DOUBLE STRAND BREAK MISREJOINING IN MAMMALIAM CELLS

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DNA double-strand breaks constitute the most important primary damage produced by ionizing radiation and are presumed to account for its high lethality, clastogenicity, and predisposition for malignant transformation. They are also formed as initiating steps in normal recombination events, including meiotic exchange and rearrangements to generate immunoglobulin diversity. Correct repair of these lesions is essential for maintenance of genomic integrity. Mammalian cells primarily employ non-homologous end joining for repair of double-strand breaks induced by ionizing radiation as well as for integration of foreign DNA. However, mammalian homologs of yeast recombinational repair genes have recently been identified, and there is increasing evidence for a contribution of homologous recombination to cellular radioresistance during late S/G2 when sister chromatids are present, although their participation in double-strand break repair has yet to be directly established. There is also the possibility that signaling pathways responsive to DNA damage may affect the operation of one or both of these processes in double-strand break rejoining. The contribution of each of these pathways to overall rejoining of radiation-induced double-strand breaks and to the probability of misrejoining will be examined using a novel approach for direct quantitation of correctly rejoined breaks in defined regions of the genome. This approach will be employed in studies of double-strand break repair in selected radiosensitive mammalian cells in order to test the hypotheses that different pathways involving non-homologous or homologous recombination mechanisms contribute to rejoining in mammalian cells in different phases of the cell cycle and that they differ in misrejoining frequency. Based on evidence for a class of breaks that are not subject to misrejoining, it is further proposed that either the primary or higher order chromatin structure in the vicinity of a break affects its probability of misrejoining. For studying the effect of the location of the break within the nucleosome, misrejoining after treatment with ionizing radiation will be compared to misrejoining after treatment with bleomycin, which induces breaks mainly in the linkers. The effect of higher order chromatin structure will be examined by comparing misrejoining frequency and dose response for misrejoining in active X chromosomes vs. heterochromatic supernumerary X and in heterochromatic vs. euchromatic regions of the Y chromosome. In both cases, molecular measurements will be related to cytogenetic observations to test the corollary idea that chromosomal breaks comprise the class of DNA breaks that are available for misrejoining. Taken together, the proposed studies will advance understanding of mechanisms for repair of double-strand breaks in mammalian cells and of factors resulting in their misrejoining, which can give rise to chromosomal rearrangements or loss of genetic information that lead to carcinogenesis.