New insights into selectivity of DNA polymerases : a combinatorial approach

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2007
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Rudinger, Nicolas Zackes
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Änderung der Selektivität von DNA-Polymerasen - Ein kombinatorischer Ansatz
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Abstract
DNA polymerase fidelity is of immense biological importance due to the fundamental requirement for accurate DNA synthesis in both replicative and repair processes. Subtle hydrogen bonding networks between DNA polymerases and their primer/template substrates are believed to have impact on DNA polymerase selectivity.
Summerer showed that increased DNA polymerase mismatch extension fidelity is feasible by modulating the steric and functional properties of a conserved enzyme motif, namely motif C. Specifically abolishing hydrogen bonding to the minor groove resulted in enhanced polymerase fidelity.
In this work it could be demonstrated that these effects can be transferred into a thermostable member of the family A DNA polymerases, namely Taq. The mutations led to an enzyme that displays significantly enhanced mismatch extension fidelity. Although the mutations were introduced into the highly conserved polymerase motif C, it is of note that KF exo- and Taq do not share a high protein sequence homology (30%). These results indicate a new mechanism of mismatch sensing for family A DNA polymerases. Furthermore, wide structural conservation of motif C may reflect generality of this mechanism regarding the function of a wider range of nucleotidyl transferases. Hence, motif C variation potentially represents a general approach to modulate the fidelity of most nucleotidyl transferases for various biotechnological applications.
In order to further substantiate the generality of the enhanced fidelity that results from altered properties of motif C in family A DNA polymerases, mutations were introduced into motif C of a thermophilic family B DNA polymerase, namely Pfu DNA polymerase. Apparently, the DNA polymerase of phage RB69 is the sole B family DNA polymerase of which the structure of a ternary complex is available. Although belonging to the same DNA polymerase family, Pfu and RB69 DNA polymerases share a sequence homology of only 16%. Based on structural data of RB69 DNA polymerase, rationally designed mutations were introduced into Pfu DNA polymerase.
Herein, deleting H-bonding capability by rationally designed hydrophobic substitution mutations without significantly altering sterical demand, results in a more selective enzyme. Furthermore, a single atom replacement within the DNA substrate through chemical modification (2-thiothymidine), which leads to an altered H-bonding acceptor potential and steric demand of the DNA substrate, further increased the selectivity of the tested polymerases. The results presented in this work give new insights into fidelity mechanisms of family B high fidelity DNA polymerases and describe for the first time the increase of the selectivity of a family B DNA polymerase.
The impact of hydrophobic modifications on DNA polymerase selectivity - enzyme- and substrate wise -, further highlights the influence of shape and hydrogen bonding capability on DNA polymerase selectivity.
Taken together, the mutant polymerases described by Summerer and in this work that carry hydrophobic mutations within motif C display enhanced mismatch extension fidelity. The succsessfulf transfer of these hydrophobic modifications from a mesophilic to a thermophilic DNA polymerase, namely Taq, suggested a general fidelity mechanism for motif C. Furthermore, it is shown that by rational design of hydrophobic modifications in motif C of a family B DNA polymerase, namely Pfu, mismatch extension fidelity can also be enhanced. Additionally, modification of the substrate at a specific hydrogen bonding acceptor position (O-2 of the thymine base) for a motif C residue also and additionally leads to increased selectivity of the polymerases tested. Thus, it seems that modifications leading to loss or attenuation of the H-bonding interactions between motif C and the DNA minor groove can enhance the fidelity of both family A and B DNA polymerases and make a general fidelity mechanism for motif C very likely.
Apart from the results on the influence of motif C on mismatch extension selectivity, other mechanisms that influence or are responsible for mismatch extension selectivity in archaeal B family DNA polymerases have not been explored. In order to explore new mechanisms that influence selectivity, randomised mutagenesis of the Pfu DNA polymerase ORF by epPCR was employed. Two mutants displaying higher mismatch extension selectivity in qualitative assays were found. Both mutants carry mutations at remote positions from the active site. The mutations do not alter charge and polarity, but mostly sterical demand and solvent accessibility of the respective residues. Accordingly, the altered side chains of the mutated residues could result in enhanced or decreased flexibility of the respective loop. It seems that these mutations somehow feed through the enzyme and thus have an influence on polymerase activity and selectivity.
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Die genaue Synthese während der Reparatur und bei replikativen Prozessen ist besonders wichtig.
Fein strukturierten Wasserstoffbrückennetzwerken zwischen der DNA-Polymerase und dem Primer/Templatkomplex wird eine wichtige Rolle in Hinsicht auf die Selektivität von DNA-Polymerasen zugesprochen.
Summerer konnte zeigen, dass erhöhte Fehlpaarungsverlängerungsgenauigkeit durch die Modulation der sterischen und funktionellen Eigenschaften eines konservierten Enzymmotivs, speziell Motiv C, möglich ist. Insbesondere das Entfernen von Wasserstoffbrückenbindungen mit der kleinen Furche der DNA führte zu erhöhter Genuaigkeit der Polymerase.
Im Verlauf dieser Arbeit konnte nun gezeigt werden, dass diese Effekte von einer mesophilen DNA-Polymerase in eine thermostabile DNA-Polymerase, speziell Taq, übertragen werden können. Die Eingeführten Mutationen führten zu einem Enzym, dass eine signifikant erhöhte Fehlpaarungsverlängerungsgenauigkeit zeigt. Obwohl die Mutationen in das hoch konservierte Polymerasemotiv C eingeführt wurden, ist zu beachten, dass die DNA-Polymerasen KF und Taq nicht sehr homolog (30% Proteinsequenzhomologie) sind.
Diese Ergebnisse sprechen für einen generellen Mechanismus der Fehlpaarungserkennung in DNA-Polymerasen der Familie A. Des Weiteren spricht die hohe strukturelle Konservierung von Motiv C für über einen weiten Bereich von Nukleotidyltransferasen, ebenfalls für einen generellen Mechanismus. Es wäre daher möglich, die Genauigkeit von vielen Nukleotidyltransferasen durch Veränderungen in Motiv C für spezifische biotechnologische Anwendungen anzupassen.
Um diese Erkenntnisse über die Funktion von Motiv C weiter zu verdeutlichen, wurden rational geplante Mutationen in Motiv C einer thermophilen DNA-Polymerase der Familie B, speziell Pfu eingeführt. Als Modellpolymerase für den rationalen Übertrag, diente die DNA-Polymerase RB69, da es die einzige DNA-Polymerase der Familie B ist, von der eine Kristallstruktur im ternären Komplex vorliegt. Obwohl Pfu und RB69 Mitglied der gleichen Polymerasefamilie sind, zeigen beide eine sehr niedrige Sequenzhomologie von nur 16% (Proteinsequenz). Basierend auf strukturellen Daten von RB69, wurden die gewählten Mutationen in Pfu übertagen.
Hierbei führte das Entfernen der Wasserstoffbrückenbildungsmöglichkeit durch hydrophobe Aminosäureaustausche, ohne den sterischen Anspruch signifikant zu ändern, zu einem selektiveren Enzym. Zusätzlich führte ein spezifischer Einzelatomaustausch im DNA-Substrat durch chemische Modifikation (2-Thiothymidin), der in einem veränderten Wasserstoffbrücken-Akzeptorpotential und sterischem Anspruch resultierte, zu erhöhter Selektivität der getesteten Polymerasen. Die Ergebnisse dieser Arbeit geben neue Einsicht in Selektivitätsmechanismen von hochgenauen DNA-Polymerasen der Familie B. Des Weiteren beschreiben die Ergebnisse das erste Mal eine Selektivitätserhöhung einer DNA-Polymerase der Familie B.
Zusammengefasst zeigt sich, dass die Mutanten, die durch Summerer und in dieser Arbeit beschrieben wurden und hydrophobe Mutationen in Motiv C tragen (Mit Ausnahme der Mutante KF exo- PLQ), erhöhte Fehlpaarungsverlängerungsgenauigkeit haben. Der erfolgreiche Transfer dieser hydrophoben Modifikationen von einer mesophilen in eine thermophile DNA-Polymerase, speziell Taq, legten einen generellen Selektivitätsmechanismus für Motiv C nahe. Des Weiteren, konnten diese Effekte auch in eine DNA-Polymerase der Familie B, speziell Pfu, übertragen werden. Außerdem, führte eine chemische Modifikation des Substrats an einer spezifischen Wasserstoffbrücken-Akzeptorposition (O-2 der Thyminbase) für einen Aminosäurerest des Motiv C zu einer zusätzlichen Erhöhung der Selektivität. Daher scheint es, dass Modifikationen, die einen Verlust oder eine Abschwächung von Wasserstoffbrückenbildung des Motiv C mit der kleinen Furche der DNA bewirken, zu einer erhöhten Selektivität von DNA-Polymerasen aus den Familien A und B führen. Diese Effekte machen einen generellen Selektivitätsmechanismus für das DNA-Polymerasemotiv C sehr wahrscheinlich.
Um neue Mechanismen, die die Selektivität beeinflussen zu erforschen, wurde eine randomisierte Mutagenese der Pfu DNA-Polymerase durchgeführt. Es konnten hierbei zwei Mutanten gefunden werden, die in qualitativen Reaktionen eine höhere Fehlpaarungsverlängerungsgenauigkeit zeigen. Beide Mutanten tragen Mutationen, die weit vom reaktiven Zentrum der Polymerase entfernt sind. Diese Mutationen führen nicht zur Änderung von Ladung oder Polarität, sondern verändern hauptsächlich den sterischen Anspruch und den Zugang des Lösungsmittels (H2O) an den entsprechenden Aminosäureresten. Daher könnten die veränderten Seitenketten zu einer erhöhten oder verminderten Flexibilität der entsprechenden Schleife führen. Es scheint, dass diese Mutationen durch das Enzym wirken und daher einen Einfluss auf die Aktivität und Selektivität haben.
Subject (DDC)
500 Natural Sciences
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DNA polymerase selectivity,hydrophobic mutations,motif C,directed evolution
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Review
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ISO 690RUDINGER, Nicolas Zackes, 2007. New insights into selectivity of DNA polymerases : a combinatorial approach [Dissertation]. Konstanz: University of Konstanz
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  year={2007},
  title={New insights into selectivity of DNA polymerases : a combinatorial approach},
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  address={Konstanz},
  school={Universität Konstanz}
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Although the mutations were introduced into the highly conserved polymerase motif C, it is of note that KF exo- and Taq do not share a high protein sequence homology (30%). These results indicate a new mechanism of mismatch sensing for family A DNA polymerases. Furthermore, wide structural conservation of motif C may reflect generality of this mechanism regarding the function of a wider range of nucleotidyl transferases. Hence, motif C variation potentially represents a general approach to modulate the fidelity of most nucleotidyl transferases for various biotechnological applications.&lt;br /&gt;In order to further substantiate the generality of the enhanced fidelity that results from altered properties of motif C in family A DNA polymerases, mutations were introduced into motif C of a thermophilic family B DNA polymerase, namely Pfu DNA polymerase. 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Additionally, modification of the substrate at a specific hydrogen bonding acceptor position (O-2 of the thymine base) for a motif C residue also and additionally leads to increased selectivity of the polymerases tested. Thus, it seems that modifications leading to loss or attenuation of the H-bonding interactions between motif C and the DNA minor groove can enhance the fidelity of both family A and B DNA polymerases and make a general fidelity mechanism for motif C very likely.&lt;br /&gt;Apart from the results on the influence of motif C on mismatch extension selectivity, other mechanisms that influence or are responsible for mismatch extension selectivity in archaeal B family DNA polymerases have not been explored. In order to explore new mechanisms that influence selectivity, randomised mutagenesis of the Pfu DNA polymerase ORF by epPCR was employed. Two mutants displaying higher mismatch extension selectivity in qualitative assays were found. 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