DOUBLE STRAND BREAK MISREJOINING IN MAMMALIAM CELLS

DNA双链断裂是电离辐射产生的最重要的初级损伤，并被认为是其高致死率、致裂性和恶性转化易感性的原因。它们也作为正常重组事件的起始步骤形成，包括减数分裂交换和重排以产生免疫球蛋白多样性。正确修复这些损伤对于维持基因组完整性至关重要。哺乳动物细胞主要采用非同源末端连接来修复电离辐射诱导的双链断裂以及整合外源DNA。然而，酵母重组修复基因的哺乳动物同源物最近已经被确定，并且有越来越多的证据表明，在S/G2晚期，当姐妹染色单体存在时，同源重组对细胞辐射抗性的贡献，尽管它们参与双链断裂修复尚未直接确定。还有一种可能性是，响应DNA损伤的信号通路可能影响双链断裂重新连接过程中的一个或两个过程的操作。这些途径对辐射诱导的双链断裂的总体重新连接和错误重新连接的可能性的贡献将使用一种新的方法来直接定量基因组定义区域中正确重新连接的断裂。该方法将被应用于选定的辐射敏感哺乳动物细胞的双链断裂修复研究，以验证涉及非同源或同源重组机制的不同途径有助于哺乳动物细胞在细胞周期的不同阶段重新连接，以及它们在错误重新连接频率上的差异的假设。基于一类断裂不受错接影响的证据，进一步提出断裂附近的初级或高阶染色质结构会影响其错接的概率。为了研究断裂在核小体内位置的影响，将电离辐射处理后的误连接与博来霉素处理后的误连接进行比较，博来霉素主要在连接体中诱导断裂。高阶染色质结构的影响将通过比较活性X染色体与异色多余X染色体以及Y染色体的异色区与常色区错接的频率和剂量反应来检验。在这两种情况下，分子测量将与细胞遗传学观察相关联，以验证染色体断裂包括可用于错误重新连接的DNA断裂这一推论。综上所述，拟议的研究将促进对哺乳动物细胞双链断裂修复机制的理解，以及导致它们错误重新连接的因素，这些因素可能导致染色体重排或导致致癌的遗传信息丢失。

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.