RADIATION INDUCTION OF GENOMIC REARRANGEMENTS IN YEAST

DNA损伤会产生重组双链断裂，诱导 表达损伤诱导基因(DIN基因)，并阻止细胞 在明确定义的细胞周期检查点进行循环。基因组的存在 白血病和淋巴瘤中的重排及其相互关系 暴露于电离辐射和急性 白血病，需要更好地理解重组基因制剂如何 刺激染色体间重排。总体目标是 了解重组损伤是如何修复的，以便基因组 保持正直。这项建议研究的是损伤诱导的反应。 在酿酒酵母(酵母)中首次关注遗传 损伤诱导的有丝分裂重组的调控 分散的重复DNA序列(异位重组)。结果是 在这些研究中得到的可能支持关于以下四个重要假设 所有生物体的基因组稳定性：1)升高的 重组酶与有丝分裂发生率的增加有关 重新安排，2)错配修复系统在流产过程中很重要 不同序列之间的重组中间体和 重组基因可以绕过这种控制，3)拓扑异构酶和 解旋酶在将重组中间体引导到 一条不会产生染色体重排的途径，以及4) 在指定的细胞周期检查点停止细胞周期是必要的，以便 这些机制的作用。第一个目标是使用his3重组 用于量化辐射修复中突变的影响的底物， 错配修复、拓扑异构酶、损伤诱导的姐妹染色单体 重组和细胞周期调控对自发性和 损伤诱导的染色体间重组。第二个目标决定了 是否增强辐射诱导基因的表达，包括 RAD51基因，与高重组相关。第三个目标是 构建重组底物以使异位频率 发生在任何两个完全相同的DNA重复之间的重组可以是 这些底物将被放置在非同源染色体上 这样就可以测量易位。重复的序列将是 研究包括出现在酵母末端的三角洲元素 反转录转座子Ty1和Alu序列约300bp序列 在酵母上克隆的人DNA中平均每6kb出现一次 人工染色体(YAC)。然后重组化验将确定 不同重复序列之间有丝分裂的异位重组是否可以 受到DNA损伤剂的刺激。第四个目标是确定 错配修复途径中的突变体是否表现出更高的频率 这些序列之间的重组以及是否过度表达 RAD51重组酶将促进这些重复序列之间的重组。 这项研究的结果将有助于理解遗传控制的 酵母基因组的稳定性，有利于外源DNA的保持 在YAC上的酵母中引入，并提供对 DNA损伤剂在真核系统中的重组性。在……里面 此外，为酵母设计的新型重组底物将是 对有兴趣创造类似重组基因的遗传学家来说是有益的 高等真核生物的底物。

DNA damage generates recombinogenic double-strand breaks, induces the expression of damage-inducible genes (DIN genes), and arrests the cell cycle at well defined cell-cycle checkpoints. The presence of genomic rearrangements in leukemias and lymphomas, and the correlation between exposure to ionizing radiation and the elevated frequencies of acute leukemia, require a better understanding of how recombinogenic agents can stimulate interchromosomal rearrangements. The general goal is to understand how recombinogenic lesions are repaired so that genomic integrity is maintained. This proposal studies damage-inducible responses in Saccharomyces cerevisae (yeast) by first focusing on the genetic regulation of damage-induced mitotic recombination occurring between dispersed repeated DNA sequences (ectopic recombination). The results obtained in these studies may support four important hypotheses concerning genomic stability in all organisms: 1) that elevated levels of recombinases are correlated with the increased incidence of mitotic rearrangements, 2) that mismatch repair systems are important in aborting recombinational intermediates between divergent sequences and recombinogens may circumvent this control, 3) that topoisomerases and helicases are important in channelling recombinational intermediates into a pathway that does not generate chromosomal rearrangements, and 4) that cell cycle arrest at defined cell cycle checkpoints is necessary to allow these mechanisms to act. The first aim uses the his3 recombinational substrates to quantitate effects of mutations in radiation repair, mismatch repair, topoisomerases, damage-induced sister chromatid recombination and cell cycle control on the rates of spontaneous and damage-induced interchromosomal recombination. The second aim determines whether enhanced expression of radiation-inducible genes, including the RAD51 gene, is correlated with elevated recombination. The third aim is to construct recombinational substrates so that the frequencies of ectopic recombination occurring between any two identical DNA repeats can be quantitated; these substrates will be placed on non-homologous chromosomes so that translocations can be measured. Repeated sequences that will be studied include delta elements occurring at the end of the yeast retrotransposon Ty1 and Alu sequences approximately 300 bp sequence occurring once every 6 kb on average in human DNA cloned on yeast artificial chromosomes (YACs). Recombination assays will then determine whether mitotic, ectopic recombination between non-identical repeats can be stimulated by DNA damaging agents. The fourth aim is to determine whether mutants in the mismatch repair pathways exhibit higher frequencies of recombination between these sequences and whether over-expression of the RAD51 recombinase will enhance recombination between these repeats. Results from this study will aid in understanding the genetic control of genomic stability in yeast, facilitate the maintenance of foreign DNA introduced in yeast on YACs, and provide insights into the recombinogenicity of DNA damaging agents in eukaryotic systems. In addition, the novel recombinational substrates designed for yeast will be of benefit to geneticists interested in creating similar recombinational substrates for higher eukaryotes.