5-methylcytosine-sensitive variants of<i>Thermococcus kodakaraensis</i>DNA polymerase

Huber, Claudia; Watzdorf, Janina von; Marx, Andreas

doi:10.1093/nar/gkw812

Cited by 16 publications

(21 citation statements)

References 59 publications

(69 reference statements)

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“…Significant differences in fidelity were observed with AMV reverse transcriptase, but not with any of the investigated family A or family B DNA polymerases. Three previous studies from our group [ 37 , 49 , 51 ] as well as the presented work clearly show the influence of cytosine methylation on DNA polymerase fidelity. The addition of the methyl group might alter substrate recognition of the enzyme, thus changing the mismatch extension properties.…”

Section: Discussionsupporting

confidence: 78%

“…After cloning, the KOD pol libraries were expressed, protein production was checked by SDS gel analysis to ensure little variations in protein levels and heat-treated bacterial lysates were screened for PCR activity. For this, a fragment of the human NANOG gene was amplified with corresponding primers [ 51 ] and DNA double strands were visualized in real time PCR reactions utilizing SYBR Green I. The resulting amplification curves gave direct evidence whether a given mutation resulted in a PCR-active or -inactive enzyme variant.…”

Section: Resultsmentioning

confidence: 99%

“…The DNA polymerase from Thermococcus kodakaraensis is a prominent member of sequence family B and our recent studies have shown the potential of engineering this DNA polymerase to be able to recognize mismatched base pairs [ 49 ]. Some of our previous works have shown that the combination of site specific mutagenesis and screening techniques in order to generate tailor-made enzymes to explore the scope of altered DNA polymerase selectivity for both biotechnological as well as diagnostic applications is a promising approach [ 37 , 49 – 51 ].…”

Section: Introductionmentioning

confidence: 99%

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Variants of sequence family B Thermococcus kodakaraensis DNA polymerase with increased mismatch extension selectivity

Huber

Marx

2017

PLoS ONE

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Fidelity and selectivity of DNA polymerases are critical determinants for the biology of life, as well as important tools for biotechnological applications. DNA polymerases catalyze the formation of DNA strands by adding deoxynucleotides to a primer, which is complementarily bound to a template. To ensure the integrity of the genome, DNA polymerases select the correct nucleotide and further extend the nascent DNA strand. Thus, DNA polymerase fidelity is pivotal for ensuring that cells can replicate their genome with minimal error. DNA polymerases are, however, further optimized for more specific biotechnological or diagnostic applications. Here we report on the semi-rational design of mutant libraries derived by saturation mutagenesis at single sites of a 3'-5'-exonuclease deficient variant of Thermococcus kodakaraensis DNA polymerase (KOD pol) and the discovery for variants with enhanced mismatch extension selectivity by screening. Sites of potential interest for saturation mutagenesis were selected by their proximity to primer or template strands. The resulting libraries were screened via quantitative real-time PCR. We identified three variants with single amino acid exchanges-R501C, R606Q, and R606W-which exhibited increased mismatch extension selectivity. These variants were further characterized towards their potential in mismatch discrimination. Additionally, the identified enzymes were also able to differentiate between cytosine and 5-methylcytosine. Our results demonstrate the potential in characterizing and developing DNA polymerases for specific PCR based applications in DNA biotechnology and diagnostics.

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Section: Discussionsupporting

confidence: 78%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Variants of sequence family B Thermococcus kodakaraensis DNA polymerase with increased mismatch extension selectivity

Huber

Marx

2017

PLoS ONE

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“…35 These approaches have yielded polymerases with useful properties including increased NTP incorporation, 36,37 RNA reverse transcriptase (RT) activity, 38 or 2′SeMeUTP ( 24 ) incorporation, 39 improved discrimination against mismatches 40 or epigenetic methylation marks, 41 or, as a result of screening variants of T7 RNA polymerase, a mutant efficient at transcribing all four 2′OMethyl ( 21 ) (2′OMe) nucleotides. 42 …”

Section: Overcoming Polymerase Substrate Specificitymentioning

confidence: 99%

Exploring the Chemistry of Genetic Information Storage and Propagation through Polymerase Engineering

2017

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ConspectusNucleic acids are a distinct form of sequence-defined biopolymer. What sets them apart from other biopolymers such as polypeptides or polysaccharides is their unique capacity to encode, store, and propagate genetic information (molecular heredity). In nature, just two closely related nucleic acids, DNA and RNA, function as repositories and carriers of genetic information. They therefore are the molecular embodiment of biological information. This naturally leads to questions regarding the degree of variation from this seemingly ideal “Goldilocks” chemistry that would still be compatible with the fundamental property of molecular heredity.To address this question, chemists have created a panoply of synthetic nucleic acids comprising unnatural sugar ring congeners, backbone linkages, and nucleobases in order to establish the molecular parameters for encoding genetic information and its emergence at the origin of life. A deeper analysis of the potential of these synthetic genetic polymers for molecular heredity requires a means of replication and a determination of the fidelity of information transfer. While non-enzymatic synthesis is an increasingly powerful method, it currently remains restricted to short polymers. Here we discuss efforts toward establishing enzymatic synthesis, replication, and evolution of synthetic genetic polymers through the engineering of polymerase enzymes found in nature.To endow natural polymerases with the ability to efficiently utilize non-cognate nucleotide substrates, novel strategies for the screening and directed evolution of polymerase function have been realized. High throughput plate-based screens, phage display, and water-in-oil emulsion technology based methods have yielded a number of engineered polymerases, some of which can synthesize and reverse transcribe synthetic genetic polymers with good efficiency and fidelity.The inception of such polymerases demonstrates that, at a basic level at least, molecular heredity is not restricted to the natural nucleic acids DNA and RNA, but may be found in a large (if finite) number of synthetic genetic polymers. And it has opened up these novel sequence spaces for investigation. Although largely unexplored, first tentative forays have yielded ligands (aptamers) against a range of targets and several catalysts elaborated in a range of different chemistries. Finally, taking the lead from established DNA designs, simple polyhedron nanostructures have been described.We anticipate that further progress in this area will expand the range of synthetic genetic polymers that can be synthesized, replicated, and evolved providing access to a rich sequence, structure, and phenotypic space. “Synthetic genetics”, that is, the exploration of these spaces, will illuminate the chemical parameter range for en- and decoding information, 3D folding, and catalysis and yield novel ligands, catalysts, and nanostructures and devices for applications in biotechnology and medicine.

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“…To date, a large number of studies pursuing different biotechnological goals have documented in vitro evolution of DNA polymerases. Accordingly, DNA polymerases with expanded recognition of natural ( 8 – 11 ) or unnatural ( 12 – 19 ) substrates, increased processivity ( 20 – 22 ), altered fidelity ( 23 – 26 ), enhanced resistance to inhibitors ( 27 , 28 ) or thermostability ( 29 ) have been engineered by different methods. In most of these cases, researches have taken advantage of the thermostability or processivity traits of polymerases, allowing the use of exponential amplification or directed selection techniques to find desired polymerase variants ( 30 ).…”

Section: Introductionmentioning

confidence: 99%

Engineering human PrimPol into an efficient RNA-dependent-DNA primase/polymerase

Agudo

Calvo

Martínez‐Jiménez

et al. 2017

Nucleic Acids Research

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We have developed a straightforward fluorometric assay to measure primase-polymerase activity of human PrimPol (HsPrimPol). The sensitivity of this procedure uncovered a novel RNA-dependent DNA priming-polymerization activity (RdDP) of this enzyme. In an attempt to enhance HsPrimPol RdDP activity, we constructed a smart mutant library guided by prior sequence-function analysis, and tested this library in an adapted screening platform of our fluorometric assay. After screening less than 500 variants, we found a specific HsPrimPol mutant, Y89R, which displays 10-fold higher RdDP activity than the wild-type enzyme. The improvement of RdDP activity in the Y89R variant was due mainly to an increased in the stabilization of the preternary complex (protein:template:incoming nucleotide), a specific step preceding dimer formation. Finally, in support of the biotechnological potential of PrimPol as a DNA primer maker during reverse transcription, mutant Y89R HsPrimPol rendered up to 17-fold more DNA than with random hexamer primers.

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5-methylcytosine-sensitive variants ofThermococcus kodakaraensisDNA polymerase

Cited by 16 publications

References 59 publications

Variants of sequence family B Thermococcus kodakaraensis DNA polymerase with increased mismatch extension selectivity

Variants of sequence family B Thermococcus kodakaraensis DNA polymerase with increased mismatch extension selectivity

Exploring the Chemistry of Genetic Information Storage and Propagation through Polymerase Engineering

Engineering human PrimPol into an efficient RNA-dependent-DNA primase/polymerase

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