New insights into selectivity of DNA polymerases : a combinatorial approach

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RUDINGER, Nicolas Zackes, 2007. New insights into selectivity of DNA polymerases : a combinatorial approach

@phdthesis{Rudinger2007insig-9712, title={New insights into selectivity of DNA polymerases : a combinatorial approach}, year={2007}, author={Rudinger, Nicolas Zackes}, address={Konstanz}, school={Universität Konstanz} }

application/pdf Rudinger, Nicolas Zackes 2007 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.<br />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.<br />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.<br />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.<br />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.<br />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.<br />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.<br />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. deposit-license New insights into selectivity of DNA polymerases : a combinatorial approach Änderung der Selektivität von DNA-Polymerasen - Ein kombinatorischer Ansatz 2011-03-24T18:13:53Z 2011-03-24T18:13:53Z Rudinger, Nicolas Zackes eng

Dateiabrufe seit 01.10.2014 (Informationen über die Zugriffsstatistik)

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