The Mechanism of Spindle Assembly Checkpoint Control in DNA Damage Response

描述（由申请人提供）：这项拟议研究的长期目标是了解真核细胞在受到DNA损伤挑战时如何维持基因组稳定性。基因组稳定性要求基因组复制、修复和分离过程的保真度，这是通过修复途径和细胞周期控制（包括细胞周期检查点）的协调作用来实现的。DNA损伤检查点（DDC）在DNA复制后受损时触发G2/M阻滞，而这种阻滞的维持需要纺锤体组装检查点（SAC）。在动物中，在DNA损伤的一些着丝点上发现了SAC蛋白，但SAC在DNA损伤反应中的控制机制尚不清楚。本研究旨在揭示两个高度保守的细胞周期检查点SAC和DDC之间的串扰机制，利用出芽酵母酿酒酵母。Aim 1的目标是通过以下方式确定SAC如何响应DNA双链断裂（DSB）的信号：(1)确定不可修复的DSB是否通过破坏着丝点-微管附着将SAC蛋白招募到着丝点，(2)确定DSB形成后影响SAC活性的因素。DSB对着丝点微管附着和SAC复合体在断裂染色体上向着丝点募集的影响将在活细胞（4-D）显微镜下进行研究。同时，DSB形成后SAC活性相对于DDC活性的模式将使用SAC活性（Mad1磷酸化）和DDC活性（Rad53和Chk1磷酸化）的分子标记进行分析。了解DSB诱导的G2/M阻滞过程中SAC何时变得重要以及影响这一时机的因素，将限制DSB形成后SAC调节的可能机制。Aim 2旨在识别在DNA损伤反应中通过SAC起作用的蛋白质。第一种方法将是对DDC和SAC基因进行靶向遗传分析，以揭示DDC蛋白是否在DSB诱导的G2/M阻滞过程中控制SAC蛋白。相比之下，第二种方法是无偏见的，旨在确定SAC对DNA损伤调节的新因素。维持DSB诱导的G2/M阻滞所需的基因突变将通过在多余染色体上产生DSB来选择，这样DSB诱导的染色体丢失在筛选过程中不会造成致死。通过应用Aim 1中使用的策略来表征Aim 2中确定的SAC调节因子，本研究将揭示DNA损伤反应过程中SAC控制的机制。由于DDC和SAC的失调发生在许多癌症中，了解它们的调节相互作用不仅将促进我们对真核细胞在DNA损伤挑战时如何防止增殖的理解，而且还将突出潜在的诊断和治疗靶点。

DESCRIPTION (provided by applicant): The long-term goal of this proposed research is to understand how eukaryotic cells maintain genome stability when challenged by DNA damage. Genome stability requires fidelity in the processes involved in genome replication, repair, and segregation, and this is achieved by coordinated action of repair pathways and cell cycle controls including the cell cycle checkpoints. The DNA Damage Checkpoint (DDC) triggers G2/M arrest when DNA is damaged after replication, and the maintenance of this arrest requires the Spindle Assembly Checkpoint (SAC). In animals, SAC proteins have been found at some kinetochores upon DNA damage, however how SAC is controlled in DNA damage response is unknown. This research aims to uncover the mechanism of crosstalk between two highly- conserved cell cycle checkpoints, the SAC and the DDC, using the budding yeast Saccharomyces cerevisiae. The goal of Aim 1 is to identify how the SAC responds to signals from DNA double- stranded breaks (DSBs) by: (1) determining whether an unrepairable DSB recruits SAC proteins to kinetochores through disruption of kinetochore-microtubule attachment, (2) identifying factors that affect SAC activity following DSB formation. Effects of DSB on kinetochore-microtubule attachment and SAC complex recruitment to kinetochores on the broken chromosome will be examined using live cell (4-D) microscopy. In parallel, the pattern of SAC activity relative to DDC activity following DSB formation will be analyzed using molecular markers for SAC activity (Mad1 phosphorylation) and for DDC activity (Rad53 and Chk1 phosphorylation). Knowledge about when SAC becomes important during DSB-induced G2/M arrest and factors affecting this timing will constrain the possible mechanisms of SAC regulation after DSB formation. Aim 2 is designed to identify proteins that act through the SAC in the DNA damage response. The first approach will be a targeted genetic analysis of DDC and SAC genes that will reveal whether DDC proteins control SAC proteins during DSB- induced G2/M arrest. In contrast, the second, unbiased, approach aims to identify novel factors involved in SAC regulation upon DNA damage. Mutations in genes required to maintain DSB-induced G2/M arrest will be selected by generating a DSB in a supernumerary chromosome so that DSB- induced chromosome loss will not cause lethality during screening. By applying strategies used in Aim 1 to characterize SAC regulators identified in Aim 2, the proposed research will uncover the mechanism of SAC control during the DNA damage response. Since DDC and SAC misregulation occurs in many cancers, understanding their regulatory interactions will not only advance our understanding of how eukaryotic cells prevent proliferation when challenged by DNA damage, but will also highlight potential diagnostic and therapeutic targets.