DNA-dependent protein kinase DNA-PK is a serine-threonine kinase that consists of a 465-kDA catalytic subunit, DNA-PKcs, and a 70 kDA and an 86-kDa heterodimeric DNA-targeting component, Ku70 and Ku80. Based on our recent pilot studies of Ku70- /- and Ku80-/- mice and earlier studies of SCID mice by others, it is postulated that each of the three components of DNA-PK may have distinct yet overlapping roles. The proposed studies aim at a test of this hypothesis and the elucidation of the physiological roles of the individual components of DNA-PK in DNA double- strand break (DSB) repair and V(D)J recombination, in lymphocyte development and lymphomagenesis. There are two specific aims.
Specific Aim 1 focuses on the physiological role(s) of the individual subunits in DSB repair, V(D)J recombination and lymphocyte development. We plan to generate mutant mice and cell lines deficient in one or more of these polypeptides, and use them to deduce the roles of individual components of DNA-PK during T-and B-cell development, and in the repair of radiation-induced DSB and the associated effects on radiation sensitivity. In addition, we will examine the effect of low (non-lethal) does of ionizing radiation on V(D)J recombination in these mutant mice and evaluate whether T- and B-cell development can be restored by X- irradiation and whether such restoration enhances the development of lymphoma.
Specific Aim II focuses on the role(s) of Ku70, Ku80 and DNA-PKcs in lymphomagenesis and tumorigenesis. We will test a hypothesis, inferred from our preliminary studies, that the loss of Ku70 enhances illegitimate recombination and leads to the development of lymphoma. Using various mutant mice and cell lines, spontaneous tumor development and tumor induction by ionizing radiation will be studied in vivo, and neoplastic transformation in vitro. In addition, the induction of chromosome damage (chromosome aberrations and sister chromatid exchanges) with and without X-irradiation will be examined with a view to understand the roles of the individual subunits of DNA-PK in the maintenance of genomic stability. The proposed study should add new information and insight regarding the physiological functions of DNA-PK and its individual subunits in programmed gene rearrangement and the maintenance of genomic stability. Furthermore, our studies should verify whether there is a role of Ku70 in lymphomagenesis, and specifically whether Ku70 may be considered as a candidate tumor suppressor gene.
