Thesis (Ph. D.)--University of Rochester. Dept. of Biology, 2014.
Aging is a combination of progressive decline in function and fitness of an organism. Its complexity arises from numerous genetic and environmental pressures that influence the aging process, rendering it stochastic and pleiotropic. Sustaining genome integrity has been postulated to play a key role in longevity. The most harmful of all genome lesions are DNA double strand breaks (DSBs) and their efficient repair is critical for cell survival. This repair is primarily facilitated by the Non-Homologous End Joining (NHEJ) pathway in vertebrates. Despite extensive evidence associating faulty NHEJ with increasing age and studies with numerous knockout mice, there is no mouse model that can analyze age-related NHEJ repair events in vivo.
To this end, we have designed and generated a novel R26NHEJ mouse model to study NHEJ repair in a tissue-specific and age-associated manner. This model consists of a chromosomally integrated NHEJ reporter cassette under the ubiquitously expressed ROSA26 promoter. Targeted generation of DSBs in this cassette is achieved by means of the I-SceI endonuclease. Successful NHEJ repair of these DSBs is then detected and quantified by the expression of Green Fluorescent Protein (GFP). To confirm the ubiquitous expression and working of the R26NHEJ construct, protocols were developed and standardized in R26NHEJ mouse colonies for aging research.
Employing the R26NHEJ mouse model as a powerful DNA repair research tool, the efficiency of NHEJ repair was found to significantly decline in brain astrocytes as well as heart, kidney, lung, and skin fibroblasts of 24 month old mice. Following the analysis of over 300 clones of NHEJ repair sites, a highly dynamic NHEJ profile consisting of tissue-specific repair-associated deletions and insertions of DNA sequences was discovered. Regardless of age, mice were found to utilize microhomology sequences during end-joining repair at a significantly higher than expected frequency. This highlights a stark contrast between human and mouse NHEJ repair, since senescent human fibroblasts have been shown to have impaired utilization of microhomology during NHEJ. These data point to the possibility that mice inherently possess inefficient NHEJ, which further deteriorates with age and may be a crucial factor contributing to their short lifespan and increased cancer prevalence.
Research with the R26NHEJ mouse model concurs with our hypothesis that DNA repair is essential for longevity. The age-related decrease in DNA repair capacity coupled with age-associated accumulation of DNA damage and the variation in repair protein levels, leads to a cyclic pattern of initial damage causing more genomic instability, culminating in functional decline and demise of an organism. The R26NHEJ mouse is expected to have significant implications in the field of aging, cancer, and genome repair research by providing valuable insight into the role of NHEJ repair in mechanisms that influence health and longevity, for eg. caloric restriction, rapamycin, exercise, and diet.
