DNA Polymerase Selectivity Research Articles

Characterization and engineering of a DNA polymerase reveals a single amino-acid substitution in the fingers subdomain to increase strand-displacement activity of A-family prokaryotic DNA polymerases

BackgroundThe discovery of thermostable DNA polymerases such as Taq DNA polymerase revolutionized amplification of DNA by polymerase chain reaction methods that rely on thermal cycling for strand separation. These methods are widely used in the laboratory for medical research, clinical diagnostics, criminal forensics and general molecular biology research. Today there is a growing demand for on-site molecular diagnostics; so-called ‘Point-of-Care tests’. Isothermal nucleic acid amplification techniques do not require a thermal cycler making these techniques more suitable for performing Point-of-Care tests at ambient temperatures compared to traditional polymerase chain reaction methods. Strand-displacement activity is essential for such isothermal nucleic acid amplification; however, the selection of DNA polymerases with inherent strand-displacement activity that are capable of performing DNA synthesis at ambient temperatures is currently limited.ResultsWe have characterized the large fragment of a DNA polymerase I originating from the marine psychrophilic bacterium Psychrobacillus sp. The enzyme showed optimal polymerase activity at pH 8–9 and 25–110 mM NaCl/KCl. The polymerase was capable of performing polymerase as well as robust strand-displacement DNA synthesis at ambient temperatures (25–37 °C). Through molecular evolution and screening of thousand variants we have identified a single amino-acid exchange of Asp to Ala at position 422 which induced a 2.5-fold increase in strand-displacement activity of the enzyme.Transferring the mutation of the conserved Asp residue to corresponding thermophilic homologues from Ureibacillus thermosphaericus and Geobacillus stearothermophilus also resulted in a significant increase in the strand-displacement activity of the enzymes.ConclusionsSubstituting Asp with Ala at positon 422 resulted in a significant increase in strand-displacement activity of three prokaryotic A-family DNA polymerases adapted to different environmental temperatures i.e. being psychrophilic and thermophilic of origin. This strongly indicates an important role for the 422 position and the O1-helix for strand-displacement activity of DNA polymerase I. The D422A variants generated here may be highly useful for isothermal nucleic acid amplification at a wide temperature scale.

Preliminary analysis of New Zealand scampi (Metanephrops challengeri) diet using metabarcoding.

Deep sea lobsters are highly valued for seafood and provide the basis of important commercial fisheries in many parts of the world. Despite their economic significance, relatively little is known about their natural diets. Microscopic analyses of foregut content in some species have suffered from low taxonomic resolution, with many of the dietary items difficult to reliably identify as their tissue is easily digested. DNA metabarcoding has the potential to provide greater taxonomic resolution of the diet of the New Zealand scampi (Metanephrops challengeri) through the identification of gut contents, but a number of methodological concerns need to be overcome first to ensure optimum DNA metabarcoding results. In this study, a range of methodological parameters were tested to determine the optimum protocols for DNA metabarcoding, and provide a first view of M. challengeri diet. Several PCR protocols were tested, using two universal primer pairs targeting the 18S rRNA and COI genes, on DNA extracted from both frozen and ethanol preserved samples for both foregut and hindgut digesta. The selection of appropriate DNA polymerases, buffers and methods for reducing PCR inhibitors (including the use of BSA) were found to be critical. Amplification from frozen or ethanol preserved gut contents appeared similarly dependable. The COI gene was found to be more effective than 18S rRNA gene for identifying large eukaryotic taxa from the digesta; however, it was less successfully amplified. The 18S rRNA gene was more easily amplified, but identified mostly smaller marine organisms such as plankton and parasites. This preliminary analysis of the diet of M. challengeri identified a range of species (13,541 reads identified as diet), which included the ghost shark (Hydrolagus novaezealandiae), silver warehou (Seriolella punctata), tall sea pen (Funiculina quadrangularis) and the salp (Ihlea racovitzai), suggesting that they have a varied diet, with a high reliance on scavenging a diverse range of pelagic and benthic species from the seafloor.

Compartmentalized Self-Replication for Evolution of a DNA Polymerase.

Compartmentalized self-replication (CSR) is an emulsion PCR-based method for the selection of DNA polymerases. E. coli host cells expressing a library of DNA polymerases are emulsified so that no more than a single cell is present in a single emulsion droplet. In a subsequent emulsion PCR step, the DNA polymerase protein, as well as the plasmid encoding it are released into the emulsion droplet and the genes that created the most active or abundant polymerase variants are exponentially amplified and can be passed to the next round of CSR. CSR is a powerful method for engineering of polymerases since it allows selection under a variety of conditions, including the use of non-standard substrates. In this unit, we provide a step-by-step procedure for the selection of polymerases, using as an example the selection of reverse transcriptase activity starting from a library of Thermococcus kodakaraensis (KOD) DNA polymerase variants. © 2018 by John Wiley & Sons, Inc.

Rapid evolution of acetic acid-detoxifying Escherichia coli under phosphate starvation conditions requires activation of the cryptic PhnE permease and induction of translesion synthesis DNA polymerases.

Escherichia coli incubated in phosphate-limiting minimal medium dies during prolonged incubation as a result of the production of acetic acid. Variants that consume acetic acid generally sweep through the population after three serial cultures. Evolvability may primarily result from induction of the potentially mutagenic LexA DNA damage response or from growth of preexisting mutants. Cells starved of phosphate induce the LexA regulon through a unique mechanism based on an increase in the internal pH at the approach of the stationary phase. Evolved cells resume growth on phosphorylated products as a result of the activation of the cryptic PhnE permease. Here, it is shown that first PhnE-expressing revertants swept through starved populations independently of the expression of the LexA regulon. Induction of the LexA regulon and especially of the translesion synthesis DNA polymerases Pol IV and Pol V was, however, absolutely required for the ultimate evolution of acetic acid-detoxifying mutant strains. Both growth under selection and induction of translesion synthesis DNA polymerases are therefore required for adaptive evolution under phosphate starvation conditions.

Andreas Marx

Andreas Marx

KlenTaq DNA Polymerase Adopts Unique Recognition States when Encountering Matched, Mismatched, and Abasic Template Sites: An NMR Study

The overall fidelity of DNA synthesis, and thus the accuracy of genome replication in general, is governed by the fundamental intrinsic fidelity of DNA polymerases. ) These enzymes catalyze the template-directed addition of nucleotides to the 3'-end of a DNA primer strand with high fidelity.(2) In the process, DNA polymerases contribute actively to nucleotide selection by introducing only correct nucleotides into the growing primer strand with high efficiencies. A currently used model for DNA polymerase selectivity is based on geometric complementarity of the nascent nucleobase pair. Correctly matching nucleotides are readily accepted and arranged for phosphodiester bond formation whereas incorrect 2'-deoxynucleoside triphosphates (dNTPs) are processed with lower efficiency. The discrimination in the early steps of nucleotide incorporation is thus governed by a series of conformational alterations leading from a conformation with an open, solvent-exposed active site to a closed conformation.) DNA polymerases are also characterized by their varying abilities to tolerate DNA template modifications and lesions.) $0 far, X-ray crystallography and fluorescencebased studies have predominantly been used to gain insight into these processesP) Although these methods have been very valuable for our current understanding of DNA polymerase mechanisms, there are still some drawbacks. Fluorescence-based studies, for instance, require the incorporation of bulky fluorescent probes that are susceptible to hydrophobic or stacking interactions, potentially introducing non-natural effects and masking natural interactions. Additionally, with each pair of fluorescent probes only a restricted subset of dynamic properties out of a highly complex overall enzymatic process can be studied. Crystallization trials, on the other hand, are performed under non-natural conditions and crystal packing effects can contribute to the nature of the 3D structure. Additionally, structural heterogeneity might prevent crystallization of certain states completely. In conclusion, for our understanding of DNA polymerase mechanisms and to minimize the risk of misinterpretation it is absolutely crucial to combine a whole set of methods. Interestingly, NMR has been exploited only sparsely for studying nucleotide polymerase mechanisms.IS) However, residue-specific isotopic labeling retains an enzyme in its native, unperturbed state with multiple probes for investigating global and local dynamics simultar)eously. Further-

Structures of DNA Polymerases Caught Processing Size-Augmented Nucleotide Probes

The integrity of the genome relies primarily on the ability of DNA polymerases to efficiently catalyze selective DNA synthesis according to the Watson–Crick rule in a templatedirected manner during DNA replication, repair, and recombination. Remarkably, some DNA polymerases achieve selective information transfer to the offspring in line with the Watson–Crick rule with intrinsic error rates as low as one mistake per one million synthesized nucleotides. This is far below the value that would be expected based on the energetic differences between canonical (i.e. Watson–Crick) and noncanonical nucleobase pairing. Geometric factors are widely cited to govern DNA polymerase selectivity. Thus, high-fidelity DNA polymerases are believed to mostly select the canonical nucleotide based on close steric complementarity of the nascent base pair to the geometry of the active site of the enzyme. Synthetic nucleotide analogues with tailored new properties are widely used to investigate the mechanisms that govern DNA polymerase selectivity. In this context nucleotides with hydrophobic nucleobase isosteres were developed to study the contribution of hydrogen bonding to DNA polymerase selectivity. 3] To investigate the steric effects in interactions between DNA polymerase and its substrate, we have developed 4’-alkyl-modified nucleotides with increasing steric demand. The modifications were introduced in the sugar residues since structural and functional data of DNA polymerases show that the sugar residues of nucleotides are involved in substrate recognition. These interactions may provide the enzymes with additional paths for achieving selectivity besides editing nucleobase geometry. Alkyl groups were employed since their effects on the hydrogen-bonding patterns and conformations of the nucleotides would be minimized. By employing these size-augmented nucleotides in functional DNA polymerase studies we could evaluate varied steric effects on DNA polymerase selectivity. Herein we report the first crystal structures of size-augmented 4’methylated and 4’-ethylated thymidine triphosphates in complex with a DNA polymerase. Significant mechanistic insights into nucleotide incorporation during DNA polymerization were derived from the high-resolution crystal structures reported by Waksman and colleagues of KlenTaq, an N-terminally truncated form of DNA polymerase from Thermus aquaticus. KlenTaq is a member of the family A DNA polymerases that play a role in prokaryotic and eukaryotic DNA replication and repair. Since data on the action of KlenTaq on 4’-alkylated thymidine-5’-triphosphates (dTTPs) is lacking, we first studied nucleotide incorporation by employing transient kinetic analysis with quench–flow technology (Table 1). Our data

Opposed Steric Constraints in Human DNA Polymerase β and E. coli DNA Polymerase I

DNA polymerase selectivity is crucial for the survival of any living species, yet varies significantly among different DNA polymerases. Errors within DNA polymerase-catalyzed DNA synthesis result from the insertion of noncanonical nucleotides and extension of misaligned DNA substrates. The substrate binding characteristics among DNA polymerases are believed to vary in properties such as shape and tightness of the binding pocket, which might account for the observed differences in fidelity. Here, we employed 4'-alkylated nucleotides and primer strands bearing 4'-alkylated nucleotides at the 3'-terminal position as steric probes to investigate differential active site properties of human DNA polymerase beta (Pol beta) and the 3'-->5'-exonuclease-deficient Klenow fragment of E. coli DNA polymerase I (KF(exo-)). Transient kinetic measurements indicate that both enzymes vary significantly in active site tightness at both positions. While small 4'-methyl and -ethyl modifications of the nucleoside triphosphate perturb Pol beta catalysis, extension of modified primer strands is only marginally affected. Just the opposite was observed for KF(exo-). Here, incorporation of the modified nucleotides is only slightly reduced, whereas size augmentation of the 3'-terminal nucleotide in the primer reduces the catalytic efficiency by more than 7000- and 260,000-fold, respectively. NMR studies support the notion that the observed effects derive from enzyme substrate interactions rather than inherent properties of the modified substrates. These findings are consistent with the observed differential capability of the investigated DNA polymerases in fidelity such as processing misaligned DNA substrates. The results presented provide direct evidence for the involvement of varied steric effects among different DNA polymerases on their fidelity.

Varied Active‐Site Constraints in the Klenow Fragment of E. coli DNA Polymerase I and the Lesion‐Bypass Dbh DNA Polymerase

We report on comparative pre-steady-state kinetic analyses of exonuclease-deficient Escherichia coli DNA polymerase I (Klenow fragment, KF-) and the archaeal Y-family DinB homologue (Dbh) of Sulfolobus solfataricus. We used size-augmented sugar-modified thymidine-5'-triphosphate (T(R)TP) analogues to test the effects of steric constraints in the active sites of the polymerases. These nucleotides serve as models for study of DNA polymerases exhibiting both relatively high and low intrinsic selectivity. Substitution of a hydrogen atom at the 4'-position in the nucleotide analogue by a methyl group reduces the maximum rate of nucleotide incorporation by about 40-fold for KF- and about twelve fold for Dbh. Increasing the size to an ethyl group leads to a further twofold reduction in the rates of incorporation for both enzymes. Interestingly, the affinity of KF- for the modified nucleotides is only marginally affected, which would indicate no discrimination during the binding step. Dbh even has a higher affinity for the modified analogues than it does for the natural substrate. Misincorporation of either TTP or T(Me)TP opposite a G template causes a drastic decline in incorporation rates for both enzymes. At the same time, the binding affinities of KF- for these nucleotides drop by about 16- and fourfold, respectively, whereas Dbh shows only a twofold reduction. Available structural data for ternary complexes of relevant DNA polymerases indicate that both enzymes make close contacts with the sugar moiety of the dNTP. Thus, the varied proficiencies of the two enzymes in processing the size-augmented probes indicate varied flexibility of the enzymes' active sites and support the notion of active site tightness being a criterion for DNA polymerase selectivity.

Chemical Biology of DNA Polymerases: From Selectivity to New Functions

DNA polymerases are involved in all DNA synthesis occurring in nature. Furthermore, DNA polymerases are the workhorses in numerous important molecular biological core technologies like the ubiquitous polymerase chain reaction (PCR), cDNA cloning, genome sequencing and nucleic acids based diagnostics. In order to identify DNA polymerase mutants with altered properties, we set up an efficient high through put setup to rapidly screen libraries of DNA polymerase mutants in automated parallel fashion and identified entities with significantly increased selectivity. Furthermore, our results indicate a rational to generally increase DNA polymerase selectivity as we demonstrate for several enzymes from different DNA polymerase families. Additionally, we show that the generation of a new DNA polymerase function is achievable through iterative screening of small libraries of DNA polymerase derived by randomization of the respective genes. We demonstrate that the identified mutants find immediate applications and provide the basis for the development of new means for diagnostic technologies.

Directed DNA Polymerase Evolution: Effects of Mutations in Motif C on the Mismatch‐Extension Selectivity of Thermus aquaticus DNA Polymerase

The selectivity of DNA polymerases for processing the canonical nucleotide and DNA substrate in favor of the noncanonical ones is the key to the integrity of the genome of every living species and to many biotechnological applications. The inborn ability of most DNA polymerases to abort efficient extension of mismatched DNA substrates adds to the overall DNA polymerase selectivity. DNA polymerases have been grouped into families according to their sequence. Within family A DNA polymerases, six motifs that come into contact with the substrates and form the active site have been discovered to be evolutionary highly conserved. Here we present results obtained from amino acid randomization within one motif, motif C, of thermostable Thermus aquaticus DNA polymerase. We have identified several distinct mutation patterns that increase the selectivity of mismatch extension. These results might lead to direct applications such as allele-specific PCR, as demonstrated by real-time PCR experiments and add to our understanding of DNA polymerase selectivity.

Hydrophobic Amino Acid and Single-Atom Substitutions Increase DNA Polymerase Selectivity

DNA polymerase fidelity is of immense biological importance due to the fundamental requirement for accurate DNA synthesis in both replicative and repair processes. Subtle hydrogen-bonding networks between DNA polymerases and their primer/template substrates are believed to have impact on DNA polymerase selectivity. We show that deleting defined interactions of that kind by rationally designed hydrophobic substitution mutations can result in a more selective enzyme. Furthermore, a single-atom replacement within the DNA substrate through chemical modification, which leads to an altered acceptor potential and steric demand of the DNA substrate, further increased the selectivity of the developed systems. Accordingly, this study about the impact of hydrophobic alterations on DNA polymerase selectivity--enzyme and substrate wise--further highlights the relevance of shape complementary and polar interactions on DNA polymerase selectivity.

4′C‐Modified Nucleotides as Chemical Tools for Investigation and Modulation of DNA Polymerase Function

AbstractFor Abstract see ChemInform Abstract in Full Text.

4′C-Modified Nucleotides as Chemical Tools for Investigation and Modulation of DNA Polymerase Function

DNA polymerases catalyze the entire biological DNA synthesis in DNA replication, repair and recombination. A DNA polymerase is presented with a pool of four structurally similar dNTPs from which it must select the sole correct substrate for incorporation into the growing DNA strand. The mechanisms how these remarkable enzymes achieve this tremendous task is a matter of ­current interest and intensive discussion. Valuable new insights into complex DNA polymerase selectivity mechanisms were gained through employment of 4′C-alkylated nucleotide analogues in ­functional enzyme studies. First investigations indicate that the 4′C-modified substrates are promising for the development of faithful methods for genome diagnostics.

DNA Polymerase Template Interactions Probed by Degenerate Isosteric Nucleobase Analogs

The development of novel artificial nucleobases and detailed X-ray crystal structures for primer/template/DNA polymerase complexes provide opportunities to assess DNA-protein interactions that dictate specificity. Recent results have shown that base pair shape recognition in the context of DNA polymerase must be considered a significant component. The isosteric azole carboxamide nucleobases (compounds 1–5; Figure 1) differ only in the number and placement of nitrogen atoms within a common shape and therefore present unique electronic distributions that are shown to dictate the selectivity of template-directed nucleotide incorporation by DNA polymerases. The results demonstrate how nucleoside triphosphate substrate selection by DNA polymerase is a complex phenomenon involving electrostatic interactions in addition to hydrogen bonding and shape recognition. These azole nucleobase analogs offer unique molecular tools for probing nonbonded interactions dictating substrate selection and fidelity of DNA polymerases.

Substrates for Investigation of DNA Polymerase Function: Synthesis and Properties of 4′‐C‐Alkylated Oligonucleotides

AbstractIn addition to their potential as diagnostic and therapeutic agents, modified oligonucleotides have also been shown to be highly valuable tools for examination of complex biological processes. Carefully designed nucleotide analogues have therefore found considerable application in investigations of DNA polymerase function and mechanism. To examine the contribution of primarily steric constraints on DNA polymerase selectivity, we have developed a new functional strategy based on the use of modified nucleotide analogues that differ primarily in their steric demand. Here we report the efficient synthesis of modified thymidine analogues bearing 4′‐alkyl groups with varying steric demand, the effects of 4′‐alkylation on sugar puckering, and the incorporation of these analogues into oligonucleotides by use of automated solid‐phase DNA synthesis. We also studied the pairing properties of 4′‐alkylated oligonucleotide duplexes in relation to those of their natural counterparts under a range of buffer conditions. In general, our studies indicate that 4′‐alkylation of thymidines has little effect on nucleoside and oligonucleotide conformation. These results have relevance to the previously reported action of 4′‐alkylated nucleotides and oligonucleotides as probes of DNA polymerase function and mechanism. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)

Selection of novel eukaryotic DNA polymerases by mutagenesis and genetic complementation of yeast.

AbstractDNA-directed DNA polymerases have been broadly classified into seven families based on their sequence homology (1). It is surprising to learn that enzymes such as DNA polymerases, which carry out pivotal role during DNA replication, repair, and recombination, are poorly conserved amongst different families, but within a given family, all the members are highly conserved. These observations have profound implications and suggest that DNA polymerases have been plastic during evolution, but can tolerate multiple mutations (2). The mutability of DNA polymerases has been utilized extensively in our studies and has shed light on structure-function relationships of each domain. Any single amino acid residue or the entire domain can be randomly mutagenized and the active mutants can be selected by genetic complementation. Here we describe the complementation of Saccharomyces cerevisiae Pol3 (Pol δ) by utilizing a common technique in yeast genetics known as “plasmid shuffling,” where the wild-type copy of the Pol3 present in a Ura3 selective marker plasmid is exchanged or genetically complemented for in vitro mutated version(s) of Pol3 in the domain-of-interest. Since Pol3p is essential for viability of yeast, only those mutants that genetically complement the loss of wild-type Pol3p survive. KeywordsYeast StrainMultiple Cloning SiteGenetic ComplementationPlasmid RescueUra3 MarkerThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Implications of active site constraints on varied DNA polymerase selectivity.

DNA polymerase selectivity often varies significantly depending on the DNA polymerase. The origin of this varying error propensity is elusive. It is assumed that DNA polymerases form nucleotide binding pockets that differ in properties such as shape and tightness. We tested this prediction and studied HIV-1 RT by employment of size-augmented nucleotides and site-directed mutagenesis of the enzyme. New valuable insights into the mechanism of DNA polymerase fidelity were obtained. The presented study provides experimental evidence that variations of steric constraints within the nucleotide binding pocket of at least two DNA polymerases cause variations in nucleotide incorporation selectivity. Thus, our results support the concept of active site tightness as a causative in differential fidelity among DNA polymerases.

DNA Polymerase Selectivity: Sugar Interactions Monitored with High-Fidelity Nucleotides This work was supported by a grant from the Deutsche Forschungsgemeinschaft. We thank Professor Dr. Michael Famulok for his continuing support.

Do enzyme–sugar interactions contribute to DNA-polymerase fidelity? With the novel chemical probes 1 (R=CH3, CH2CH3, CH(CH3)2, CH2CH(CH3)2), which are used by a DNA polymerase more selectively than the natural counterpart (R=H), insights are obtained into the influence of enzyme–sugar interactions on the selectivity of the enzyme.

Selection of bacteriophage T4 antimutator DNA polymerases: a link between proofreading and sensitivity to phosphonoacetic acid

During DNA replication, DNA polymerases alternate between DNA synthesis and proofreading the newly synthesized DNA. In order to understand the molecular details of how DNA polymerases determine the balance between polymerase and proofreading activities, it would be useful to have mutants which switch between the two activities either more or less frequently. Antimutator DNA polymerases switch more frequently and thus have more opportunity for proofreading. We have observed that mutant DNA polymerases which proofread less frequently have a mutator phenotype and are inhibited by the pyrophosphate analogue phosphonoacetic acid. Sensitivity to phosphonoacetic acid can be used to isolate second-site suppressor mutations. These suppressor mutations encode amino acid substitutions which produce antimutator DNA polymerases.