This work will investigate transcription, and the p53-directed response to DNA damage, as factors that affect the processing of double strand breaks (DSB) into mutations. The work is based is based being able to specifically target restriction sites for the extremely rare-cutting yeast SceI nuclease to the endogenous thymidine kinase (tk) gene in human lymphoblast cells. The mutagenic and apoptotic effects of DSB induced at those sites can be determined. Hypothesis to be explored are as follows. (i) DSB are more effective at inducing mutation or apoptosis when occurring in a gene undergoing transcription than in a non-transcribed region. (ii) DSB are much more efficient mediators of deletion mutations in p53 mutant cells. (iii) Two DSB within a few kilobases will have synergistic effects. (iv) X-rays will act synergistically with DSB to lead to large-scale mutations. (v) Recombination between Alu sequences will not stimulate mutagenesis in p53 wild type cells, but they may in p53 mutants. (vi) Apoptosis will not decrease mutagenesis unless a major premutagenic lesion is also a key signal for apoptosis.
Aim 1 will determine the effect of specific DSB on mutagenesis in the tk gene and on apoptosis. Lymphoblast lines will be constructed that will respond to an exogenous inducer by transcribing the SceI nuclease. Second, gene targeting vectors will be constructed and utilized to introduce SceI restriction sites to distinct positions in the active tk allele of TK6 heterozygotes. The effectiveness of nuclease-generated DSB alone or with a dose of ionizing radiation in inducing gene mutation and apoptosis will be determined.
Aim 2 will examine th4e effect of transcription level on mutagenesis and apoptosis induced by DSB. Studies will indicate whether cells actively transcribing a gene with a DSB are more sensitive to mutations in that gene and/or to apoptosis. A tk transactivator will be added to the cells, allowing levels of transcription of tk RNA to be modulated. Other experiments will explore the relationship between cycle position and mutagenesis.
Aim 3 will examine the effect of p53 status on apoptosis and mutagenesis induced by DSB. Cells will be transfected with a dominant-0negative p53, and the effects on mutagenesis and apoptosis, under normal and high levels of transcription.
