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损伤的挑战时如何保持基因组稳定性。基因组稳定性需要基因组复制、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期阻滞，而维持这种阻滞需要纺锤体组装检查点（SAC）。在动物中，SAC蛋白被发现存在于DNA损伤后的某些动粒中，但SAC在DNA损伤反应中的调控机制尚不清楚。本研究的目的是利用芽殖酵母酿酒酵母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。目的1的目标是通过以下方式鉴定SAC如何响应来自DNA双链断裂（DSB）的信号：（1）确定不可修复的DSB是否通过破坏着丝粒-微管附着将SAC蛋白募集到着丝粒，（2）鉴定DSB形成后影响SAC活性的因素。将使用活细胞（4-D）显微镜检查DSB对着丝粒-微管附着和SAC复合物募集到断裂染色体上的着丝粒的影响。同时，将使用SAC活性（Mad 1磷酸化）和DDC活性（Rad 53和Chk 1磷酸化）的分子标记物分析DSB形成后SAC活性相对于DDC活性的模式。关于SAC何时在DSB诱导的G2/M期阻滞过程中变得重要以及影响该时间的因素的知识将限制DSB形成后SAC调节的可能机制。目的2旨在鉴定在DNA损伤反应中通过SAC起作用的蛋白质。第一种方法将是DDC和SAC基因的靶向遗传分析，其将揭示DDC蛋白在DSB诱导的G2/M停滞期间是否控制SAC蛋白。相比之下，第二，公正的，方法的目的是确定新的因素参与SAC调节DNA损伤。维持DSB诱导的G2/M停滞所需的基因突变将通过在额外染色体中产生DSB来选择，使得DSB诱导的染色体丢失在筛选期间不会导致致死性。通过应用目标1中使用的策略来表征目标2中确定的SAC调节剂，所提出的研究将揭示DNA损伤反应期间SAC控制的机制。由于DDC和SAC的错误调节发生在许多癌症中，了解它们的调节相互作用不仅会促进我们对真核细胞在受到DNA损伤的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.