Impact of Cell Cycle Checkpoints on DNA Repair

描述 这项合作提案将主要在乌拉圭作为一个 延长美国国立卫生研究院#RO 1-CA87381号拨款。研究相互作用的主要目标是 芽期细胞周期关卡停滞与DNA链断裂修复的关系 酿酒酵母特别强调检查点蛋白 RAD17.具体地说，建议 1.RAD17突变体的DNA双链断裂修复研究 细胞周期进程。这将通过孵化经辐射的 非生长条件下的二倍体菌株及其整合性分析 脉冲场琼脂糖凝胶中的染色体。因此，经常辩论的问题 将解决检查点突变体的已知辐射敏感性 例如RAD17完全是由于维修时间窗口不足(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。双突变分析将指示修复途径 这是受Rad17的影响。 2.rad17和chk1突变体的DNA双链断裂修复特性 在将激活正常细胞中的细胞周期检查点的条件下。在……里面 为了达到这个目的，它将被调查如果未能在检查站逮捕 (特别是在G2/M检查点)在生长中的细胞转化为 断股修复不完整。因此，通常假设的 将探索辐射敏感性、检查站逮捕和修复能力。 对rad17和最近分离的chk1突变体进行比较会很有意义。 在这方面，由于G2/M停滞和虚拟 在后者中发现野生型对DNA损伤剂的敏感性 变种人。链后存活菌落细胞的染色体图谱 将分析rad17突变体中的断裂损伤和 我们将研究染色体的异常情况。 3.确定RAD17基因突变版本在修复过程中的作用 非生长和生长条件。 总而言之，该项目解决了检查点缺陷的后果 DNA修复和遗传稳定性的控制。遗传不稳定性一直是 被认为是导致多步骤事件序列的关键过程 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.