Arrest, Recovery, and Adaptation from DNA Damage

描述：增强它们修复双链染色体断裂的能力 (DSB)，真核细胞激活DNA损伤检查点，导致细胞 停滞在细胞周期的G2/M交界处，在有丝分裂之前。故障： 检查点调节是癌细胞和染色体的标志 由于不能修复断裂的染色体而引起的重排 准确的时尚在晚期癌症中也很明显。虽然很多人 蛋白质，包括一系列蛋白激酶，都与此有关。 检查点过程，目前还没有确定直接识别哪些蛋白质 损伤以及细胞如何在损伤发生后恢复和恢复生长 修好了。遭受不可修复的DNA损伤的酿酒酵母细胞 逮捕了很长一段时间，但有能力适应和恢复 成长。该实验室的研究已经证实，细胞能够 适应性取决于5‘到3’产生的单链DNA(SsDNA)的程度 DSB核酸外切结束。当单链DNA的数量增加时，细胞 永久逮捕。单链DNA结合复合体RPA的突变抑制了 这次永久逮捕。这个项目的一个目标是确定如何适应 发生。建议进行实验以确定适应性细胞是否下调 DNA切除率或单链DNA的持久性，或如果细胞 对单链DNA的持续存在不敏感。营业额和 将测量检查点蛋白激酶的去磷酸化。几个新的 适应缺陷突变最近已被鉴定为 实验室，并将寻求更多的突变。这些特性将在 它们对单链DNA形成的影响及其与DNA的相互作用 RPA。与突变的RPA亚单位相互作用的蛋白质监测程度 将通过高拷贝抑制子筛选来鉴定单链DNA。第二个目标将是 评估DNA损伤诱导基因在DNA效率中的作用 修理。第三个目标是理解适应和适应之间的关系 以及“恢复”，即DNA损伤可以修复的时候。一种新设计的野生型 将使用菌株，其中单个DSB可以修复，但只能在6 HR，此时细胞已经停滞在G2/M期。 在损伤诱导和修复之间，将有可能评估 许多检查点蛋白的贡献。

DESCRIPTION: To enhance their ability to repair double-strand chromosome breaks (DSBs), eukaryotic cells invoke a DNA damage checkpoint, causing cells to arrest at the G2/M boundary of the cell cycle, prior to mitosis. Failures of checkpoint regulation are a hallmark of cancer cells, and chromosomal rearrangements arising by the failure to repair broken chromosomes in an accurate fashion are also evident in late-stage cancers. Although many proteins, including a cascade of protein kinases, have been implicated in this checkpoint process, it has not been established what proteins directly identify the damage and how cells recover and resume growth when that damage has been repaired. Saccharomyces cerevisiae cells that suffer unrepairable DNA damage arrest for a long time, but are capable of adaptation and the resumption of growth. Work from this laboratory has established that the ability of cells to adapt depends on the extent of single-stranded DNA (ssDNA) produced by 5' to 3' exonuclease resection of DSB ends. When the amount of ssDNA increases, cells arrest permanently. A mutation in the ssDNA-binding complex, RPA, suppresses this permanent arrest. One aim of this project is to determine how adaptation occurs. Experiments are proposed to determine if adapting cells down-regulate the rate of DNA resection or the persistence of ssDNA, or if cells become insensitive to the continued presence of ssDNA. The turnover and dephosphorylation of checkpoint protein kinases will be measured. Several new adaptation-defective mutations have recently been identified by this laboratory, and more mutations will be sought. These will be characterized in terms of their effect on the formation of ssDNA and their interactions with RPA. Proteins that interact with a mutant RPA subunit in monitoring the extent of ssDNA will be identified by a high-copy suppressor screen. A second aim will be to assess the role of DNA-damage inducible genes in the efficiency of DNA repair. A third objective is to understand the relationship between adaptation and "recovery," when DNA damage can be repaired. A newly-designed wild type strain will be used, in which a single DSB can be repaired, but only after 6 hr, by which time cells have arrested in G2/M. By spreading out the time between the induction of damage and repair, it will be possible to assess the contributions of many checkpoint proteins.