The goal of this project is to investigate the roles of mouse Mre11 in lymphocyte development and general DNA repair. Yeast Mre11 is a member of a pathway called non-homologous end joining (NHEJ), which repairs chromosomes with DNA double strand breaks. In mammals, other members of this pathway are essential for V(D)J recombination and class switch recombination, which generate the diverse repertoire of antibodies and T cell receptors produced by lymphocytes. In Humans, defects in these processes result in immunodeficiency and malignancy. Mre11 is evolutionarily conserved, and is known to be an endo/exonuclease which forms a complex with at least two other protein, Rad50 and NBS. No mouse model deficient in Mre11 exists, hindering the study of this protein in mammals. The construction of such a mouse is proposed here. This project entails the following specific aims: (1) To generate mouse mre11 alleles which cripple biochemical activity but maintain the ability to form protein complexes. (2) To engineer these alleles into the genome of mouse embryonic stem cells. (3) To generate mice from these stem cells. The cells and mice resulting from this work will allow us to determine Mre11's role in VDJ recombination, class switching, and general DNA repair. This application is for a Mentored Clinical Scientist Development Award (K08) to an applicant who has received graduate training in molecular biology, and residency training in Pathology. The candidate's long term goals are to establish and direct an independent research laboratory studying the basic and clinical aspects of the immune system and DNA repair. While experienced in microbial genetics and recombinant DNA, the candidate requires this development award to gain experience in immunology and the use of mouse as a model system. This essential training will be conducted under the sponsorship of Dr. Frederick Alt in the Center for Blood Research at the Harvard Medical School.
