DOUBLE STRAND BREAK MISREJOINING IN MAMMALIAM CELLS

DNA双链断裂是电离辐射造成的最重要的原发性损伤，据推测是电离辐射致死率高、致染色体断裂和易发生恶性转化的原因。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。这些损伤的正确修复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，哺乳动物同源的酵母重组修复基因最近已被确定，有越来越多的证据表明，同源重组的贡献，细胞辐射抗性在晚S/G2时，姐妹染色单体存在，虽然他们参与双链断裂修复尚未直接建立。还有一种可能性是，对DNA损伤有反应的信号通路可能会影响双链断裂重新连接中这些过程中的一个或两个的操作。这些途径中的每一个辐射诱导的双链断裂的整体重新连接和错误重新连接的概率的贡献将使用一种新的方法进行检查，在基因组的定义区域的正确重新连接的断裂直接定量。这种方法将采用在选定的放射敏感性哺乳动物细胞的双链断裂修复的研究，以测试的假设，涉及非同源或同源重组机制的不同途径有助于重新加入在哺乳动物细胞在细胞周期的不同阶段，他们在misrejoining频率不同。基于一类断裂不受误连接影响的证据，进一步提出断裂附近的初级或高级染色质结构影响其误连接的概率。为了研究核小体内断裂位置的影响，将用电离辐射处理后的错误重新连接与用博来霉素处理后的错误重新连接进行比较，博来霉素主要在接头中诱导断裂。 将通过比较活性X染色体与异染色质多余X染色体以及Y染色体异染色质与常染色质区域中的错接频率和错接的剂量反应，检查高阶染色质结构的影响。 在这两种情况下，分子测量将与细胞遗传学观察相关，以测试染色体断裂包括可用于错误接合的DNA断裂的类别的推论。总之，拟议的研究将促进对哺乳动物细胞中双链断裂修复机制的理解，以及对导致其错误连接的因素的理解，这种错误连接可能导致染色体重排或遗传信息丢失，从而导致致癌。

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.