Cell Cycle Checkpoint in Response to DNA Damage

描述(由申请人提供)：细胞周期检查点确保从一个复制细胞周期到下一个复制细胞周期基因组的完整性。在基因组遭受灾难性破坏的情况下，多细胞真核生物的细胞可以进行凋亡，可能是为了将它们从细胞群中消除，并降低繁殖遗传不稳定细胞的风险。或者，细胞可能会通过细胞周期的短暂停滞来应对DNA损伤，这与它们在DNA损伤剂中生存的能力有关。这种反应需要DNA损伤检查点途径；如果受到突变或药物治疗的影响，细胞将进入有丝分裂，DNA受损并死亡。裂解酵母是一种非常有价值的系统，用于识别和表征DNA损伤检查点的成分。事实上，目前已知在哺乳动物细胞中发挥检查点途径功能的许多蛋白质，仅仅是根据它们与酵母中从基因和功能上鉴定的蛋白质的序列同源性来鉴定的。因此，很明显，利用经典遗传学的力量鉴定酵母中的蛋白质是鉴定和洞察哺乳动物对应蛋白功能的一种有效和富有成效的手段。这项提案中描述的实验将继续研究蛋白激酶Chk1，它是真核细胞中检查点的关键调节因子。此外，我们还将重点介绍一种新的裂解酵母蛋白Msc1。Msc1与哺乳动物RbBP2有结构域同源性，RbBP2是一种蛋白质，凭借其结合肿瘤抑制蛋白Rb的能力而被识别，并与PLU-1，PLU-1是乳腺癌细胞上调基因的产物。Msc1之所以被克隆，是因为它可以补偿Chk1功能的丧失。Msc1蛋白似乎是染色质的重要或组蛋白修饰，对于基因组的稳定性和DNA损伤后的生存是重要的。这项建议中描述的实验旨在剖析Msc1蛋白在分裂酵母中的作用，为了解人类同源物的功能奠定基础。

DESCRIPTION (provided by applicant): Cell cycle checkpoints ensure the integrity of the genome from one replicative cell cycle to the next. In the event of catastrophic damage to the genome, cells of multicellular eukaryotes can undergo apoptosis, presumably to eliminate them from the cell population and reduce the risk of propagating genetically unstable cells. Alternatively, cells may respond to DNA damage by undergoing a transient arrest of the cell cycle, which correlates with their ability to survive exposure to DNA damaging agents. This response requires the DNA damage checkpoint pathway; if compromised by mutation or drug treatment, cells will enter mitosis with damaged DNA and die. The fission yeast has been an extremely valuable system for identifying and characterizing components of the DNA damage checkpoint. Indeed, many proteins that are now known to function in the checkpoint pathway in mammalian cells were identified solely based on their sequence homology to proteins that were identified genetically and functionally in yeast. Thus, it is clear that the identification of proteins in yeast using the power of classical genetics is a valid and productive means of identifying and gaining insight into the function of mammalian counterparts. Experiments described in this proposal will continue to investigate the protein kinase, Chk1, a key regulator of the checkpoint in eukaryotic cells. In addition, we will focus on a novel fission yeast protein, Msc1. Msc1 shares structural domain homology with mammalian RbBP2, a protein identified by virtue of its ability to bind the tumor suppressor protein Rb and with PLU-1, the product of a gene that is up regulated in breast cancer cells. Msc1 was cloned because it can compensate for the loss of function Chk1. The Msc1 protein appears to be important or histone modifications of chromatin, for genomic stability and for survival after DNA damage. Experiments described in this proposal aim to dissect the role of the Msc1 protein in fission yeast, to lay the groundwork for understanding the functions of the human homologues.