This Program Project reflects a confluence of interests of faculty members at the University of Southern California Main Campus and Medical School. The Program is tightly focused on exploring an important human genomic problem, the biochemical and genetic basis for expansions of trinucleotide repeats associated with neurological diseases. Many of the specific aims offer a novel perspective which we believe is unique to the group of investigators at USC. A significant advantage of this Project is the breadth of expertise of the investigators in studying genetic instability from a variety of different perspectives. The Program Project contains three research projects and three core facilities: a Biochemical-Analytical Core, a Mouse core and an Administrative Core. The goal of Project 1 is to investigate the dynamics of how trinucleotide repeat sequences expand over time, using an experimental in vitro system containing """"""""seed"""""""" repeat sequences and different combinations of replication proteins, and a convenient in vivo system in which large. biologically relevant, repeat expansions can be monitored in wild type and mutant procaryotic cells. Insight into the area of expansion dynamics is crucial to understanding this process at the molecular level. Unique to our approach is a combination of expansion kinetics data, using experimental model systems with """"""""state-of the art"""""""" theoretical analysis involving molecular dynamics calculations. Project I will use the facilities of """"""""Biochemical-Analytical"""""""" Core. The goals of Project 2 are first to examine the role of the enzyme FEN-1 in trinucleotide repeat instability in an animal model. Recently, experiments in yeast carrying large trinucleotide repeat tracts showed that cells defective in the Okazaki fragment processing enzyme FEN-1 had a sizable fraction of expansion mutations compared to wild type cells. Genetic instability at microsatellite loci in mice homozygous for a knockout mutation of FEN-1 will be studied using single molecule analysis of DNA from somatic and germline cells. The second goal of Project 2 is to examine the detailed biochemical properties of FEN-1 and other Okazaki fragment processing enzymes to determine their possible roles in genetic instability. Project 2 will rely heavily on both the Mouse core and the Biochemical-Analytical Core. Project 3 will generate the targeted deletion of the murine FEN-1 gene and/or replacement with an inducible version. The impact of FEN-1 deletion on chromosome stability will be particularly interesting in light of chromosomal changes with age in humans and mice. The impact at the tissue and organismal level of mutant animals will be examined. Finally, the effect of mutations at other loci involved in nonhomologous DNA end joining (NHEJ) will be examined for their effect on genetic instability including triplet repeats. The chromosomal, tissue and organismal effects of these mutations will also be examined in animals as a function of age. Project 3 will require the use of both the Mouse core and the Biochemical-Analytical Core.
