The correlation between defective DNA repair pathways and cancer susceptibility has been well documented in studies of several human hereditary diseases and in mice carrying mutations in different genes involved in DNA repair. While it is clear that enzymes implicated in the DNA double strand break repair (dsbr) pathway are involved, little is known about the mechanisms used by mammals to protect themselves from and/or repair this kind of damage. Reports that correlate increased DNA repair with drug resistance highlights the need for studies that will further contribute to our understanding of DNA repair in higher eukaryotes. By studying a crucial reaction involved in genomic recombination events necessary for lymphocyte differentiation, the principal investigator has demonstrated a direct link between antigen receptor assembly and the dsbr pathway. These studies culminated in the identification of three gene products [DSBR / V(D)J] shared by these two systems, two of which are known components of the DNA-dependent protein kinase (DNA-PK) complex, Ku80 and DNA-PK catalytic subunit (DNA-PKcs). The overall goal of this project is to understand the role these proteins in V(D)J recombination and dsbr.
The specific aims of this proposal are: i) to unravel the role that the DNA-PKcs plays in V(D)J recombination/dsbr. This will be accomplished by gene ablation in ES by homologous recombination, and assaying for its impact on V(D)J recombination and dsbr. These cell lines will also be employed to identify in vivo substrates for this kinase and to generates a mouse disease model; ii) to define critical regions of the Ku80 protein that are involved in its interaction with other components of the DNA-PK complex. To accomplish this, a mutational analysis of the Ku80 gene will be pursued and mutants assayed by their ability to complement V(D)J/dsbr defects display by the hamster cell line xrs-6. iii) to identify new genes involved in V(D)J recombination. This will involve the use of other existing DNA repair cell mutants as well as fibroblasts from severe combined immune deficient (SCID) patients. A molecular characterization of these activities could potentially contribute important insights into biological relationships between processes that lead to chromosomal instability and translocation, and thus cancer susceptibility, as well as to provide murine models for the study of human SCID.
