Application of Pulsed Field Gel Electrophoresis to Determine γ-ray-induced Double-strand Breaks in Yeast Chromosomal Molecules

The frequency of DNA double-strand breaks (dsb) was determined in yeast cells exposed to γ-rays under anoxic conditions. Genomic DNA of treated cells was separated by pulsed field gel electrophoresis, and two different approaches for the evaluation of the gels were employed: (1) The DNA mass distribution profile obtained by electrophoresis was compared to computed profiles, and the number of DSB per unit length was then derived in terms of a fitting procedure; (2) hybridization of selected chromosomes was performed, and a comparison of the hybridization signals in treated and untreated samples was then used to derive the frequency of dsb

Is DNA double strand breaks recognition related to longevity?

In mammals, species lifespan can vary by more than 100 fold (shrew 2 years, bowhead whale 211 years). Despite considerable research, the cellular mechanisms that make this variation possible remain unclear. In regard to these mechanisms, several predictions can be made. First, they must impact fundamental biochemical processes. Second, they would be expected to be related to structural differences between species at the cellular level. Furthermore, the goal would be to find significant correlation between cellular differences and the life span magnitude. As a tool to investigate these mechanisms, we have developed a series of skin fibroblast cell lines derived from mammalian species with a wide variation in lifespan (man, cow, bat, dog, mouse etc.). Using these lines, we have previously shown that the reported dependence of replicative capacity on longevity1 is most likely due to the dependence of replicative capacity on body mass, which is itself correlated with longevity2. Therefore, comparative studies of longevity must address the influence of body mass. The fact that DNA-PKcs and Ku 80 ablation in mice reduces average lifespan approximately 25% and 50% respectively and that Ku 80 null mice display symptoms of premature aging supports the potential role of these nuclear proteins in the aging process. DNA-PKcs and Ku are key proteins in double strand damage recognition. So we tested the capacity of skin fibroblast nuclear extracts from different mammalian species to bind DNA double strand breaks using an electrophoresis super-shift method that we have previously developed and that is now widely used in the field of DNA damage/repair3. Our results indicate that Ku-dependant DNA double strand break recognition increases exponentially with longevity and suggest that an enhanced ability to detect critical DNA damage may be a key requirement for longevity

Significant Correlation of Species Longevity with DNA Double Strand Break-Recognition but not with Telomere Length

The identification of the cellular mechanisms responsible for the wide differences in species lifespan remains one of the major unsolved problems of the biology of aging. We measured the capacity of nuclear protein to recognize DNA double strand breaks (DSB) and telomere length of skin fibroblasts derived from mammalian species that exhibit wide differences in longevity. Our results indicate DNA DSB recognition increases exponentially with longevity. Further, an analysis of the level of Ku80 protein in human, cow, and mouse suggests that Ku levels vary dramatically between species and these levels are strongly correlated with longevity. In contrast mean telomere length appears to decrease with increasing longevity of the species, although not significantly. These findings suggest that an enhanced ability to bind to DNA-ends may be important for longevity. A number of possible roles for increased levels of Ku and DNA-PKcs are discussed