Mutation S543N in the thumb subdomain of the Taq DNA polymerase large fragment suppresses pausing associated with the template structure.
TL;DR: Substitution of Asn for the conserved Ser543 in the thumb subdomain of the Taq DNA polymerase large fragment (KlentaqDNA polymerase) prevents pausing during DNA synthesis and allows the enzyme to circumvent template regions with a complex structure.
About: This article is published in FEBS Letters.The article was published on 1999-04-02 and is currently open access. It has received 13 citations till now. The article focuses on the topics: Hot start PCR & DNA clamp.
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TL;DR: DNA is not only a repository of genetic information for life, it is also a unique polymer with remarkable properties: it associates according to well-defined rules, it can be assembled into diverse nanostructures of defined geometry and it can serve as a supramolecular scaffold to arrange chemical groups in space.
86 citations
TL;DR: The antifungal potency of malting barley grain ns-LTP was examined on Saccharomyces cerevisiae, Candida albicans and the plant pathogen Fusarium solani growth in vitro and the importance of ns- LTP for barley grain protection from fungal diseases has been discussed.
Abstract: A basic protein (pi < 9) was isolated to homogeneity from a domestic cultivar of malting barley grain (Hordeum vulgare). In its unreduced form it exists as a dimer of a 9 kDa protomer with four disulphide bridges. These characteristics together with protein sequence data revealed that the isolated protein belongs to the class of ns-LTP. The antifungal potency of malting barley grain ns-LTP was examined on Saccharomyces cerevisiae, Candida albicans and the plant pathogen Fusarium solani growth in vitro. It was found that ns-LTP inhibits Saccharomyces and Fusarium growth; the concentration required for 50% inhibition after 24 h of incubation (IC 50 ) was 100 and 80 μg/mL, respectively. On the basis of these results, the importance of ns-LTP for barley grain protection from fungal diseases has been discussed.
28 citations
TL;DR: A method for studying polymerase errors with exceptional resolution is described, which combines unique molecular identifier tagging and high-throughput sequencing and demonstrates that the detected high-frequency PCR errors are highly recurrent and that the position in the template sequence and polymerase-specific substitution preferences are among the major factors influencing the observed PCR error rate.
Abstract: The accuracy with which DNA polymerase can replicate a template DNA sequence is an extremely important property that can vary by an order of magnitude from one enzyme to another. The rate of nucleotide misincorporation is shaped by multiple factors, including PCR conditions and proofreading capabilities, and proper assessment of polymerase error rate is essential for a wide range of sensitive PCR-based assays. In this paper, we describe a method for studying polymerase errors with exceptional resolution, which combines unique molecular identifier tagging and high-throughput sequencing. Our protocol is less laborious than commonly-used methods, and is also scalable, robust and accurate. In a series of nine PCR assays, we have measured a range of polymerase accuracies that is in line with previous observations. However, we were also able to comprehensively describe individual errors introduced by each polymerase after either 20 PCR cycles or a linear amplification, revealing specific substitution preferences and the diversity of PCR error frequency profiles. We also demonstrate that the detected high-frequency PCR errors are highly recurrent and that the position in the template sequence and polymerase-specific substitution preferences are among the major factors influencing the observed PCR error rate.
25 citations
TL;DR: A wide range of effects of predicted residues are found, including some mutations that abolish damage bypass and most of the DinB variants constructed are unable to carry out the extension step of lesion bypass.
Abstract: DinB is one of two Y family polymerases in E. coli and is involved in copying damaged DNA. DinB is specialized to bypass deoxyguanosine adducts that occur at the N2 position, with its cognate lesion being the furfuryl adduct. Active site residues have been identified that make contact with the substrate and carry out deoxynucleotide triphosphate (dNTP) addition to the growing DNA strand. In DNA polymerases, these include negatively charged aspartate and glutamate residues (D8, D103, and E104 in E. coli DNA polymerase IV DinB). These residues position the essential magnesium ions correctly to facilitate nucleophilic attack by the primer hydroxyl group on the α-phosphate group of the incoming dNTP. To study the contribution of DinB residues to lesion bypass, the computational methods THEMATICS and POOL were employed. These methods correctly predict the known active site residues, as well as other residues known to be important for activity. In addition, these methods predict other residues involved in substrate binding as well as more remote residues. DinB variants with mutations at the predicted positions were constructed and assayed for bypass of the N2-furfuryl-dG lesion. We find a wide range of effects of predicted residues, including some mutations that abolish damage bypass. Moreover, most of the DinB variants constructed are unable to carry out the extension step of lesion bypass. The use of computational prediction methods represents another tool that will lead to a more complete understanding of translesion DNA synthesis. Mol. Mutagen. 2012. © 2012 Wiley Periodicals, Inc.
21 citations
Cites background from "Mutation S543N in the thumb subdoma..."
...…Thermus aquaticus DNA polymerase I can impact its ability to replicate DNA; S543N in the thumb region can hinder template-dependent pausing during replication [Ignatov et al., 1999] and a number of mutations along the O helix (F667L, A661E, I665T) change the mutation spectrum [Suzuki et al., 2000]....
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...Distal mutations in two separate domains in Thermus aquaticus DNA polymerase I can impact its ability to replicate DNA; S543N in the thumb region can hinder template-dependent pausing during replication [Ignatov et al., 1999] and a number of mutations along the O helix (F667L, A661E, I665T) change the mutation spectrum [Suzuki et al....
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TL;DR: This chapter describes some of the CSR protocols used successfully to evolve variants of T. aquaticus Pol I (Taq) polymerase with novel and useful properties, such as increased thermostability or resistance to the potent inhibitor, heparin, from a repertoire of randomly mutated Taq polymerase genes.
Abstract: Compartmentalized self-replication (CSR) is a novel method for the directed evolution of enzymes and, in particular, polymerases. In its simplest form, CSR consists of a simple feedback loop involving a polymerase that replicates only its own encoding gene (self-replication). Self-replication occurs in discrete, spatially separate, noncommunicating compartments formed by a heat-stable water-in-oil emulsion. Compartmentalization ensures the linkage of phenotype and genotype (i.e., it ensures that each polymerase replicates only its own encoding gene to the exclusion of those in the other compartments). As a result, adaptive gains by the polymerase directly (and proportionally) translate into genetic amplification of the encoding polymerase gene. CSR has proven to be a useful strategy for the directed evolution of polymerases directly from diverse repertoires of polymerase genes. In this chapter, we describe some of the CSR protocols used successfully to evolve variants of T. aquaticus Pol I (Taq) polymerase with novel and useful properties, such as increased thermostability or resistance to the potent inhibitor, heparin, from a repertoire of randomly mutated Taq polymerase genes.
19 citations
References
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TL;DR: The base-pair fidelity, the ability to use PCR products as primers, and the maximum yield of target fragment were increased and improvements were achieved by the combination of an exonuclease-free, N-terminal deletion mutant of Taq DNA polymerase, Klentaq1.
Abstract: A target length limitation to PCR amplification of DNA has been identified and addressed. Concomitantly, the base-pair fidelity, the ability to use PCR products as primers, and the maximum yield of target fragment were increased. These improvements were achieved by the combination of a high level of an exonuclease-free, N-terminal deletion mutant of Taq DNA polymerase, Klentaq1, with a very low level of a thermostable DNA polymerase exhibiting a 3'-exonuclease activity (Pfu, Vent, or Deep Vent). At least 35 kb can be amplified to high yields from 1 ng of lambda DNA template.
1,185 citations
TL;DR: The 3.3-Å resolution crystal structure of the large proteolytic fragment of Escherichia coli DNA polymerase I complexed with deoxythymidine monophosphate consists of two domains, the smaller of which binds zinc-deoxythmidine monophile and the most striking feature of the larger domain is a deep crevice of the appropriate size and shape for binding double-stranded B-DNA.
Abstract: The 33-A resolution crystal structure of the large proteolytic fragment of Escherichia coli DNA polymerase I complexed with deoxythymidine monophosphate consists of two domains, the smaller of which binds zinc-deoxythymidine monophosphate The most striking feature of the larger domain is a deep crevice of the appropriate size and shape for binding double-stranded B-DNA A flexible subdomain may allow the enzyme to surround completely the DNA substrate, thereby allowing processive nucleotide polymerization without enzyme dissociation
834 citations
TL;DR: The ability to amplify DNA sequences of 10-40 kb will bring the speed and simplicity of PCR to genomic mapping and sequencing and facilitate studies in molecular genetics.
Abstract: We have used the polymerase chain reaction (PCR) to amplify up to 22 kb of the beta-globin gene cluster from human genomic DNA and up to 42 kb from phaga lambda DNA. We have also amplified 91 human genomic inserts of 9-23 kb directly from recombinant lambda plaques. To do this, we increased pH, added glycerol and dimethyl sulfoxide, decreased denaturation times, increased extension times, and used a secondary thermostable DNA polymerase that possesses a 3'-to 5'-exonuclease, or "proofreading," activity. Our "long PCR" protocols maintain the specificity required for targets in genomic DNA by using lower levels of polymerase and temperature and salt conditions for specific primer annealing. The ability to amplify DNA sequences of 10-40 kb will bring the speed and simplicity of PCR to genomic mapping and sequencing and facilitate studies in molecular genetics.
727 citations
TL;DR: Betaine improves the co-amplification of the two alternatively spliced variants of the prostate-specific membrane antigen mRNA as well as the amplification of the coding cDNA region of c-jun.
Abstract: Betaine improves the co-amplification of the two alternatively spliced variants of the prostate-specific membrane antigen mRNA as well as the amplification of the coding cDNA region of c-jun. It is suggested that betaine improves the amplification of these genes by reducing the formation of secondary structure caused by GC-rich regions and, therefore, may be generally applicable to ameliorate the amplification of GC-rich DNA sequences.
550 citations
TL;DR: Although this cocrystal structure appears to be an editing complex, it suggests that the primer strand approaches the catalytic site of the polymerase from the direction of the 3' to 5' exonuclease domain and that the duplex DNA product may bend to enter the cleft that contains the polypeptide catalyst site.
Abstract: Klenow fragment of Escherichia coli DNA polymerase I, which was cocrystallized with duplex DNA, positioned 11 base pairs of DNA in a groove that lies at right angles to the cleft that contains the polymerase active site and is adjacent to the 3' to 5' exonuclease domain. When the fragment bound DNA, a region previously referred to as the "disordered domain" became more ordered and moved along with two helices toward the 3' to 5' exonuclease domain to form the binding groove. A single-stranded, 3' extension of three nucleotides bound to the 3' to 5' exonuclease active site. Although this cocrystal structure appears to be an editing complex, it suggests that the primer strand approaches the catalytic site of the polymerase from the direction of the 3' to 5' exonuclease domain and that the duplex DNA product may bend to enter the cleft that contains the polymerase catalytic site.
490 citations