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Strategies to Potentiate the Cellular Poly(ADP-ribosyl)ation Response to DNA Damage

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Strategies to Potentiate the Cellular Poly(ADP-ribosyl)ation Response to DNA Damage

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KUNZMANN, Andrea, 2009. Strategies to Potentiate the Cellular Poly(ADP-ribosyl)ation Response to DNA Damage [Dissertation]. Konstanz: University of Konstanz

@phdthesis{Kunzmann2009Strat-8669, title={Strategies to Potentiate the Cellular Poly(ADP-ribosyl)ation Response to DNA Damage}, year={2009}, author={Kunzmann, Andrea}, address={Konstanz}, school={Universität Konstanz} }

Kunzmann, Andrea Strategien zur Potenzierung der zellulären poly(ADP-ribosyl)ierung als Antwort auf DNA Schädigung Strategies to Potentiate the Cellular Poly(ADP-ribosyl)ation Response to DNA Damage application/pdf eng Kunzmann, Andrea 2009 terms-of-use Poly(ADP-ribosyl)ation is a posttranslational modification of cellular proteins, which is mainly catalyzed by poly(ADP-ribose) polymerase 1 (PARP1) by using NAD+ as substrate. The catalytic activity of PARP1 is known to be triggered by the binding of PARP1 to broken DNA via its two aminoterminal zinc finger motifs. DNA strand break-induced poly(ADP-ribosyl)ation is linked to DNA repair and maintenance of genomic stability.<br />Up to now, little information exists on the biological consequences of an enhanced poly(ADP-ribosyl)ation response.<br />The aim of the PhD project was to identify compounds that are able to enhance cellular poly(ADP-ribosyl)ation and to investigate if enhanced cellular poly(ADPribosyl)ation improves DNA repair and leads to higher genomic stability. To address these questions, two different approaches were used. The first one is to increase cellular PARP1 activity by supplementation of the nutritional factors zinc or nicotinic acid (NA) respectively; the second one is overexpression of human PARP1.<br />The determination of PARP1 activity as function of cellular zinc revealed a positive correlation between PARP1 activity and zinc status. To avoid the rapid decrease of the cellular NAD+ pool in PBMC, which was observed concomitant with polymer formation, cellular NAD+ pools were successfully replenished by ex-vivo supplementation of PBMC with the NAD+ precursor NA. NA supplementation led to substantially increased poly(ADP-ribose) formation after X-irradiation. In parallel cell survival was increased in NA supplemented PBMC when exposed to X-irradiation.<br />By contrast, overexpression of PARP1 resulted in reduced cell viability and a delay in DNA repair. However, frequency of micronuclei was reduced in PARP1-overexpressing cells, which indicates higher genomic stability.<br />Finally, since PARP1 acts as a catalytic dimer, with one molecule catalyzing automodification of the other, enzymatic studies were performed to determine the minimal fragment of PARP1, which can operate as a partner for automodification by wt-PARP1 and restores full enzymatic activity of wt-PARP1. Various fragments were cloned, expressed in E.coli and purified. In-vitro activity assays revealed, however that none of the PARP1 fragments generated was sufficient as an interaction partner for wt-PARP1 to reconstitute full activity of the enzyme. 2011-05-04T10:02:09Z

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