Impact of Cell Cycle Checkpoints on DNA Repair

描述 这项合作建议将主要在乌拉圭进行， NIH补助金# RO 1 -CA 87381延期。主要目的是研究 细胞周期检查点停滞和DNA链断裂修复之间的联系 特别强调检查点蛋白的酿酒酵母 Rad17.具体而言，建议 1.研究一个rad 17突变体在排除DNA双链断裂后的DNA双链断裂修复。 细胞周期进程这将通过孵育辐照的 二倍体菌株在非生长条件下， 脉冲场琼脂糖凝胶中的染色体。因此，经常争论的问题 将讨论检查点突变体的已知辐射敏感性是否 例如RAD 17仅仅是由于用于修复时间窗口不足（由于 细胞周期停滞的缺陷），或者这些基因是否发挥了额外的直接作用， 在DNA修复中的作用双突变体分析将指出修复途径 受Rad 17影响的细胞 2.描述rad 17和chk 1突变体中DNA双链断裂修复的特征 在正常细胞中激活细胞周期检查点的条件下。在 为了达到这一目的，如果在检查站没有逮捕，将进行调查。 （特别是在G2/M检查点）在生长细胞中转化为 不完全链断裂修复。因此，通常假设的 将探索辐射敏感性、检查点阻滞和修复能力。 比较rad 17和最近分离的chk 1突变体将是有趣的。 在这方面，由于G2/M逮捕中不太明显缺陷， 在后者中发现了野生型对DNA损伤剂的敏感性 变种人链转移后存活集落细胞的染色体图谱 将在rad 17突变体中分析断裂损伤， 将研究染色体畸变。 3.为了确定Rad 17的突变形式在修复中的作用， 非生长和生长条件。 总之，该项目解决了缺陷检查点的后果 控制DNA修复和遗传稳定性。遗传不稳定性 被认为是一个多步骤事件序列中的关键过程， 人类癌症因此，该项目与人类健康高度相关。

DESCRIPTION This collaborative proposal will be carried out primarily in Uruguay as an extension of NIH grant # RO 1 -CA87381. The main goal to study the interactions between cell cycle checkpoint arrest and DNA strand break repair in the budding yeast Saccharornyces cerevisiae with special emphasis on the checkpoint protein Rad17. Specifically, it is proposed 1. To study DNA double-strand break repair in a rad17 mutant under exclusion of cell cycle progression. This will be accomplished by incubating irradiated diploid strains under non-growth conditions and analyzing the integrity of the chromosomes in pulsed-field agarose gels. Thus, the frequently debated issue will be addressed whether the known radiation sensitivity of checkpoint mutants such as rad17 is solely due to an insufficient time window for repair (due to a defect in cell cycle arrest) or whether these genes play an additional direct role in DNA repair. Double mutant analysis will indicate the repair pathway that is affected by Rad17. 2. To characterize DNA double-strand break repair in a rad17 and a chk1 mutant under conditions that will activate cell cycle checkpoints in normal cells. In this aim, it will be investigated if the failure to arrest at a checkpoint (specifically at the G2/M checkpoint) in growing cells translates into incomplete strand break repair. Thus, the commonly assumed correlation between radiation sensitivity, checkpoint arrest, and repair capacity will be explored. It will be of interest to compare rad17 and the recently isolated chk1 mutant in this respect since a less pronounced defect in G2/M arrest and virtually wild-type sensitivity towards DNA damaging agents was found in the latter mutant. The chromosome profiles of cells of surviving colonies following strand break damage will be analyzed in the rad17 mutant and the pattern of chromosomal aberrations will be studied. 3. To determine the effect of mutant versions of Rad17 in repair under non-growth and growth conditions. In summary, this project addresses the consequences of defective checkpoint control for DNA repair and genetic stability. Genetic instability has been recognized as a key process in the multistep sequence of events leading to human cancer. The project is therefore highly relevant to human health.