli Pol I, and T7 - because they are widely utilized in biotechnology today. The redesign of polymerases often requires knowledge of the function of specific residues in the protein, including those located in six evolutionarily conserved regions. The most well characterized of these are motifs A and B, which regulate the fidelity of replication and the incorporation of nucleotide analogs such as dideoxynucleotides. Regions that remain to be more thoroughly characterized are motif C, which is critical for catalysis, and motifs 1, 2 and 6, all of which bind to DNA primer or template. Several recently identified mutants with abilities to incorporate nucleotides with bulky adducts have mutations that are not located within conserved regions and warrant further study. Analysis of these mutants will help advance our understanding of how DNA polymerases select bases with high fidelity.     

Design of Sequence-Specific DNA Binding Ligands That Use a Two-Stranded Peptide Motif for DNA Sequence Recognition

Abstract

The design and DNA binding activity of β-structure-forming peptides and netropsin-peptide conjugates are reported. It is found that a pair of peptides - S,S'-bis(Lys-Gly-Val-Cys-Val- NH-NH-Dns) - bridged by an S-S bond binds at least 10 times more strongly to poly(dG)?poly(dC) than to poly(dA)?poly(dT). This peptide can also discriminate between 5′-GpG-3′ and 5′-GpC-3′ steps in the DNA minor groove. Based on these observations, new synthetic ligands, bis-netropsins, were constructed in which two netropsin-like fragments were attached by means of short linkers to a pair of peptides - Gly-Cys-Gly- or Val-Cys-Val - bridged by S-S bonds. These compounds possess a composite binding specificity: the peptide chains recognize 5′-GpG-3′ steps on DNA, whereas the netropsin-like fragments bind preferentially to tuns of 4 AT base pairs. Our data indicate that combining the AT-base-pair specific properties of the netropsin-type structure with the 5′-GpG-3′-specific properties of certain oligopeptides offers a new approach to the synthesis of ligands capable of recognizing mixed sequences of AT- and GC-base pairs in the DNA minor groove. These compounds are potential models for DNA-binding domains in proteins which specifically recognize base pair sequences in the minor groove of DNA.     

Characterization of DNA polymerase from the hyperthermophilic archaeon Thermococcus marinus and its application to PCR

The family B DNA polymerase gene from the archaeon Thermococcus marinus (Tma) contains a long open reading frame of 3,939 bp that encodes 1,312 amino acid residues. The gene is split by one intervening sequence that forms a continuous open reading frame with the two polymerase exteins. In this study, the Tma DNA polymerase gene both with (precursor form) and without (mature form) its intein was expressed in Escherichia coli, purified by heat treatment and HiTrap™ Heparin HP column chromatography and characterized. Primary sequence analysis of the mature Tma polymerase showed high sequence identity with DNA polymerases in the genus Thermococcus. The expressed precursor form was easily spliced during purification steps. The molecular mass of the purified Tma DNA polymerases is about 90 kDa, as estimated by SDS-PAGE. Both Tma DNA polymerases showed the same properties. PCR performed with this enzyme was found to be optimal in the presence of 50 mM Tris–HCl (pH 8.4), 40 mM KCl, 12.5 mM (NH₄)₂SO_4, 2 mM MgCl_2, 0.05% Triton X-100 and 0.0075% BSA. Furthermore, long-range PCR and time-saving PCR were performed using various specific ratios of Taq and Tma DNA polymerases (Tma plus DNA polymerase).     

The structure of a high fidelity DNA polymerase bound to a mismatched nucleotide reveals an "ajar" intermediate conformation in the nucleotide selection mechanism

To achieve accurate DNA synthesis, DNA polymerases must rapidly sample and discriminate against incorrect nucleotides. Here we report the crystal structure of a high fidelity DNA polymerase I bound to DNA primer-template caught in the act of binding a mismatched (dG:dTTP) nucleoside triphosphate. The polymerase adopts a conformation in between the previously established "open" and "closed" states. In this "ajar" conformation, the template base has moved into the insertion site but misaligns an incorrect nucleotide relative to the primer terminus. The displacement of a conserved active site tyrosine in the insertion site by the template base is accommodated by a distinctive kink in the polymerase O helix, resulting in a partially open ternary complex. We suggest that the ajar conformation allows the template to probe incoming nucleotides for complementarity before closure of the enzyme around the substrate. Based on solution fluorescence, kinetics, and crystallographic analyses of wild-type and mutant polymerases reported here, we present a three-state reaction pathway in which nucleotides either pass through this intermediate conformation to the closed conformation and catalysis or are misaligned within the intermediate, leading to destabilization of the closed conformation.     

Mutational specificity of a bacteriophage T4 DNA polymerase mutant, mel88

Summary Several bacteriophage T4 DNA polymerase mutants have been shown to increase the frequency of spontaneous mutations (Speyer et al. 1966; Freese and Freese 1967; de Vries et al. 1972; Reha-Krantz et al. 1986). In order to determine the molecular basis of the mutator phenotype, it is necessary to characterize the types of mutations produced by the mutator DNA polymerases. We show here that at least one DNA polymerase mutator mutant, mel88, induces an increased number of base substitution mutations compared with wild-type.     

Detection of Chlamydia psittaci using DNA probes and the polymerase chain reaction

Fewer than 10(5) elementary bodies of Chlamydia psittaci could be detected by using DNA hybridisation with a plasmid probe specific for avian chlamydial strains. PCR amplification of chlamydial DNA using primers specific for conserved regions of the major outer membrane protein gene enabled the detection of fewer than 10 elementary bodies. DNA could be amplified from 22 of the 24 chlamydial strains tested including avian, feline, ovine, caprine, koala and lymphogranuloma venereum strains.     

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.     

Crystal structure of Pfu, the high fidelity DNA polymerase from Pyrococcus furiosus

We have determined a 2.6 Å resolution crystal structure of Pfu DNA polymerase, the most commonly used high fidelity PCR enzyme, from Pyrococcus furiosus. Although the structures of Pfu and KOD1 are highly similar, the structure of Pfu elucidates the electron density of the interface between the exonuclease and thumb domains, which has not been previously observed in the KOD1 structure. The interaction of these two domains is known to coordinate the proofreading and polymerization activity of DNA polymerases, especially via H147 that is present within the loop (residues 144–158) of the exonuclease domain. In our structure of Pfu, however, E148 rather than H147 is located at better position to interact with the thumb domain. In addition, the structural analysis of Pfu and KOD1 shows that both the Y-GG/A and β-hairpin motifs of Pfu are found to differ with that of KOD1, and may explain differences in processivity. This information enables us to better understand the mechanisms of polymerization and proofreading of DNA polymerases.     

Kin I Kinesins: Insights into the Mechanism of Depolymerization

ABSTRACT

Kin I kinesins are members of the diverse kinesin superfamily of molecular motors. Whereas most kinesins use ATP to move along microtubules, Kin I kinesins depolymerize microtubules rather than walk along them. Functionally, this distinct subfamily of kinesins is important in regulating cellular microtubule dynamics and plays a crucial role in spindle assembly and chromosome segregation. The molecular mechanism of Kin I-induced microtubule destabilization is as yet unclear. It is generally believed that Kin Is induce a structural change on the microtubule that leads to microtubule destabilization. Recently, much progress has been made towards understanding how Kin Is may cause this structural change, and how ATPase activity is employed in the catalytic cycle.     

Isolation and cloning by a polymerase chain reaction of a genomic DNA fragment of the human slow skeletal troponin (TNNT1) gene

The genomic 3′ structure of the gene coding for the human slow skeletal troponin T (TNNT1) gene, is reported. An intron of 912 nucleotides containing an Alu-element has been identified and characterized. The complexity of the sequenced region suggests an alternative exon use. The present results may be valuable for further studies on the gene structure of TNNT1 and the related troponin gene family.     

PCR performance of the highly thermostable proof-reading B-type DNA polymerase from Pyrococcus abyssi

DNA polymerase from the archaeon Pyrococcus abyssi strain Orsay was expressed in Escherichia coli. The recombinant DNA polymerase (Pab) was purified to homogeneity by heat treatment followed by 5 steps of chromatography and characterized for PCR applications. Buffer optimization experiments indicated that Pab PCR performance and fidelity parameters were highest in the presence of 20 mM Tris-HCl, pH 9.0, 1.5 mM MgSO4, 25 mM KCl, 10 mM (NH4)2SO4 and 40 microM of each dNTP. Under these conditions, the error rate was 0.66.10(-6) mutations/nucleotide/duplication. Pab DNA polymerase, having a half life of 5 h at 100 degrees C, was demonstrated to be highly thermostable in PCR conditions compared to commercial Taq and Pfu DNA polymerases. These characteristics enable Pab to be one of the most efficient thermostable DNA polymerases described, exhibiting very high accuracy compared to other available commercial DNA polymerases and robust thermostable activity. This new DNA polymerase is currently on the market under the name Isis DNA Polymerase (Qbiogene Molecular Biology).     

Cloning, expression, and characterization of DNA polymerase I from the hyperthermophilic archaea Thermococcus fumicolans

The DNA polymerase I gene of a newly described deep-sea hydrothermal vent Archaea species, Thermococcus fumicolans, from IFREMERS's collection of hyperthermophiles has been cloned in Escherichia coli. As in Thermococcus litoralis, the gene is split by two intervening sequences (IVS) encoding inteins inserted in sites A and C of family B DNA polymerases. The entire DNA polymerase gene, containing both inteins, was expressed at 30°C in E. coli strain BL21(DE3)pLysS using the pARHS2 expression vector. The native polypeptide precursor of 170 kDa was obtained, and intein splicing as well as ligation of the three exteins was observed in vitro after heat exposure. The recombinant enzyme was purified and some of its activities were characterized: polymerization, thermostability, exonuclease activities, and fidelity. Received: September 17, 1999 / Accepted: March 21, 2000