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1
McInerney, Peter, Paul Adams, and Masood Z. Hadi. "Error Rate Comparison during Polymerase Chain Reaction by DNA Polymerase." Molecular Biology International 2014 (August 17, 2014): 1–8. http://dx.doi.org/10.1155/2014/287430.
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As larger-scale cloning projects become more prevalent, there is an increasing need for comparisons among high fidelity DNA polymerases used for PCR amplification. All polymerases marketed for PCR applications are tested for fidelity properties (i.e., error rate determination) by vendors, and numerous literature reports have addressed PCR enzyme fidelity. Nonetheless, it is often difficult to make direct comparisons among different enzymes due to numerous methodological and analytical differences from study to study. We have measured the error rates for 6 DNA polymerases commonly used in PCR applications, including 3 polymerases typically used for cloning applications requiring high fidelity. Error rate measurement values reported here were obtained by direct sequencing of cloned PCR products. The strategy employed here allows interrogation of error rate across a very large DNA sequence space, since 94 unique DNA targets were used as templates for PCR cloning. The six enzymes included in the study, Taq polymerase, AccuPrime-Taq High Fidelity, KOD Hot Start, cloned Pfu polymerase, Phusion Hot Start, and Pwo polymerase, we find the lowest error rates with Pfu, Phusion, and Pwo polymerases. Error rates are comparable for these 3 enzymes and are >10x lower than the error rate observed with Taq polymerase. Mutation spectra are reported, with the 3 high fidelity enzymes displaying broadly similar types of mutations. For these enzymes, transition mutations predominate, with little bias observed for type of transition.
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Pospiech, Helmut, and Juhani E. Syväoja. "DNA Polymerase e - More Than a Polymerase." Scientific World JOURNAL 3 (2003): 87–104. http://dx.doi.org/10.1100/tsw.2003.08.
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This paper presents a comprehensive review of the structure and function of DNA polymerase e. Together with DNA polymerases a and d, this enzyme replicates the nuclear DNA in the eukaryotic cell. During this process, DNA polymerase a lays down RNA-DNA primers that are utilized by DNA polymerases d and e for the bulk DNA synthesis. Attempts have been made to assign these two enzymes specifically to the synthesis of the leading and the lagging strand. Alternatively, the two DNA polymerases may be needed to replicate distinct regions depending on chromatin structure. Surprisingly, the essential function of DNA polymerase e does not depend on its catalytic activity, but resides in the nonenzymatic carboxy-terminal domain. This domain not only mediates the interaction of the catalytic subunit with the three smaller regulatory subunits, but also links the replication machinery to the S phase checkpoint. In addition to its role in DNA replication, DNA polymerase e fulfils roles in the DNA synthesis step of nucleotide excision and base excision repair, and has been implicated in recombinational processes in the cell.
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McDonald, John P., Agnès Tissier, Ekaterina G. Frank, Shigenori Iwai, Fumio Hanaoka, and Roger Woodgate. "DNA polymerase iota and related Rad30–like enzymes." Philosophical Transactions of the Royal Society of London. Series B: Biological Sciences 356, no. 1405 (January 29, 2001): 53–60. http://dx.doi.org/10.1098/rstb.2000.0748.
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Until recently, the molecular mechanisms of translesion DNA synthesis (TLS), a process whereby a damaged base is used as a template for continued replication, was poorly understood. This area of scientific research has, however, been revolutionized by the finding that proteins long implicated in TLS are, in fact, DNA polymerases. Members of this so–called UmuC/DinB/Rev1/Rad30 superfamily of polymerases have been identified in prokaryotes, eukaryotes and archaea. Biochemical studies with the highly purified polymerases reveal that some, but not all, can traverse blocking lesions in template DNA. All of them share a common feature, however, in that they exhibit low fidelity when replicating undamaged DNA. Of particular interest to us is the Rad30 subfamily of polymerases found exclusively in eukaryotes. Humans possess two Rad30 paralogs, Rad30A and Rad30B. The RAD30A gene encodes DNA polymerase η and defects in the protein lead to the xeroderma pigmentosum variant (XP–V) phenotype in humans. Very recently RAD30B has also been shown to encode a novel DNA polymerase, designated as Pol ι. Based upon in vitro studies, it appears that Pol ι has the lowest fidelity of any eukaryotic polymerase studied to date and we speculate as to the possible cellular functions of such a remarkably error–prone DNA polymerase.
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MIZUSHINA, Yoshiyuki, Akira IIDA, Keisuke OHTA, Fumio SUGAWARA, and Kengo SAKAGUCHI. "Novel triterpenoids inhibit both DNA polymerase and DNA topoisomerase." Biochemical Journal 350, no. 3 (September 8, 2000): 757–63. http://dx.doi.org/10.1042/bj3500757.
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As described previously, we found that new triterpenoid compounds, designated fomitellic acids A and B, which selectively inhibit the activities of mammalian DNA polymerases α and β [Mizushina, Tanaka, Kitamura, Tamai, Ikeda, Takemura, Sugawara, Arai, Matsukage, Yoshida and Sakaguchi (1998) Biochem. J. 330, 1325–1332; Tanaka, Kitamura, Mizushina, Sugawara and Sakaguchi (1998) J. Nat. Prod. 61, 193–197] and that a known triterpenoid, ursolic acid, is an inhibitor of human DNA topoisomerases I and II (A. Iida, Y. Mizushina and K. Sakaguchi, unpublished work). Here we report that all of these triterpenoids are potent inhibitors of calf DNA polymerase α, rat DNA polymerase β and human DNA topoisomerases I and II, and show moderate inhibitory effects on plant DNA polymerase II and human immunodeficiency virus reverse transcriptase. However, these compounds did not influence the activities of prokaryotic DNA polymerases such as Escherichia coli DNA polymerase I or other DNA metabolic enzymes such as human telomerase, T7 RNA polymerase and bovine deoxyribonuclease I. These triterpenoids were not only mammalian DNA polymerase inhibitors but also inhibitors of DNA topoisomerases I and II even though the enzymic characteristics of DNA polymerases and DNA topoisomerases, including their modes of action, amino acid sequences and three-dimensional structures, differed markedly. These triterpenoids did not bind to DNA, suggesting that they act directly on these enzymes. Because the three-dimensional structures of fomitellic acids were shown by computer simulation to be very similar to that of ursolic acid, the DNA-binding sites of both enzymes, which compete for the inhibitors, might be very similar. Fomitellic acid A and ursolic acid prevented the growth of NUGC cancer cells, with LD50 values of 38 and 30µM respectively.
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Wright, G. E. "Nucleotide probes of DNA polymerases." Acta Biochimica Polonica 43, no. 1 (March 31, 1996): 115–24. http://dx.doi.org/10.18388/abp.1996_4522.
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The modified nucleotides, N2-(p-n-butylphenyl)dGTP and 2-(p-n-butylanilino) dATP and related compounds have been developed as inhibitor-probes of B family DNA polymerases. Synthetic approaches to these compounds are summarized. The nucleotides are potent, non-substrate inhibitors of DNA polymerase a. In contrast, they inhibit other members of the family with less potency but act as substrates for these enzymes. Modelling of the inhibitor: enzyme binding mechanism has been done based on the known structure of E. coli DNA polymerase I, and site-directed mutagenesis experiments to evaluate this mechanism are proposed.
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Makioka, A., B. Stavros, J. T. Ellis, and A. M. Johnson. "Detection and characterization of DNA polymerase activity in Toxoplasma gondii." Parasitology 107, no. 2 (August 1993): 135–39. http://dx.doi.org/10.1017/s0031182000067238.
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SUMMARYA DNA polymerase activity has been detected and characterized in crude extracts from tachzoites of Toxoplasma gondii. The enzyme has a sedimentation coefficient of 6·4 S, corresponding to an approximate molecular weight of 150000 assuming a globular shape. Like mammalian DNA polymerase α, the DNA polymerase of T. gondii was sensitive to N-ethylmaleimide and inhibited by high ionic strength. However, the enzyme activity was not inhibited by aphidicolin which is an inhibitor of mammalian DNA polymerases α, δ and ε and also cytosine-β-D-arabinofuranoside-5′-triphosphate which is an inhibitor of α polymerase. The activity was inhibited by 2′,3′-dideoxythymidine-5′-triphosphate which is an inhibitor of mammalian DNA polymerase β and γ. Magnesium ions (Mg2+) were absolutely required for activity and its optimal concentration was 6 mM. The optimum potassium (K+) concentration was 50 mM and a higher concentration of K+ markedly inhibited the activity. Activity was optimal at pH 8. Monoclonal antibodies against human DNA polymerase did not bind to DNA polymerase of T. gondii. Thus the T. gondii enzyme differs from the human enzymes and may be a useful target for the design of toxoplasmacidal drugs.
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Mattila, P., J. Korpela, T. Tenkanen, and K. Pitkämem. "Fidelity of DNA synthesis by the Thermococcus litoralis DNA polymerase—an extremely heat stable enzyme with proofreading activity." Nucleic Acids Research 19, no. 18 (September 25, 1991): 4967–73. http://dx.doi.org/10.1093/nar/19.18.4967.
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Abstract We demonstrate that the DNA polymerase isolated from Thermococcus litoralis (VentTM DNA polymerase) is the first thermostable DNA polymerase reported having a 3′—5′ proofreading exonuclease activity. This facilitates a highly accurate DNA synthesis in vitro by the polymerase. Mutational frequencies observed in the base substitution fidelity assays were in the range of 30×10−6. These values were 5–10 times lower compared to other thermostable DNA polymerases lacking the proofreading activity. All classes of DNA polymerase errors (transitions, transversions, frameshift mutations) were assayed using the forward mutational assay (1). The mutation frequencies of Thermococcus litoralis DNA polymerase varied between 15−35×10−4 being 2 – 4 times lower than the respective values obtained using enzymes without proofreading activity. We also noticed that the fidelity of the DNA polymerase from Thermococcus litoralis responds to changes in dNTP concentration, units of enzyme used per one reaction and the concentration of MgSO4 relative to the total concentration of dNTPs present in the reaction. The high fidelity DNA synthesis In vitro by Thermococcus litoralis DNA polymerase provides good possibilities for maintaining the genetic information of original target DNA sequences intact in the DNA amplification applications.
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Pezo, Valerie, Faten Jaziri, Pierre-Yves Bourguignon, Dominique Louis, Deborah Jacobs-Sera, Jef Rozenski, Sylvie Pochet, et al. "Noncanonical DNA polymerization by aminoadenine-based siphoviruses." Science 372, no. 6541 (April 29, 2021): 520–24. http://dx.doi.org/10.1126/science.abe6542.
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Bacteriophage genomes harbor the broadest chemical diversity of nucleobases across all life forms. Certain DNA viruses that infect hosts as diverse as cyanobacteria, proteobacteria, and actinobacteria exhibit wholesale substitution of aminoadenine for adenine, thereby forming three hydrogen bonds with thymine and violating Watson-Crick pairing rules. Aminoadenine-encoded DNA polymerases, homologous to the Klenow fragment of bacterial DNA polymerase I that includes 3′-exonuclease but lacks 5′-exonuclease, were found to preferentially select for aminoadenine instead of adenine in deoxynucleoside triphosphate incorporation templated by thymine. Polymerase genes occur in synteny with genes for a biosynthesis enzyme that produces aminoadenine deoxynucleotides in a wide array of Siphoviridae bacteriophages. Congruent phylogenetic clustering of the polymerases and biosynthesis enzymes suggests that aminoadenine has propagated in DNA alongside adenine since archaic stages of evolution.
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Kropp, Heike Maria, Simon Leonard Dürr, Christine Peter, Kay Diederichs, and Andreas Marx. "Snapshots of a modified nucleotide moving through the confines of a DNA polymerase." Proceedings of the National Academy of Sciences 115, no. 40 (September 17, 2018): 9992–97. http://dx.doi.org/10.1073/pnas.1811518115.
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DNA polymerases have evolved to process the four canonical nucleotides accurately. Nevertheless, these enzymes are also known to process modified nucleotides, which is the key to numerous core biotechnology applications. Processing of modified nucleotides includes incorporation of the modified nucleotide and postincorporation elongation to proceed with the synthesis of the nascent DNA strand. The structural basis for postincorporation elongation is currently unknown. We addressed this issue and successfully crystallized KlenTaq DNA polymerase in six closed ternary complexes containing the enzyme, the modified DNA substrate, and the incoming nucleotide. Each structure shows a high-resolution snapshot of the elongation of a modified primer, where the modification “moves” from the 3′-primer terminus upstream to the sixth nucleotide in the primer strand. Combining these data with quantum mechanics/molecular mechanics calculations and biochemical studies elucidates how the enzyme and the modified substrate mutually modulate their conformations without compromising the enzyme’s activity significantly. The study highlights the plasticity of the system as origin of the broad substrate properties of DNA polymerases and facilitates the design of improved systems.
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Bebenek, Anna, Geraldine T. Carver, Holly Kloos Dressman, Farid A. Kadyrov, Joseph K. Haseman, Vasiliy Petrov, William H. Konigsberg, Jim D. Karam, and John W. Drake. "Dissecting the Fidelity of Bacteriophage RB69 DNA Polymerase: Site-Specific Modulation of Fidelity by Polymerase Accessory Proteins." Genetics 162, no. 3 (November 1, 2002): 1003–18. http://dx.doi.org/10.1093/genetics/162.3.1003.
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Abstract Bacteriophage RB69 encodes a replicative B-family DNA polymerase (RB69 gp43) with an associated proofreading 3′ exonuclease. Crystal structures have been determined for this enzyme with and without DNA substrates. We previously described the mutation rates and kinds of mutations produced in vivo by the wild-type (Pol+ Exo+) enzyme, an exonuclease-deficient mutator variant (Pol+ Exo-), mutator variants with substitutions at Tyr567 in the polymerase active site (PolM Exo+), and the double mutator PolM Exo-. Comparing the mutational spectra of the Pol+ Exo- and Pol+ Exo+ enzymes revealed the patterns and efficiencies of proofreading, while Tyr567 was identified as an important determinant of base-selection fidelity. Here, we sought to determine how well the fidelities of the same enzymes are reflected in vitro. Compared to their behavior in vivo, the three mutator polymerases exhibited modestly higher mutation rates in vitro and their mutational predilections were also somewhat different. Although the RB69 gp43 accessory proteins exerted little or no effect on total mutation rates in vitro, they strongly affected mutation rates at many specific sites, increasing some rates and decreasing others.
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Gill, Joshua P., Jun Wang, and David P. Millar. "DNA polymerase activity at the single-molecule level." Biochemical Society Transactions 39, no. 2 (March 22, 2011): 595–99. http://dx.doi.org/10.1042/bst0390595.
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DNA polymerases are essential enzymes responsible for replication and repair of DNA in all organisms. To replicate DNA with high fidelity, DNA polymerases must select the correct incoming nucleotide substrate during each cycle of nucleotide incorporation, in accordance with the templating base. When an incorrect nucleotide is sometimes inserted, the polymerase uses a separate 3′→5′ exonuclease to remove the misincorporated base (proofreading). Large conformational rearrangements of the polymerase–DNA complex occur during both the nucleotide incorporation and proofreading steps. Single-molecule fluorescence spectroscopy provides a unique tool for observation of these dynamic conformational changes in real-time, without the need to synchronize a population of DNA–protein complexes.
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Coggins, Si'Ana A., Bijan Mahboubi, Raymond F. Schinazi, and Baek Kim. "Mechanistic cross-talk between DNA/RNA polymerase enzyme kinetics and nucleotide substrate availability in cells: Implications for polymerase inhibitor discovery." Journal of Biological Chemistry 295, no. 39 (July 31, 2020): 13432–43. http://dx.doi.org/10.1074/jbc.rev120.013746.
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Enzyme kinetic analysis reveals a dynamic relationship between enzymes and their substrates. Overall enzyme activity can be controlled by both protein expression and various cellular regulatory systems. Interestingly, the availability and concentrations of intracellular substrates can constantly change, depending on conditions and cell types. Here, we review previously reported enzyme kinetic parameters of cellular and viral DNA and RNA polymerases with respect to cellular levels of their nucleotide substrates. This broad perspective exposes a remarkable co-evolution scenario of DNA polymerase enzyme kinetics with dNTP levels that can vastly change, depending on cell proliferation profiles. Similarly, RNA polymerases display much higher Km values than DNA polymerases, possibly due to millimolar range rNTP concentrations found in cells (compared with micromolar range dNTP levels). Polymerases are commonly targeted by nucleotide analog inhibitors for the treatments of various human diseases, such as cancers and viral pathogens. Because these inhibitors compete against natural cellular nucleotides, the efficacy of each inhibitor can be affected by varying cellular nucleotide levels in their target cells. Overall, both kinetic discrepancy between DNA and RNA polymerases and cellular concentration discrepancy between dNTPs and rNTPs present pharmacological and mechanistic considerations for therapeutic discovery.
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Amir, Muhammad, Sabeera Afzal, and Alia Ishaq. "PrimPol (Primase/Polymerase), replicating enzyme – A mini review." Pak-Euro Journal of Medical and Life Sciences 2, no. 4 (April 3, 2020): 89–92. http://dx.doi.org/10.31580/pjmls.v2i4.956.
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Polymerases were revealed first in 1970s. Most important to the modest perception the enzyme responsible for nuclear DNA replication that was pol , for DNA repair pol and for mitochondrial DNA replication pol DNA construction and renovation done by DNA polymerases, so directing both the constancy and discrepancy of genetic information. Replication of genome initiate with DNA template-dependent fusion of small primers of RNA. This preliminary phase in replication of DNA demarcated as de novo primer synthesis which is catalyzed by specified polymerases known as primases. Sixteen diverse DNA-synthesizing enzymes about human perspective are devoted to replication, reparation, mutilation lenience, and inconsistency of nuclear DNA. But in dissimilarity, merely one DNA polymerase has been called in mitochondria. It has been suggest that PrimPol is extremely acting the roles by re-priming DNA replication in mitochondria to permit an effective and appropriate way replication to be accomplished. Investigations from a numeral of test site have significantly amplified our appreciative of the role, recruitment and regulation of the enzyme during DNA replication. Though, we are simply just start to increase in value the versatile roles that play PrimPol in eukaryote.
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Blanca, Giuseppina, Emmanuelle Delagoutte, Nicolas Tanguy le gac, Neil P. Johnson, Giuseppe Baldacci, and Giuseppe Villani. "Accessory proteins assist exonuclease-deficient bacteriophage T4 DNA polymerase in replicating past an abasic site." Biochemical Journal 402, no. 2 (February 12, 2007): 321–29. http://dx.doi.org/10.1042/bj20060898.
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Replicative DNA polymerases, such as T4 polymerase, possess both elongation and 3′–5′ exonuclease proofreading catalytic activities. They arrest at the base preceding DNA damage on the coding DNA strand and specialized DNA polymerases have evolved to replicate across the lesion by a process known as TLS (translesion DNA synthesis). TLS is considered to take place in two steps that often require different enzymes, insertion of a nucleotide opposite the damaged template base followed by extension from the inserted nucleotide. We and others have observed that inactivation of the 3′–5′ exonuclease function of T4 polymerase enables TLS across a single site-specific abasic [AP (apurinic/apyrimidinic)] lesion. In the present study we report a role for auxiliary replicative factors in this reaction. When replication is performed with a large excess of DNA template over DNA polymerase in the absence of auxiliary factors, the exo− polymerase (T4 DNA polymerase deficient in the 3′–5′ exonuclease activity) inserts one nucleotide opposite the AP site but does not extend past the lesion. Addition of the clamp processivity factor and the clamp loader complex restores primer extension across an AP lesion on a circular AP-containing DNA substrate by the exo− polymerase, but has no effect on the wild-type enzyme. Hence T4 DNA polymerase exhibits a variety of responses to DNA damage. It can behave as a replicative polymerase or (in the absence of proofreading activity) as a specialized DNA polymerase and carry out TLS. As a specialized polymerase it can function either as an inserter or (with the help of accessory proteins) as an extender. The capacity to separate these distinct functions in a single DNA polymerase provides insight into the biochemical requirements for translesion DNA synthesis.
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Mohapatra, Satish C., and James T. Hsu. "Kinetics of deactivation for thermostable DNA polymerase enzymes." Biotechnology Techniques 10, no. 8 (August 1996): 569–72. http://dx.doi.org/10.1007/bf00157363.
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Murphy, Kelly, Hariyanto Darmawan, Amy Schultz, Elizabeth Fidalgo da Silva, and Linda J. Reha-Krantz. "A method to select for mutator DNA polymerase δs in Saccharomyces cerevisiae." Genome 49, no. 4 (April 1, 2006): 403–10. http://dx.doi.org/10.1139/g05-106.
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Proofreading DNA polymerases share common short peptide motifs that bind Mg2+ in the exonuclease active center; however, hydrolysis rates are not the same for all of the enzymes, which indicates that there are functional and likely structural differences outside of the conserved residues. Since structural information is available for only a few proofreading DNA polymerases, we developed a genetic selection method to identify mutant alleles of the POL3 gene in Saccharomyces cerevisiae, which encode DNA polymerase δ mutants that replicate DNA with reduced fidelity. The selection procedure is based on genetic methods used to identify "mutator" DNA polymerases in bacteriophage T4. New yeast DNA polymerase δ mutants were identified, but some mutants expected from studies of the phage T4 DNA polymerase were not detected. This would indicate that there may be important differences in the proofreading pathways catalyzed by the two DNA polymerases.Key words: DNA polymerase proofreading, genetic selection for mutator mutants, fidelity of DNA replication, yeast.
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Jozwiakowski, Stanislaw K., Sandra Kummer, and Kerstin Gari. "Human DNA polymerase delta requires an iron–sulfur cluster for high-fidelity DNA synthesis." Life Science Alliance 2, no. 4 (July 5, 2019): e201900321. http://dx.doi.org/10.26508/lsa.201900321.
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Replication of eukaryotic genomes relies on the family B DNA polymerases Pol α, Pol δ, and Pol ε. All of these enzymes coordinate an iron–sulfur (FeS) cluster, but the function of this cofactor has remained largely unclear. Here, we show that the FeS cluster in the catalytic subunit of human Pol δ is coordinated by four invariant cysteines of the C-terminal CysB motif. FeS cluster loss causes a partial destabilisation of the four-subunit enzyme, a defect in double-stranded DNA binding, and compromised polymerase and exonuclease activities. Importantly, complex stability, DNA binding, and enzymatic activities are restored in the presence of proliferating cell nuclear antigen. We further show that also more subtle changes to the FeS cluster-binding pocket that do not abolish FeS cluster binding can have repercussions on the distant exonuclease domain and render the enzyme error prone. Our data, hence, suggest that the FeS cluster in human Pol δ is an important cofactor that despite its C-terminal location, it has an impact on both DNA polymerase and exonuclease activities and can influence the fidelity of DNA synthesis.
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Jackson, Lynnette N., Nicholas Chim, Changhua Shi, and John C. Chaput. "Crystal structures of a natural DNA polymerase that functions as an XNA reverse transcriptase." Nucleic Acids Research 47, no. 13 (June 6, 2019): 6973–83. http://dx.doi.org/10.1093/nar/gkz513.
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Abstract Replicative DNA polymerases are highly efficient enzymes that maintain stringent geometric control over shape and orientation of the template and incoming nucleoside triphosphate. In a surprising twist to this paradigm, a naturally occurring bacterial DNA polymerase I member isolated from Geobacillus stearothermophilus (Bst) exhibits an innate ability to reverse transcribe RNA and other synthetic congeners (XNAs) into DNA. This observation raises the interesting question of how a replicative DNA polymerase is able to recognize templates of diverse chemical composition. Here, we present crystal structures of natural Bst DNA polymerase that capture the post-translocated product of DNA synthesis on templates composed entirely of 2′-deoxy-2′-fluoro-β-d-arabino nucleic acid (FANA) and α-l-threofuranosyl nucleic acid (TNA). Analysis of the enzyme active site reveals the importance of structural plasticity as a possible mechanism for XNA-dependent DNA synthesis and provides insights into the construction of variants with improved activity.
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Guo, Jingxu, Wenling Zhang, Alun R. Coker, Steve P. Wood, Jonathan B. Cooper, Shazeel Ahmad, Syed Ali, Naeem Rashid, and Muhummad Akhtar. "Structure of the family B DNA polymerase from the hyperthermophilic archaeonPyrobaculum calidifontis." Acta Crystallographica Section D Structural Biology 73, no. 5 (April 26, 2017): 420–27. http://dx.doi.org/10.1107/s2059798317004090.
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The family B DNA polymerase fromPyrobaculum calidifontis(Pc-polymerase) consists of 783 amino acids and is magnesium-ion dependent. It has an optimal pH of 8.5, an optimal temperature of 75°C and a half-life of 4.5 h at 95°C, giving it greater thermostability than the widely usedTaqDNA polymerase. The enzyme is also capable of PCR-amplifying larger DNA fragments of up to 7.5 kb in length. It was shown to have functional, error-correcting 3′–5′ exonuclease activity, as do the related high-fidelity DNA polymerases fromPyrococcus furiosus,Thermococcus kodakarensisKOD1 andThermococcus gorgonarius, which have extensive commercial applications.Pc-polymerase has a quite low sequence identity of approximately 37% to these enzymes, which, in contrast, have very high sequence identity to each other, suggesting that theP. calidifontisenzyme is distinct. Here, the structure determination ofPc-polymerase is reported, which has been refined to anRfactor of 24.47% and anRfreeof 28.81% at 2.80 Å resolution. The domains of the enzyme are arranged in a circular fashion to form a disc with a narrow central channel. One face of the disc has a number of connected crevices in it, which allow the protein to bind duplex and single-stranded DNA. The central channel is thought to allow incoming nucleoside triphosphates to access the active site. The enzyme has a number of unique structural features which distinguish it from other archaeal DNA polymerases and may account for its high processivity. A model of the complex with the primer-template duplex of DNA indicates that the largest conformational change that occurs upon DNA binding is the movement of the thumb domain, which rotates by 7.6° and moves by 10.0 Å. The surface potential of the enzyme is dominated by acidic groups in the central region of the molecule, where catalytic magnesium ions bind at the polymerase and exonuclease active sites. The outer regions are richer in basic amino acids that presumably interact with the sugar-phosphate backbone of DNA. The large number of salt bridges may contribute to the high thermal stability of this enzyme.
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Saxowsky, Tina T., Gunjan Choudhary, Michele M. Klingbeil, and Paul T. Englund. "Trypanosoma bruceiHas Two Distinct Mitochondrial DNA Polymerase β Enzymes." Journal of Biological Chemistry 278, no. 49 (September 8, 2003): 49095–101. http://dx.doi.org/10.1074/jbc.m308565200.
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Blanco, L., A. Bernad, and M. Salas. "MIP1 DNA polymerase of S.cerevisiae: structural similarity with theE.coliDNA polymerase I-type enzymes." Nucleic Acids Research 19, no. 4 (1991): 955. http://dx.doi.org/10.1093/nar/19.4.955.
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Dorawa, Sebastian, Magdalena Plotka, Anna-Karina Kaczorowska, Olafur H. Fridjonsson, Gudmundur O. Hreggvidsson, Arnthor Aevarsson, and Tadeusz Kaczorowski. "Characterization of DNA Polymerase from Thermus thermophilus MAT72 Phage Tt72." Proceedings 50, no. 1 (June 11, 2020): 38. http://dx.doi.org/10.3390/proceedings2020050038.
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Thermophilic phages are recognized as an untapped source of thermostable enzymes relevant in biotechnology; however, their biology is poorly explored. This has led us to start a project aimed at investigating thermophilic phages isolated from geothermal areas of Iceland. In this study, we present a structural and functional analysis of the DNA polymerase of phage Tt72, which infects thermophilic bacterium Thermus thermophilus MAT72. An in silico analysis of the Tt72 phage genome revealed the presence of a 2112-bp open reading frame (ORF) encoding protein homologous to the members of the A family of DNA polymerases. It contains a conserved nucleotidyltransferase domain and a 3′ → 5′ exonuclease domain but lacks the 5′ → 3′ exonuclease domain. The amino acid sequence of Tt72 DNA polymerase shows high similarity to two as yet uncharacterized DNA polymerases of T. thermophilus phages: ΦYS40 (91%) and ΦTMA (90%). The gene coding for Tt72 DNA polymerase was cloned and overexpressed in E. coli. The Tt72 polA gene is composed of 2112 nucleotides. The overall G+C content of this gene is 31.58%, which is lower than the G+C content of T. thermophilus genomic DNA (69.49%). The Tt72 polA gene codes for a 703-aa protein with a predicted molecular weight of 80,477. The enzyme was overproduced in E. coli, purified by heat treatment, followed by HiTrap TALON column and HiTrap Heparin HP column chromatography, then biochemically characterized. The optimum activity was found at 55 °C, pH 8.5, 25 mM KCl, and 0.5 mM Mg2+. Furthermore, the Tt72 DNA polymerase shows strong 3′ → 5′ exonucleolytic activity.
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Yoder, Kristine E., and Frederic D. Bushman. "Repair of Gaps in Retroviral DNA Integration Intermediates." Journal of Virology 74, no. 23 (December 1, 2000): 11191–200. http://dx.doi.org/10.1128/jvi.74.23.11191-11200.2000.
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ABSTRACT Diverse mobile DNA elements are believed to pirate host cell enzymes to complete DNA transfer. Prominent examples are provided by retroviral cDNA integration and transposon insertion. These reactions initially involve the attachment of each element 3′ DNA end to staggered sites in the host DNA by element-encoded integrase or transposase enzymes. Unfolding of such intermediates yields DNA gaps at each junction. It has been widely assumed that host DNA repair enzymes complete attachment of the remaining DNA ends, but the enzymes involved have not been identified for any system. We have synthesized DNA substrates containing the expected gap and 5′ two-base flap structure present in retroviral integration intermediates and tested candidate enzymes for the ability to support repair in vitro. We find three required activities, two of which can be satisfied by multiple enzymes. These are a polymerase (polymerase beta, polymerase delta and its cofactor PCNA, or reverse transcriptase), a nuclease (flap endonuclease), and a ligase (ligase I, III, or IV and its cofactor XRCC4). A proposed pathway involving retroviral integrase and reverse transcriptase did not carry out repair under the conditions tested. In addition, prebinding of integrase protein to gapped DNA inhibited repair reactions, indicating that gap repair in vivo may require active disassembly of the integrase complex.
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Steitz, Thomas A., and Y. Whitney Yin. "Accuracy, lesion bypass, strand displacement and translocation by DNA polymerases." Philosophical Transactions of the Royal Society of London. Series B: Biological Sciences 359, no. 1441 (January 29, 2004): 17–23. http://dx.doi.org/10.1098/rstb.2003.1374.
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The structures of DNA polymerases from different families show common features and significant differences that shed light on the ability of these enzymes to accurately copy DNA and translocate. The structure of a B family DNA polymerase from phage RB69 exhibits an active–site closing conformational change in the fingers domain upon forming a ternary complex with primer template in deoxynucleoside triphosphate. The rotation of the fingers domain α–helices by 60° upon dNTP binding is analogous to the changes seen in other families of polymerases. When the 3' terminus is bound to the editing 3' exonuclease active site, the orientation of the DNA helix axis changes by 40° and the thumb domain re–orients with the DNA. Structures of substrate and product complexes of T7 RNA polymerase, a structural homologue of T7 DNA polymerase, show that family polymerases use the rotation conformational change of the fingers domain to translocate down the DNA. The fingers opening rotation that results in translocation is powered by the release of the product pyrophosphate and also enables the Pol I family polymerases to function as a helicase in displacing the downstream non–template strand from the template strand.
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Harris, P. V., O. M. Mazina, E. A. Leonhardt, R. B. Case, J. B. Boyd, and K. C. Burtis. "Molecular cloning of Drosophila mus308, a gene involved in DNA cross-link repair with homology to prokaryotic DNA polymerase I genes." Molecular and Cellular Biology 16, no. 10 (October 1996): 5764–71. http://dx.doi.org/10.1128/mcb.16.10.5764.
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Mutations in the Drosophila mus308 gene confer specific hypersensitivity to DNA-cross-linking agents as a consequence of defects in DNA repair. The mus308 gene is shown here to encode a 229-kDa protein in which the amino-terminal domain contains the seven conserved motifs characteristic of DNA and RNA helicases and the carboxy-terminal domain shares over 55% sequence similarity with the polymerase domains of prokaryotic DNA polymerase I-like enzymes. This is the first reported member of this family of DNA polymerases in a eukaryotic organism, as well as the first example of a single polypeptide with homology to both DNA polymerase and helicase motifs. Identification of a closely related gene in the genome of Caenorhabditis elegans suggests that this novel polypeptide may play an evolutionarily conserved role in the repair of DNA damage in eukaryotic organisms.
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Kramata, Pavel, and Ivan Votruba. "Enzymes of Human Herpesviruses." Collection of Czechoslovak Chemical Communications 57, no. 8 (1992): 1577–612. http://dx.doi.org/10.1135/cccc19921577.
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The properties of human herpesvirus-encoded enzymes are reviewed and the importance of sequence analysis of viral genomes as well as the experiments on characteristics of enzymes isolated from infected cell cultures are emphasized. The following enzymes are described in detail: DNA replication complex consisting of DNA polymerase, DNA helicase-primase, single-stranded DNA binding protein and origin binding protein, further thymidine kinase, ribonucleotide reductase, deoxyuridine triphosphatase as well as uracil-DNA-glycosylase, deoxyribonuclease and protein kinase. The importance of these enzymes from the point of view of antiviral chemotherapy is discussed.
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Majerník, A. I., E. R. Jenkinson, and J. P. J. Chong. "DNA replication in thermophiles." Biochemical Society Transactions 32, no. 2 (April 1, 2004): 236–39. http://dx.doi.org/10.1042/bst0320236.
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DNA replication enzymes in the thermophilic Archaea have previously attracted attention due to their obvious use in methods such as PCR. The proofreading ability of the Pyrococcus furiosus DNA polymerase has resulted in a commercially successful product (Pfu polymerase). One of the many notable features of the Archaea is the fact that their DNA processing enzymes appear on the whole to be more like those found in eukaryotes than bacteria. These proteins also appear to be simpler versions of those found in eukaryotes. For these reasons, archaeal organisms make potentially interesting model systems to explore the molecular mechanisms of processes such as DNA replication, repair and recombination. Why archaeal DNA-manipulation systems were adopted over bacterial systems by eukaryotic cells remains a most interesting question that we suggest may be linked to thermophily.
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Nash, Kevin, Weijun Chen, William F. McDonald, Xiaohuai Zhou, and Nicholas Muzyczka. "Purification of Host Cell Enzymes Involved in Adeno-Associated Virus DNA Replication." Journal of Virology 81, no. 11 (March 14, 2007): 5777–87. http://dx.doi.org/10.1128/jvi.02651-06.
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ABSTRACT Adeno-associated virus (AAV) replicates its DNA by a modified rolling-circle mechanism that exclusively uses leading strand displacement synthesis. To identify the enzymes directly involved in AAV DNA replication, we fractionated adenovirus-infected crude extracts and tested them in an in vitro replication system that required the presence of the AAV-encoded Rep protein and the AAV origins of DNA replication, thus faithfully reproducing in vivo viral DNA replication. Fractions that contained replication factor C (RFC) and proliferating cell nuclear antigen (PCNA) were found to be essential for reconstituting AAV DNA replication. These could be replaced by purified PCNA and RFC to retain full activity. We also found that fractions containing polymerase δ, but not polymerase ε or α, were capable of replicating AAV DNA in vitro. This was confirmed when highly purified polymerase δ complex purified from baculovirus expression clones was used. Curiously, as the components of the DNA replication system were purified, neither the cellular single-stranded DNA binding protein (RPA) nor the adenovirus-encoded DNA binding protein was found to be essential for DNA replication; both only modestly stimulated DNA synthesis on an AAV template. Also, in addition to polymerase δ, RFC, and PCNA, an as yet unidentified factor(s) is required for AAV DNA replication, which appeared to be enriched in adenovirus-infected cells. Finally, the absence of any apparent cellular DNA helicase requirement led us to develop an artificial AAV replication system in which polymerase δ, RFC, and PCNA were replaced with T4 DNA polymerase and gp32 protein. This system was capable of supporting AAV DNA replication, demonstrating that under some conditions the Rep helicase activity can function to unwind duplex DNA during strand displacement synthesis.
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Yudkina, Anna V., Anton V. Endutkin, Eugenia A. Diatlova, Nina A. Moor, Ivan P. Vokhtantsev, Inga R. Grin, and Dmitry O. Zharkov. "Displacement of Slow-Turnover DNA Glycosylases by Molecular Traffic on DNA." Genes 11, no. 8 (July 30, 2020): 866. http://dx.doi.org/10.3390/genes11080866.
Full textAbstract:
In the base excision repair pathway, the initiating enzymes, DNA glycosylases, remove damaged bases and form long-living complexes with the abasic DNA product, but can be displaced by AP endonucleases. However, many nuclear proteins can move along DNA, either actively (such as DNA or RNA polymerases) or by passive one-dimensional diffusion. In most cases, it is not clear whether this movement is disturbed by other bound proteins or how collisions with moving proteins affect the bound proteins, including DNA glycosylases. We have used a two-substrate system to study the displacement of human OGG1 and NEIL1 DNA glycosylases by DNA polymerases in both elongation and diffusion mode and by D4, a passively diffusing subunit of a viral DNA polymerase. The OGG1–DNA product complex was disrupted by DNA polymerase β (POLβ) in both elongation and diffusion mode, Klenow fragment (KF) in the elongation mode and by D4. NEIL1, which has a shorter half-life on DNA, was displaced more efficiently. Hence, both possibly specific interactions with POLβ and nonspecific collisions (KF, D4) can displace DNA glycosylases from DNA. The protein movement along DNA was blocked by very tightly bound Cas9 RNA-targeted nuclease, providing an upper limit on the efficiency of obstacle clearance.
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Tori, Kazuo, Megumi Kimizu, Sonoko Ishino, and Yoshizumi Ishino. "DNA Polymerases BI and D from the Hyperthermophilic Archaeon Pyrococcus furiosus Both Bind to Proliferating Cell Nuclear Antigen with Their C-Terminal PIP-Box Motifs." Journal of Bacteriology 189, no. 15 (May 11, 2007): 5652–57. http://dx.doi.org/10.1128/jb.00073-07.
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ABSTRACT Proliferating cell nuclear antigen (PCNA) is the sliding clamp that is essential for the high processivity of DNA synthesis during DNA replication. Pyrococcus furiosus, a hyperthermophilic archaeon, has at least two DNA polymerases, polymerase BI (PolBI) and PolD. Both of the two DNA polymerases interact with the archaeal P. furiosus PCNA (PfuPCNA) and perform processive DNA synthesis in vitro. This phenomenon, in addition to the fact that both enzymes display 3′-5′ exonuclease activity, suggests that both DNA polymerases work in replication fork progression. We demonstrated here that both PolBI and PolD functionally interact with PfuPCNA at their C-terminal PIP boxes. The mutant PolBI and PolD enzymes lacking the PIP-box sequence do not respond to the PfuPCNA at all in an in vitro primer extension reaction. This is the first experimental evidence that the PIP-box motif, located at the C termini of the archaeal DNA polymerases, is actually critical for PCNA binding to form a processive DNA-synthesizing complex.
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Forterre, Patrick, Christiane Eue, Mouldy Sioud, and Abdellah Hamal. "Studies on DNA polymerases and topoisomerases in archaebacteria." Canadian Journal of Microbiology 35, no. 1 (January 1, 1989): 228–33. http://dx.doi.org/10.1139/m89-035.
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We have isolated DNA polymerases and topoisomerases from two thermoacidophilic archaebacteria: Sulfolobus acidocaldarius and Thermoplasma acidophilum. The DNA polymerases are composed of a single polypeptide with molecular masses of 100 and 85 kDa, respectively. Antibodies against Sulfolobus DNA polymerase did not cross react with Thermoplasma DNA polymerase. Whereas the major DNA topoisomerase activity in S. acidocaldarius is an ATP-dependent type I DNA topoisomerase with a reverse gyrase activity, the major DNA topoisomerase activity in T. acidophilum is a ATP-independent relaxing activity. Both enzymes resemble more the eubacterial than the eukaryotic type I DNA topoisomerase. We have found that small plasmids from halobacteria are negatively supercoiled and that DNA topoisomerase II inhibitors modify their topology. This suggests the existence of an archaebacterial type II DNA topoisomerase related to its eubacterial and eukaryotic counterparts. As in eubacteria, novobiocin induces positive supercoiling of halobacterial plasmids, indicating the absence of a eukaryotic-like type I DNA topoisomerase that relaxes positive superturns.Key words: archaebacteria, DNA topoisomerases, DNA polymerases, DNA topology, gyrase.
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Adamiec, R., Z. Szewczyk, and J. Szopa. "DNA-dependent RNA polymerase enzymes affected in uraemic lymphocyte cells." International Urology and Nephrology 25, no. 1 (January 1993): 97–104. http://dx.doi.org/10.1007/bf02552261.
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Earnshaw, D. L., T. J. C. Beebee, and W. E. Gutteridge. "Demonstration of RNA polymerase multiplicity in Trypanosoma brucei. Characterization and purification of α-amanitin-resistant and -sensitive enzymes." Biochemical Journal 241, no. 3 (February 1, 1987): 649–55. http://dx.doi.org/10.1042/bj2410649.
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We have isolated, characterized and substantially purified two distinct RNA polymerase activities from the flagellate protozoan parasite Trypanosoma brucei. RNA polymerases from this organism were resolved poorly on DEAE-Sephadex, but could be separated with CM-Sephadex. One form was totally resistant to alpha-amanitin, whereas the second was 50% inhibited by 10-20 micrograms of the drug/ml. The enzymes had different salt optima, but both were of high Mr (greater than 480,000) and demonstrated the template preference: poly[d(A-T)] greater than denatured DNA greater than native DNA, and both were more active with Mn2+ than with Mg2+. The amanitin-resistant enzyme, polymerase R, was partially purified by chromatography on CM-Sephadex, DEAE-Sephadex and heparin-Sepharose. This enzyme was very labile, and activity yields were around 9%; after purification, one or two protein bands could be discerned after electrophoresis under non-denaturing conditions, but about 20 polypeptides were resolved on denaturing gels, including a major component (not thought to be part of the enzyme) of Mr 65,000. Polymerase S, sensitive to low alpha-amanitin concentrations, was more extensively purified, with an 18% recovery, and yielded a single major band with two minor ones after native gel electrophoresis. Analysis under denaturing conditions permitted a possible subunit structure for this enzyme to be ascribed.
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Birkuš, Gabriel, Ivan Votruba, Miroslav Otmar, and Antonín Holý. "Interactions of 1-[(S)-3-Hydroxy-2-(phosphonomethoxy)propyl]cytosine (Cidofovir) Diphosphate with DNA Polymerases α, δ and ε*." Collection of Czechoslovak Chemical Communications 66, no. 11 (2001): 1698–706. http://dx.doi.org/10.1135/cccc20011698.
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The inhibitory and/or substrate activity of 1-[(S)-3-hydroxy-2-(phosphonomethoxy)propyl]cytosine [(S)-HPMPC, cidofovir, Vistide™] diphosphate towards eukaryotic DNA polymerases α, δ and ε* was examined. Cidofovir diphosphate is a weak competitive inhibitor of the above enzymes, approximately 3 to 7 times weaker than its adenine analogue (S)-HPMPApp. The enzymes also catalyze incorporation of (S)-HPMPC into DNA; after insertion of one (S)-HPMPC residue into DNA, another dNMP residue may incorporate. DNA polymerase δ and ε* can successively accommodate in the growing chain two (S)-HPMPC residues at the maximum, whereas pol α up to three residues.
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Broyles, S. S., and B. Moss. "Sedimentation of an RNA polymerase complex from vaccinia virus that specifically initiates and terminates transcription." Molecular and Cellular Biology 7, no. 1 (January 1987): 7–14. http://dx.doi.org/10.1128/mcb.7.1.7.
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A high-molecular-weight protein complex that is capable of accurate transcription initiation and termination of vaccinia virus early genes without additional factors was demonstrated. The complex was solubilized by disruption of purified virions, freed of DNA by passage through a DEAE-cellulose column, and isolated by glycerol gradient sedimentation. All detectable RNA polymerase activity was associated with the transcription complex, whereas the majority of enzymes released from virus cores including mRNA (nucleoside-2'-O)methyltransferase, poly(A) polymerase, topoisomerase, nucleoside triphosphate phosphohydrolase II, protein kinase, and single-strand DNase sedimented more slowly. Activities corresponding to two enzymes, mRNA guanylyltransferase (capping enzyme) and nucleoside triphosphate phosphohydrolase I (DNA-dependent ATPase), partially sedimented with the complex. Silver-stained polyacrylamide gels, immunoblots, and autoradiographs confirmed the presence of subunits of vaccinia virus RNA polymerase, mRNA guanylyltransferase, and nucleoside triphosphate phosphohydrolase I, as well as additional unidentified polypeptides, in fractions with transcriptase activity. A possible role for the DNA-dependent ATPase was suggested by studies with ATP analogs with gamma-S or nonhydrolyzable beta-gamma-phosphodiester bonds. These analogs were used by vaccinia virus RNA polymerase to nonspecifically transcribe single-stranded DNA templates but did not support accurate transcription of early genes by the complex. Transcription also was sensitive to high concentrations of novobiocin; however, this effect could be attributed to inhibition of RNA polymerase or ATPase activities rather than topoisomerase.
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Broyles, S. S., and B. Moss. "Sedimentation of an RNA polymerase complex from vaccinia virus that specifically initiates and terminates transcription." Molecular and Cellular Biology 7, no. 1 (January 1987): 7–14. http://dx.doi.org/10.1128/mcb.7.1.7-14.1987.
Full textAbstract:
A high-molecular-weight protein complex that is capable of accurate transcription initiation and termination of vaccinia virus early genes without additional factors was demonstrated. The complex was solubilized by disruption of purified virions, freed of DNA by passage through a DEAE-cellulose column, and isolated by glycerol gradient sedimentation. All detectable RNA polymerase activity was associated with the transcription complex, whereas the majority of enzymes released from virus cores including mRNA (nucleoside-2'-O)methyltransferase, poly(A) polymerase, topoisomerase, nucleoside triphosphate phosphohydrolase II, protein kinase, and single-strand DNase sedimented more slowly. Activities corresponding to two enzymes, mRNA guanylyltransferase (capping enzyme) and nucleoside triphosphate phosphohydrolase I (DNA-dependent ATPase), partially sedimented with the complex. Silver-stained polyacrylamide gels, immunoblots, and autoradiographs confirmed the presence of subunits of vaccinia virus RNA polymerase, mRNA guanylyltransferase, and nucleoside triphosphate phosphohydrolase I, as well as additional unidentified polypeptides, in fractions with transcriptase activity. A possible role for the DNA-dependent ATPase was suggested by studies with ATP analogs with gamma-S or nonhydrolyzable beta-gamma-phosphodiester bonds. These analogs were used by vaccinia virus RNA polymerase to nonspecifically transcribe single-stranded DNA templates but did not support accurate transcription of early genes by the complex. Transcription also was sensitive to high concentrations of novobiocin; however, this effect could be attributed to inhibition of RNA polymerase or ATPase activities rather than topoisomerase.
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Brown, Jessica A., Lindsey R. Pack, Jason D. Fowler, and Zucai Suo. "Pre-Steady-State Kinetic Analysis of the Incorporation of Anti-HIV Nucleotide Analogs Catalyzed by Human X- and Y-Family DNA Polymerases." Antimicrobial Agents and Chemotherapy 55, no. 1 (November 15, 2010): 276–83. http://dx.doi.org/10.1128/aac.01229-10.
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ABSTRACTNucleoside reverse transcriptase inhibitors (NRTIs) are an important class of antiviral drugs used to manage infections by human immunodeficiency virus, which causes AIDS. Unfortunately, these drugs cause unwanted side effects, and the molecular basis of NRTI toxicity is not fully understood. Putative routes of NRTI toxicity include the inhibition of human nuclear and mitochondrial DNA polymerases. A strong correlation between mitochondrial toxicity and NRTI incorporation catalyzed by human mitochondrial DNA polymerase has been established bothin vitroandin vivo. However, it remains to be determined whether NRTIs are substrates for the recently discovered human X- and Y-family DNA polymerases, which participate in DNA repair and DNA lesion bypassin vivo. Using pre-steady-state kinetic techniques, we measured the substrate specificity constants for human DNA polymerases β, λ, η, ι, κ, and Rev1 incorporating the active, 5′-phosphorylated forms of tenofovir, lamivudine, emtricitabine, and zidovudine. For the six enzymes, all of the drug analogs were incorporated less efficiently (40- to >110,000-fold) than the corresponding natural nucleotides, usually due to a weaker binding affinity and a slower rate of incorporation for the incoming nucleotide analog. In general, the 5′-triphosphate forms of lamivudine and zidovudine were better substrates than emtricitabine and tenofovir for the six human enzymes, although the substrate specificity profile depended on the DNA polymerase. Our kinetic results suggest NRTI insertion catalyzed by human X- and Y-family DNA polymerases is a potential mechanism of NRTI drug toxicity, and we have established a structure-function relationship for designing improved NRTIs.
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Washington, M. Todd, Robert E. Johnson, Louise Prakash, and Satya Prakash. "The Mechanism of Nucleotide Incorporation by Human DNA Polymerase η Differs from That of the Yeast Enzyme." Molecular and Cellular Biology 23, no. 22 (November 15, 2003): 8316–22. http://dx.doi.org/10.1128/mcb.23.22.8316-8322.2003.
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ABSTRACT DNA polymerase η (Polη) catalyzes the efficient and accurate synthesis of DNA opposite cyclobutane pyrimidine dimers, and inactivation of Polη in humans causes the cancer-prone syndrome, the variant form of xeroderma pigmentosum. Pre-steady-state kinetic studies of yeast Polη have indicated that the low level of fidelity of this enzyme results from a poorly discriminating induced-fit mechanism. Here we examine the mechanistic basis of the low level of fidelity of human Polη. Because the human and yeast enzymes behave similarly under steady-state conditions, we expected these enzymes to utilize similar mechanisms of nucleotide incorporation. Surprisingly, however, we find that human Polη differs from the yeast enzyme in several important respects. The human enzyme has a 50-fold-faster rate of nucleotide incorporation than the yeast enzyme but binds the nucleotide with an approximately 50-fold-lower level of affinity. This lower level of binding affinity might provide a means of regulation whereby the human enzyme remains relatively inactive except when the cellular deoxynucleoside triphosphate concentrations are high, as may occur during DNA damage, thereby avoiding the mutagenic consequences arising from the inadvertent action of this enzyme during normal DNA replication.
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Henneke, Ghislaine. "In vitro reconstitution of RNA primer removal in Archaea reveals the existence of two pathways." Biochemical Journal 447, no. 2 (September 26, 2012): 271–80. http://dx.doi.org/10.1042/bj20120959.
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Using model DNA substrates and purified recombinant proteins from Pyrococcus abyssi, I have reconstituted the enzymatic reactions involved in RNA primer elimination in vitro. In my dual-labelled system, polymerase D performed efficient strand displacement DNA synthesis, generating 5′-RNA flaps which were subsequently released by Fen1, before ligation by Lig1. In this pathway, the initial cleavage event by RNase HII facilitated RNA primer removal of Okazaki fragments. In addition, I have shown that polymerase B was able to displace downstream DNA strands with a single ribonucleotide at the 5′-end, a product resulting from a single cut in the RNA initiator by RNase HII. After RNA elimination, the combined activities of strand displacement DNA synthesis by polymerase B and flap cleavage by Fen1 provided a nicked substrate for ligation by Lig1. The unique specificities of Okazaki fragment maturation enzymes and replicative DNA polymerases strongly support the existence of two pathways in the resolution of RNA fragments.
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Akabayov, Barak, and Charles C. Richardson. "Binding of Mn-deoxyribonucleoside triphosphates to the active site of the DNA polymerase of bacteriophage T7." Powder Diffraction 26, no. 2 (June 2011): 159–62. http://dx.doi.org/10.1154/1.3583156.
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Divalent metal ions are crucial as cofactors for a variety of intracellular enzymatic activities. Mg2+, as an example, mediates binding of deoxyribonucleoside 5′-triphosphates followed by their hydrolysis in the active site of DNA polymerase. It is difficult to study the binding of Mg2+ to an active site because Mg2+ is spectroscopically silent and Mg2+ binds with low affinity to the active site of an enzyme. Therefore, we substituted Mg2+ with Mn2+:Mn2+ that is not only visible spectroscopically but also provides full activity of the DNA polymerase of bacteriophage T7. In order to demonstrate that the majority of Mn2+ is bound to the enzyme, we have applied site-directed titration analysis of T7 DNA polymerase using X-ray near edge spectroscopy. Here we show how X-ray near edge spectroscopy can be used to distinguish between signal originating from Mn2+ that is free in solution and Mn2+ bound to the active site of T7 DNA polymerase. This method can be applied to other enzymes that use divalent metal ions as a cofactor.
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Wolfle, William T., M. Todd Washington, Eric T. Kool, Thomas E. Spratt, Sandra A. Helquist, Louise Prakash, and Satya Prakash. "Evidence for a Watson-Crick Hydrogen Bonding Requirement in DNA Synthesis by Human DNA Polymerase κ." Molecular and Cellular Biology 25, no. 16 (August 15, 2005): 7137–43. http://dx.doi.org/10.1128/mcb.25.16.7137-7143.2005.
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ABSTRACT The efficiency and fidelity of nucleotide incorporation by high-fidelity replicative DNA polymerases (Pols) are governed by the geometric constraints imposed upon the nascent base pair by the active site. Consequently, these polymerases can efficiently and accurately replicate through the template bases which are isosteric to natural DNA bases but which lack the ability to engage in Watson-Crick (W-C) hydrogen bonding. DNA synthesis by Polη, a low-fidelity polymerase able to replicate through DNA lesions, however, is inhibited in the presence of such an analog, suggesting a dependence of this polymerase upon W-C hydrogen bonding. Here we examine whether human Polκ, which differs from Polη in having a higher fidelity and which, unlike Polη, is inhibited at inserting nucleotides opposite DNA lesions, shows less of a dependence upon W-C hydrogen bonding than does Polη. We find that an isosteric thymidine analog is replicated with low efficiency by Polκ, whereas a nucleobase analog lacking minor-groove H bonding potential is replicated with high efficiency. These observations suggest that both Polη and Polκ rely on W-C hydrogen bonding for localizing the nascent base pair in the active site for the polymerization reaction to occur, thus overcoming these enzymes' low geometric selectivity.
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Gammon, Don B., and David H. Evans. "The 3′-to-5′ Exonuclease Activity of Vaccinia Virus DNA Polymerase Is Essential and Plays a Role in Promoting Virus Genetic Recombination." Journal of Virology 83, no. 9 (February 18, 2009): 4236–50. http://dx.doi.org/10.1128/jvi.02255-08.
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ABSTRACT Poxviruses are subjected to extraordinarily high levels of genetic recombination during infection, although the enzymes catalyzing these reactions have never been identified. However, it is clear that virus-encoded DNA polymerases play some unknown yet critical role in virus recombination. Using a novel, antiviral-drug-based strategy to dissect recombination and replication reactions, we now show that the 3′-to-5′ proofreading exonuclease activity of the viral DNA polymerase plays a key role in promoting recombination reactions. Linear DNA substrates were prepared containing the dCMP analog cidofovir (CDV) incorporated into the 3′ ends of the molecules. The drug blocked the formation of concatemeric recombinant molecules in vitro in a process that was catalyzed by the proofreading activity of vaccinia virus DNA polymerase. Recombinant formation was also blocked when CDV-containing recombination substrates were transfected into cells infected with wild-type vaccinia virus. These inhibitory effects could be overcome if CDV-containing substrates were transfected into cells infected with CDV-resistant (CDVr) viruses, but only when resistance was linked to an A314T substitution mutation mapping within the 3′-to-5′ exonuclease domain of the viral polymerase. Viruses encoding a CDVr mutation in the polymerase domain still exhibited a CDV-induced recombination deficiency. The A314T substitution also enhanced the enzyme's capacity to excise CDV molecules from the 3′ ends of duplex DNA and to recombine these DNAs in vitro, as judged from experiments using purified mutant DNA polymerase. The 3′-to-5′ exonuclease activity appears to be an essential virus function, and our results suggest that this might be because poxviruses use it to promote genetic exchange.
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Earnshaw, David L., and Andrew J. Pope. "FlashPlate Scintillation Proximity Assays for Characterization and Screening of DNA Polymerase, Primase, and Helicase Activities." Journal of Biomolecular Screening 6, no. 1 (February 2001): 39–46. http://dx.doi.org/10.1177/108705710100600106.
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DNA replication proteins represent a class of extremely well-established anti-infective drug targets for which improvements in assay technology are required in order to support enzyme characterization, HTS, and structure-activity relationship studies. Replication proteins are conventionally assayed using precipitation/filtration or gelbased techniques, and are not yet all suitable for conversion into homogeneous fluorescence-based formats. We have therefore developed radiometric assays for these enzymes based upon FlashPlate technology that can be applied to a wide range of targets using a common set of reagents. This approach has allowed the rapid characterization of DNA polymerase, DNA primase, and DNA helicase activities. The resultant 96-/384-well microplate assays are suitable for primary HTS, hit selectivity determination, and/or elucidating the mechanism of action of inhibitors. In all cases, biotinylated DNA oligonucleotide substrates were tethered to streptavidin-coated scintillant-embedded FlashPlate wells. Various adaptations were employed for each enzyme activity. For DNA polymerase, a short complementary oligonucleotide primer was annealed to the longer tethered oligonucleotide, and polymerization was measured by incorporation of [3H]-dNTPs onto the growing primer 3′ end. For DNA primase, direct synthesis of short oligoribonucleotides complementary to the tethered DNA strand was measured by incorporation of [3H]-rNTPs or by subsequent polymerase extension with [3H]-dNTPs from unlabeled primers. For DNA helicase, unwinding of a [33P]-labeled oligonucleotide complementary to the tethered oligonucleotide was measured. This robust and flexible system has a number of substantial advantages over conventional assay techniques for this difficult class of enzymes.
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Parani, M., K. Rajesh, M. Lakshmi, L. Parducci, A. E. Szmidt, and A. Parida. "Species identification in seven small millet species using polymerase chain reaction - restriction fragment length polymorphism of trnS-psbC gene region." Genome 44, no. 3 (June 1, 2001): 495–99. http://dx.doi.org/10.1139/g01-023.
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The chloroplast trnS-psbC gene regions from total genomic DNA of 119 accessions from seven small millet species were amplified by polymerase chain reaction (PCR) and digested with eight restriction enzymes individually as well as in combinations of two enzymes to generate restriction fragment length polymorphism (PCRRFLP). PCRRFLP with individual enzymes revealed polymorphism between only some species. However, all the species could be distinguished by using a combination of two enzymes, specifically HaeIII and MspI. PCRRFLP of 11 to 20 accessions with the same enzyme combination showed no intraspecific variation, which established that the differential banding patterns were species specific. In contrast, the same enzyme combination was not useful for differentiating different species of the genera Cajanus, Rhyncosia, Abies, Rhizophora, Ceriops, and Bruguiera, and it also revealed intraspecies variation in three species of Abies. The present study indicated that digestion of trnS-psbC with two four-base recognizing enzymes reveals more variation than with either enzyme alone and that it may be a method of choice for species identification in some genera.Key words: small millets, PCRRFLP, trnS-psbC, chloroplast DNA.
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Srivastava, Vinod K., and David L. Busbee. "Replicative enzymes, DNA polymerase alpha (pol α), and in vitro ageing." Experimental Gerontology 38, no. 11-12 (November 2003): 1285–97. http://dx.doi.org/10.1016/j.exger.2003.09.008.
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Abellón-Ruiz, Javier, Sonoko Ishino, Yoshizumi Ishino, and Bernard A. Connolly. "Archaeal DNA Polymerase-B as a DNA Template Guardian: Links between Polymerases and Base/Alternative Excision Repair Enzymes in Handling the Deaminated Bases Uracil and Hypoxanthine." Archaea 2016 (2016): 1–8. http://dx.doi.org/10.1155/2016/1510938.
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In Archaea repair of uracil and hypoxanthine, which arise by deamination of cytosine and adenine, respectively, is initiated by three enzymes: Uracil-DNA-glycosylase (UDG, which recognises uracil); Endonuclease V (EndoV, which recognises hypoxanthine); and Endonuclease Q (EndoQ), (which recognises both uracil and hypoxanthine). Two archaeal DNA polymerases, Pol-B and Pol-D, are inhibited by deaminated bases in template strands, a feature unique to this domain. Thus the three repair enzymes and the two polymerases show overlapping specificity for uracil and hypoxanthine. Here it is demonstrated that binding of Pol-D to primer-templates containing deaminated bases inhibits the activity of UDG, EndoV, and EndoQ. Similarly Pol-B almost completely turns off EndoQ, extending earlier work that demonstrated that Pol-B reduces catalysis by UDG and EndoV. Pol-B was observed to be a more potent inhibitor of the enzymes compared to Pol-D. Although Pol-D is directly inhibited by template strand uracil, the presence of Pol-B further suppresses any residual activity of Pol-D, to near-zero levels. The results are compatible with Pol-D acting as the replicative polymerase and Pol-B functioning primarily as a guardian preventing deaminated base-induced DNA mutations.
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Hrabina, Ondrej, Viktor Brabec, and Olga Novakova. "Translesion DNA Synthesis Across Lesions Induced by Oxidative Products of Pyrimidines: An Insight into the Mechanism by Microscale Thermophoresis." International Journal of Molecular Sciences 20, no. 20 (October 10, 2019): 5012. http://dx.doi.org/10.3390/ijms20205012.
Full textAbstract:
Oxidative stress in cells can lead to the accumulation of reactive oxygen species and oxidation of DNA precursors. Oxidized nucleotides such as 2’-deoxyribo-5-hydroxyuridin (HdU) and 2’-deoxyribo-5-hydroxymethyluridin (HMdU) can be inserted into DNA during replication and repair. HdU and HMdU have attracted particular interest because they have different effects on damaged-DNA processing enzymes that control the downstream effects of the lesions. Herein, we studied the chemically simulated translesion DNA synthesis (TLS) across the lesions formed by HdU or HMdU using microscale thermophoresis (MST). The thermodynamic changes associated with replication across HdU or HMdU show that the HdU paired with the mismatched deoxyribonucleoside triphosphates disturbs DNA duplexes considerably less than thymidine (dT) or HMdU. Moreover, we also demonstrate that TLS by DNA polymerases across the lesion derived from HdU was markedly less extensive and potentially more mutagenic than that across the lesion formed by HMdU. Thus, DNA polymerization by DNA polymerase η (polη), the exonuclease-deficient Klenow fragment of DNA polymerase I (KF–), and reverse transcriptase from human immunodeficiency virus type 1 (HIV-1 RT) across these pyrimidine lesions correlated with the different stabilization effects of the HdU and HMdU in DNA duplexes revealed by MST. The equilibrium thermodynamic data obtained by MST can explain the influence of the thermodynamic alterations on the ability of DNA polymerases to bypass lesions induced by oxidative products of pyrimidines. The results also highlighted the usefulness of MST in evaluating the impact of oxidative products of pyrimidines on the processing of these lesions by damaged DNA processing enzymes.
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Xu, Xinlan, Li Xiao, Jia Zhang, Dixian Luo, Li Wang, Zhuang Peng, Duanfang Liao, Chungen Xing, Wangyang Pu, and Kai Li. "A Modified Mutation-Sensitive On/Off Switch and Its Application in B-Raf Proto-Oncogene Hotspot Mutation Assay." Nanoscience and Nanotechnology Letters 11, no. 11 (November 1, 2019): 1582–88. http://dx.doi.org/10.1166/nnl.2019.3033.
Full textAbstract:
The combination of phosphorothioate-modified primer and high-fidelity DNA polymerase constitute a mutation sensitive on/off switch. Herein, a modified mutation-sensitive on/off switch was designed with an extra point mutation at 5′ far away from the 3′ of the phosphorothioate modified primer to test the power of the new enzymes: Phusion, Q5 and Phanta UC polymerases. A modified on/off switch with the introduction of an extra point mismatched nucleotide at –8 from the 3′ terminus, and the use of Phanta UC Super-fidelity DNA polymerase has been confirmed to be highly sensitive and specific as compared to those without the 5′ artificial mismatched nucleotide, when applied in circulating tumor DNA (ctDNA) analysis of BRAF hotspot mutation.
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Peersen, Olve. "A Comprehensive Superposition of Viral Polymerase Structures." Viruses 11, no. 8 (August 13, 2019): 745. http://dx.doi.org/10.3390/v11080745.
Full textAbstract:
Nucleic acid polymerases are essential enzymes that replicate the genomes of both RNA and DNA viruses. These enzymes are generally encoded by viruses themselves so as to provide biochemical functions and control elements that differ from those of the host cell polymerases. The core active site structure used by all replicative polymerases is highly conserved and composed of two key aspartate residues from the conserved motifs A and C, but beyond this there is significant divergence among structures. These differences can make it difficult to select which portions of structures to align for comparisons, yet there are extended structural similarities within different groups of viral polymerases that should clearly be considered to generate optimal alignments. This manuscript describes a comprehensive structure-based superposition of every viral polymerase structure solved thus far based on an alignment-tree approach wherein aligned regions grow in complexity as similarity among polymerases increases. The result is a set of 646 structures that have been aligned into a single common orientation. This provides a convenient resource for directly comparing viral polymerases and illustrating structural conservation among them. It also sets the stage for detailed bioinformatics analysis to further assess common structural features. The full set of protein data bank (PDB) formatted files is publicly available via the Polymerase Structures community page at the Zenodo.org open data repository.
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Barnes, Marjorie H., Shelley D. Miller, and Neal C. Brown. "DNA Polymerases of Low-GC Gram-Positive Eubacteria: Identification of the Replication-Specific Enzyme Encoded by dnaE." Journal of Bacteriology 184, no. 14 (July 15, 2002): 3834–38. http://dx.doi.org/10.1128/jb.184.14.3834-3838.2002.
Full textAbstract:
ABSTRACT dnaE, the gene encoding one of the two replication-specific DNA polymerases (Pols) of low-GC-content gram-positive bacteria (E. Dervyn et al., Science 294:1716-1719, 2001; R. Inoue et al., Mol. Genet. Genomics 266:564-571, 2001), was cloned from Bacillus subtilis, a model low-GC gram-positive organism. The gene was overexpressed in Escherichia coli. The purified recombinant product displayed inhibitor responses and physical, catalytic, and antigenic properties indistinguishable from those of the low-GC gram-positive-organism-specific enzyme previously named DNA Pol II after the polB-encoded DNA Pol II of E. coli. Whereas a polB-like gene is absent from low-GC gram-positive genomes and whereas the low-GC gram-positive DNA Pol II strongly conserves a dnaE-like, Pol III primary structure, it is proposed that it be renamed DNA polymerase III E (Pol III E) to accurately reflect its replicative function and its origin from dnaE. It is also proposed that DNA Pol III, the other replication-specific Pol of low-GC gram-positive organisms, be renamed DNA polymerase III C (Pol III C) to denote its origin from polC. By this revised nomenclature, the DNA Pols that are expressed constitutively in low-GC gram-positive bacteria would include DNA Pol I, the dispensable repair enzyme encoded by polA, and the two essential, replication-specific enzymes Pol III C and Pol III E, encoded, respectively, by polC and dnaE.