Arrest, Recovery, and Adaptation from DNA Damage

描述：增强其修复双链染色体断裂的能力 （DSB），真核细胞调用DNA损伤检查点，导致细胞 在有丝分裂之前，停滞在细胞周期的G2/M边界。故障 检查点调节是癌细胞的标志， 由于不能修复染色体断裂而引起的重排， 精确的方式在晚期癌症中也很明显。尽管许多 蛋白质，包括蛋白激酶的级联反应，与此有关。 检查点过程中，它还没有建立什么蛋白质直接识别 以及细胞如何恢复和恢复生长时，损害已经 修复.酿酒酵母细胞遭受不可修复的DNA损伤 逮捕了很长一段时间，但能够适应和恢复 增长这个实验室的工作已经证实，细胞的能力， 适应取决于单链DNA（ssDNA）由5'到3'产生的程度。 DSB末端的核酸外切酶切除。当ssDNA的量增加时，细胞 永久逮捕。ssDNA结合复合物RPA的突变抑制了 永久逮捕这个项目的目的之一是确定如何适应 发生。实验提出，以确定是否适应细胞下调 DNA切除率或ssDNA的持久性，或者细胞是否变得 对ssDNA的持续存在不敏感。营业额及 将测量检查点蛋白激酶的去磷酸化。几个新 适应缺陷突变最近已被确定这一点 实验室，并将寻求更多的突变。这些将被描述为 它们对ssDNA形成的影响及其与 RPA。与突变型RPA亚基相互作用的蛋白质， 将通过高拷贝抑制筛选来鉴定ssDNA。第二个目标将 目的是评估DNA损伤诱导基因在DNA修复效率中的作用。 修复.第三个目标是了解适应与 和“恢复”，即DNA损伤可以修复。一种新设计的野生型 将使用菌株，其中单个DSB可以被修复，但仅在6 hr，此时细胞已停滞在G2/M期。通过分散时间 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.