EvoEPIGEN - Evolved Replication Systems of Epigenetics

Institutionen
  • FB Chemie
Publikationen
    Raghunathan, Govindan; Marx, Andreas (2019): Identification of Thermus aquaticus DNA polymerase variants with increased mismatch discrimination and reverse transcriptase activity from a smart enzyme mutant library Scientific reports. 2019, 9, 590. eISSN 2045-2322. Available under: doi: 10.1038/s41598-018-37233-y

Identification of Thermus aquaticus DNA polymerase variants with increased mismatch discrimination and reverse transcriptase activity from a smart enzyme mutant library

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DNA polymerases the key enzymes for several biotechnological applications. Obviously, nature has not evolved these enzymes to be compatible with applications in biotechnology. Thus, engineering of a natural scaffold of DNA polymerases may lead to enzymes improved for several applications. Here, we investigated a two-step approach for the design and construction of a combinatorial library of mutants of KlenTaq DNA polymerase. First, we selected amino acid sites for saturation mutagenesis that interact with the primer/template strands or are evolutionarily conserved. From this library, we identified mutations that little interfere with DNA polymerase activity. Next, these functionally active mutants were combined randomly to construct a second library with enriched sequence diversity. We reasoned that the combination of mutants that have minuscule effect on enzyme activity and thermostability, will result in entities that have an increased mutation load but still retain activity. Besides activity and thermostability, we screened the library for entities with two distinct properties. Indeed, we identified two different KlenTaq DNA polymerase variants that either exhibit increased mismatch extension discrimination or increased reverse transcription PCR activity, respectively.

Forschungszusammenhang (Projekte)

    Kropp, Heike M.; Dürr, Simon Leonard; Peter, Christine; Diederichs, Kay; Marx, Andreas (2018): Snapshots of a modified nucleotide moving through the confines of a DNA polymerase Proceedings of the National Academy of Sciences of the United States of America. 2018, 115(40), pp. 9992-9997. ISSN 0027-8424. eISSN 1091-6490. Available under: doi: 10.1073/pnas.1811518115

Snapshots of a modified nucleotide moving through the confines of a DNA polymerase

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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.

Forschungszusammenhang (Projekte)

    Huber, Claudia; Marx, Andreas (2017): Variants of sequence family B Thermococcus kodakaraensis DNA polymerase with increased mismatch extension selectivity PLoS ONE. 2017, 12(8), e0183623. eISSN 1932-6203. Available under: doi: 10.1371/journal.pone.0183623

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

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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.

Forschungszusammenhang (Projekte)

    Kropp, Heike M.; Betz, Karin; Wirth, Johannes; Diederichs, Kay; Marx, Andreas (2017): Crystal structures of ternary complexes of archaeal B-family DNA polymerases PloS ONE. 2017, 12(12), e0188005. eISSN 1932-6203. Available under: doi: 10.1371/journal.pone.0188005

Crystal structures of ternary complexes of archaeal B-family DNA polymerases

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Archaeal B-family polymerases drive biotechnology by accepting a wide substrate range of chemically modified nucleotides. By now no structural data for archaeal B-family DNA polymerases in a closed, ternary complex are available, which would be the basis for developing next generation nucleotides. We present the ternary crystal structures of KOD and 9°N DNA polymerases complexed with DNA and the incoming dATP. The structures reveal a third metal ion in the active site, which was so far only observed for the eukaryotic B-family DNA polymerase δ and no other B-family DNA polymerase. The structures reveal a wide inner channel and numerous interactions with the template strand that provide space for modifications within the enzyme and may account for the high processivity, respectively. The crystal structures provide insights into the superiority over other DNA polymerases concerning the acceptance of modified nucleotides.

Forschungszusammenhang (Projekte)

    Aschenbrenner, Joos; Marx, Andreas (2016): Direct and site-specific quantification of RNA 2′-O-methylation by PCR with an engineered DNA polymerase Nucleic Acids Research. 2016, 44(8), pp. 3495-3502. ISSN 0301-5610. eISSN 1362-4962. Available under: doi: 10.1093/nar/gkw200

Direct and site-specific quantification of RNA 2′-O-methylation by PCR with an engineered DNA polymerase

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Methylation of the 2'-hydroxyl-group of ribonucleotides is found in all major classes of RNA in eukaryotes and is one of the most abundant posttranscriptional modifications of stable RNAs. In spite of intense studies, the multiple functions of RNA 2'-O-methylation are still not understood. One major obstacle in the field are the technical demanding detection methods, which are typically laborious and do not always deliver unambiguous results. We present a thermostable KlenTaq DNA polymerase variant with significant reverse transcription activity that is able to discriminate 2'-O-methylated from unmethylated RNAs. The engineered enzyme catalyzes DNA synthesis from DNA as well as RNA templates and enables expeditious quantification of 2'-O-methylation of individual nucleotides directly from total RNA extracts by a simple qRT-PCR.

Forschungszusammenhang (Projekte)

    von Watzdorf, Janina; Leitner, Kim Michaela; Marx, Andreas (2016): Modified Nucleotides for Discrimination between Cytosine and the Epigenetic Marker 5-Methylcytosine Angewandte Chemie International Edition. 2016, 55(9), pp. 3229-3232. ISSN 1433-7851. eISSN 1521-3773. Available under: doi: 10.1002/anie.201511520

Modified Nucleotides for Discrimination between Cytosine and the Epigenetic Marker 5-Methylcytosine

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5-Methyl-2'-deoxycytosine, the most common epigenetic marker of DNA in eukaryotic cells, plays a key role in gene regulation and affects various cellular processes such as development and carcinogenesis. Therefore, the detection of 5mC can serve as an important biomarker for diagnostics. Here we describe that modified dGTP analogues as well as modified primers are able to sense the presence or absence of a single methylation of C, even though this modification does not interfere directly with Watson-Crick nucleobase pairing. By screening several modified nucleotide scaffolds, O6 -modified 2'-deoxyguanosine analogues were identified as discriminating between C and 5mC. These modified nucleotides might find application in site-specific 5mC detection, for example, through real-time PCR approaches.

Forschungszusammenhang (Projekte)

    Huber, Claudia; von Watzdorf, Janina; Marx, Andreas (2016): 5-methylcytosine-sensitive variants of Thermococcus kodakaraensis DNA polymerase Nucleic Acids Research. 2016, 44(20), pp. 9881-9890. ISSN 0305-1048. eISSN 1362-4962. Available under: doi: 10.1093/nar/gkw812

5-methylcytosine-sensitive variants of Thermococcus kodakaraensis DNA polymerase

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DNA methylation of cytosine in eukaryotic cells is a common epigenetic modification, which plays an important role in gene expression and thus affects various cellular processes like development and carcinogenesis. The occurrence of 5-methyl-2'-deoxycytosine (5mC) as well as the distribution pattern of this epigenetic marker were shown to be crucial for gene regulation and can serve as important biomarkers for diagnostics. DNA polymerases distinguish little, if any, between incorporation opposite C and 5mC, which is not surprising since the site of methylation is not involved in Watson-Crick recognition. Here, we describe the development of a DNA polymerase variant that incorporates the canonical 2'-deoxyguanosine 5'-monophosphate (dGMP) opposite C with higher efficiency compared to 5mC. The variant of Thermococcus kodakaraensis (KOD) exo- DNA polymerase was discovered by screening mutant libraries that were built by rational design. We discovered that an amino acid substitution at a single site that does not directly interact with the templating nucleobase, may alter the ability of the DNA polymerase in processing C in comparison to 5mC. Employing these findings in combination with a nucleotide, which is fluorescently labeled at the terminal phosphate, indicates the potential use of the mutant DNA polymerase in the detection of 5mC.

Forschungszusammenhang (Projekte)

    von Watzdorf, Janina; Marx, Andreas (2016): 6-Substituted 2-Aminopurine-2′-deoxyribonucleoside 5′-Triphosphates that Trace Cytosine Methylation ChemBioChem. 2016, 17(16), pp. 1532-1540. ISSN 1439-4227. eISSN 1439-7633. Available under: doi: 10.1002/cbic.201600245

6-Substituted 2-Aminopurine-2′-deoxyribonucleoside 5′-Triphosphates that Trace Cytosine Methylation

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Gene expression is extensively regulated by the occurrence and distribution of the epigenetic marker 2'-deoxy 5-methylcytosine (5mC) in genomic DNA. Because of its effects on tumorigenesis there is an important link to human health. In addition, detection of 5mC can serve as an outstanding biomarker for diagnostics as well as for disease therapy. Our previous studies have already shown that, by processing O(6) -alkylated 2'-deoxyguanosine triphosphate (dGTP) analogues, DNA polymerases are able to sense the presence of a single 5mC unit in a template. Here we present the synthesis and evaluation of an extended toolbox of 6-substituted 2-aminopurine-2'-deoxyribonucleoside 5'-triphosphates modified at position 6 with various functionalities. We found that sensing of 5-methylation by this class of nucleotides is more general, not being restricted to O(6) -alkyl modification of dGTP but also applying to other functionalities.

Forschungszusammenhang (Projekte)

    Aschenbrenner, Joos; Drum, Matthias; Topal, Hüsnü; Wieland, Markus; Marx, Andreas (2014): Direct Sensing of 5-Methylcytosine by Polymerase Chain Reaction Angewandte Chemie International Edition. 2014, 53(31), pp. 8154-8158. ISSN 1433-7851. eISSN 1521-3773. Available under: doi: 10.1002/anie.201403745

Direct Sensing of 5-Methylcytosine by Polymerase Chain Reaction

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The epigenetic control of genes by the methylation of cytosine resulting in 5-methylcytosine (5mC) has fundamental implications for human development and disease. Analysis of alterations in DNA methylation patterns is an emerging tool for cancer diagnostics and prognostics. Here we report that two thermostable DNA polymerases, namely the DNA polymerase KlenTaq derived from Thermus aquaticus and the KOD DNA polymerase from Thermococcus kodakaraensis, are able to extend 3′-mismatched primer strands more efficiently from 5 mC than from unmethylated C. This feature was advanced by generating a DNA polymerase mutant with further improved 5mC/C discrimination properties and its successful application in a novel methylation-specific PCR approach directly from untreated human genomic DNA.

Forschungszusammenhang (Projekte)

Mittelgeber
Name Finanzierungstyp Kategorie Kennziffer
Europäische Union Drittmittel Forschungsförderprogramm 805/13
Weitere Informationen
Laufzeit: 06.12.2013 – 31.01.2019