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Cloning and Characterisation of the DNA Polymerase A of the Extremely Radioresistant Organism Deinococcus radiodurans

Cloning and Characterisation of the DNA Polymerase A of the Extremely Radioresistant Organism Deinococcus radiodurans

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HEINZ, Kathrin, 2007. Cloning and Characterisation of the DNA Polymerase A of the Extremely Radioresistant Organism Deinococcus radiodurans

@phdthesis{Heinz2007Cloni-9937, title={Cloning and Characterisation of the DNA Polymerase A of the Extremely Radioresistant Organism Deinococcus radiodurans}, year={2007}, author={Heinz, Kathrin}, address={Konstanz}, school={Universität Konstanz} }

Heinz, Kathrin deposit-license application/pdf Klonierung und Charakterisierung der DNA Polymerase A aus dem strahlenresistenten Organismus Deinococcus radiodurans 2011-03-24T18:15:26Z Included in this work is the cloning and basic characterisation of the DNA polymerase A from the extremely radioresistant organism Deinococcus radiodurans.<br />According to sequence alignments and identities to Taq polymerase and DNA polymerase I from E.coli the shortened gene of polA (polA*) analog to Klentaq-1 was cloned and expressed in E. coli. A protocol for expression and purification from the t-RNA supplemented strain E. coli BL21 DE3 RIL* was established.<br />The purified polA* enzyme was tested successfully for polymerase activity. Experiments showed that polA* is not thermostable, although the tight relations of the Deinococcacae to the Thermus family.<br />It was published that D. radiodurans accumulates actively manganese ions intracellular during radiation and DNA damaging events, but the comprehensive meaning of this is still not fully understood. Experiments were conducted to show if polA* is active only with Mg2+ or also with additional manganese ions. The results showed that Mn2+ up to 1 mM can increase the enzyme activity, higher concentrations will inhibit polA*.<br />In a next set of experiments the template dependence of polA* was tested. A DNA primer-template system, an RNA system and a DNA-RNA mixed system were tested for either NTP or dNTP incorporation. Because only DNA primer-template elongation with dNTPs could be detected in quantitative yields, polA* is a DNA-dependent DNA polymerase.<br />For further characterisation the selectivity of nucleotide incorporation by polA* was tested. Qualitative experiments with all possible combinations of template and incorporated nucleotide were conducted. It was observed that under conditions with low enzyme concentrations polA* only inserts the matching nucleotide. This high selectivity can be modified by addition of manganese ions. To be able to compare these data for polA* with other polymerases steady-state kinetic were measured for all reactions with and without 1 mM Mn2+.<br />It was shown that polA* is involved in DNA repair mechanisms of D. radiodurans. That is why the next set of experiments should show the ability of polA* for DNA lesion bypass. Modifications like an abasic site, oxoAdenine and oxoGuanine were intruduced to template strands, which were incubated with either a primer for running start conditions or standing start conditions. In primer extension experiments was observed that the incorporation stops prior to the lesion on the template strand. Higher enzyme concentrations and 1 mM Mn2+ enabled further synthesis - a nucleotide was incorporated opposite to the lesion. In case of the oxo-modifications also full-lenghth elongation could be detected. Next the single nucleotide incorporation opposite to the lesions was examined. The results showed that polA* incorporates mainly dATP opposite to the abasic site and the matching nucleotide opposite to oxo-modified bases. Again steady-state kinetics were conducted for all nucleotide combinations and with and without manganese ions.<br />The wildtype enzyme of polA consists of a polymerase domain and a 5´-3´exonuclease. The latter is used to remove 5´overhangs from DNA double strains. This can be done by removing the flap directly at the beginning of the new strand or by strand displacement synthesis. To test whether polA* is able to displace an already existing DNA strand during synthesis, a primer-template system was developed which contains a single-stranded gap between two double-stranded parts. It could be shown that polA* can displace the strand with higher enzyme concentrations. Modification of the results by addition of manganese ions could not be observed.<br />To gain further insights into the action of polA, a crystal structure of the protein would be neccessary. Beside this experiments with purified and partially purified components of already known repair pathways of D. radiodurans could give more details of the involvement and action of polA. Cloning and Characterisation of the DNA Polymerase A of the Extremely Radioresistant Organism Deinococcus radiodurans 2011-03-24T18:15:26Z Heinz, Kathrin eng 2007

Dateiabrufe seit 01.10.2014 (Informationen über die Zugriffsstatistik)

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