There is increasing evidence that many of the adverse biological effects of ionizing radiation are the result of DNA double-strand breaks (DSB). However, ionizing radiation produces such a broad spectrum of DNA lesions that it has previously been impossible to relate these lesions to biologically significant endpoints such as mutations and chromosomal aberrations. Recent investigations demonstrate that restriction enzymes introduced into mammalian cells permit the induction of very specific DNA lesions (DSB at short, defined sequences). This makes it possible to determine directly the types of genetic alterations which arise from specific types of DSB. We have constructed an EBV-based shuttle vector (pHAZE) for analyzing restriction enzyme-, X-ray and radon-induced mutations. This vector is stably maintained in human lymphoblastoid cells as an episomal element and contains the entire 3.1 kb E. coli lacZ gene as a target for mutagenesis. Restriction enzymes introduced into cells containing the vector induce a wide variety of mutations including frameshifts, deletions, insertions, and inversions. The specific types of mutations, however, are related to the type of the initial DSB (i.e., blunt, 5' overhang, 3' overhang). These differences appear to be a consequence of the specific repair processes to which each type of DSB is subject. The objectives of this proposal are to better understand the biological consequences of DSB, the role of specific DNA repair processes in the production of DSB-induced chromosome aberrations and mutation, and the nature of the defects underlying the DNA repair deficiency syndromes ataxia telangiectasia (AT) and Bloom's syndrome (BS).
The specific aims are to: 1) analyze and compare the spectrum of mutations induced by restriction enzyme-produced DSB in normal and DNA repair-deficient cell lines; 2) compare the sensitivity of normal and DNA repair-deficient cell lines to restriction enzyme-induced chromosome aberrations; 3) study the effect of specific DNA repair-inhibiting compounds on the spectrum of mutations induced by restriction enzymes and 4) study the effect of specific DNA repair-inhibiting compounds on restriction enzyme-induced chromosome aberration production. Restriction enzyme-induced mutations will be analyzed in the shuttle vector pHAZE stably maintained in normal, AT, and BS lymphoblastoid cell lines. Restriction enzymes that produce specific types of DSB will be introduced into these cells by electroporation. After allowing 24 h for repair, the vector will be rescued from the mammalian cells, transformed into E. coli for screening. Mutant copies of pHAZE will be analyzed by restriction digest mapping and DNA sequencing. The clastogenic potential of specific types of DSB will be analyzed in normal, AT, and BS cell by scoring chromosome aberrations from cells treated with various restriction enzymes. Our increased knowledge of the processes involved in DSB-induced mutations and chromosome aberrations, will further our understanding how ionizing radiations produce adverse biological consequences.
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