Development of methods to investigate poly(ADP-ribose) polymerase-1 activity and DNA base-excision repair in relation to cancer and ageing

Summary The work presented here comprises the development of a new immuno-dot-blot method to detect poly(ADP-ribosyl)ation capacity in cells and the automation of the fluorescence detected alkaline DNA unwinding (FADU) assay to measure DNA strand breaks and repair. Poly(ADP-ribose) polymerase-1 (PARP-1) is involved in DNA base-excision repair and the maintenance of genomic stability, but emerges to have many more functions in cellular physiology and pathophysiology. Cellular poly(ADP-ribosyl)ation capacity is correlated with maximal life span in mammalian species and a with longevity in humans. In this work a new, non-isotopic, simple and reliable method for the assessment of poly(ADP-ribosyl)ation capacity is presented. In experiments comparing fibroblasts from wild-type mice (PARP-1+/+) with those from PARP-1 knockout mice (PARP-1-/-) we could demonstrate that the p(ADPr) formation assessed in this assay is due almost exclusively to PARP-1 and only in trace amounts to PARP-2 or other members of the PARP enzyme family. Here an automated simplified version of the FADU assay is described, that posesses very high reproducibility. To investigate if DNA base-excision repair capacity or half-life time can be correlated with the life span of mammals, a preliminary set of comparative experiments on g-irradiated human and rat blood cells was performed. The percentage of lesions repaired during the first 50 min after irradiation was significantly lower in the rat cells (17 vs 45 ). These data suggest that there might exist a correlation between DNA base excision repair and mammalian longevity. We have also performed experiments comparing DNA base excision repair in PARP-1-/- and in PARP-1+/+ fibroblasts. DNA repair was found to be dramatically impaired in PARP-1-/- cells but very active in PARP-1+/+ cells. These results represent additional clear evidence for the involvement of PARP-1 in base excision repair.

Level thesis:degree_level

thesis.doctoral

Grantor dc:publisher

Universität Heidelberg

Year

2001

Author dc:creator

• Pfeiffer, Ragen

Contributors dc:contributor

• Wießler, Manfred, Professor Doktor

Repository record source_url

http://www.ub.uni-heidelberg.de/archiv/1874

OAI identifier oai:identifier

oai:archiv.ub.uni-heidelberg.de:1874