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RADIATION INDUCTION OF GENOMIC REARRANGEMENTS IN YEAST

酵母基因组重排的辐射诱导

基本信息

批准号：
2895495
负责人：
MICHAEL Thomas FASULLO
金额：
$ 12.63万
依托单位：
ALBANY MEDICAL COLLEGE
依托单位国家：
美国
项目类别：
财政年份：
1995
资助国家：
美国
起止时间：
1995-08-01 至 2000-07-31
项目状态：
已结题

项目摘要

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.

DNA 损伤产生重组双链断裂，诱导损伤诱导基因（DIN 基因）的表达，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的重排以及两者之间的相关性暴露于电离辐射和急性发作频率升高白血病，需要更好地了解重组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，以便基因组保持完整性。该提案研究损伤诱导反应在酿酒酵母（酵母）中，首先关注遗传损伤诱导的有丝分裂重组的调节分散的重复 DNA 序列（异位重组）。结果这些研究中获得的结果可能支持四个重要的假设：所有生物体的基因组稳定性：1）提高了重组酶与有丝分裂发生率增加相关重排，2）错配修复系统对于中止很重要不同序列之间的重组中间体重组原可能会绕过这种控制，3）拓扑异构酶和解旋酶对于将重组中间体引导至不产生染色体重排的途径，4) 细胞周期在确定的细胞周期检查点处停滞是必要的这些机制发挥作用。第一个目标使用 his3 重组定量辐射修复中突变影响的底物，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控制对自发和损伤引起的染色体间重组。第二个目标决定辐射诱导基因的表达是否增强，包括 RAD51 基因与重组升高相关。第三个目标是构建重组底物，使异位的频率任何两个相同的 DNA 重复序列之间发生的重组可以是定量；这些底物将被放置在非同源染色体上以便可以测量易位。重复的序列将是研究包括酵母末端出现的δ元素逆转录转座子 Ty1 和 Alu 序列约 300 bp 序列在酵母上克隆的人类 DNA 中平均每 6 kb 出现一次人工染色体（YAC）。然后重组测定将确定不同重复序列之间的有丝分裂、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 重组酶将增强这些重复之间的重组。这项研究的结果将有助于了解基因控制酵母基因组稳定性，有利于外源DNA的维持在 YAC 上引入酵母，并提供对真核系统中 DNA 损伤剂的重组原性。在此外，为酵母设计的新型重组底物将是对有兴趣创造类似重组的遗传学家有好处高等真核生物的底物。