Arrest, Recovery, and Adaptation from DNA Damage

描述（由申请人提供）：DNA损伤反应（DDR）的诱导和维持在保护基因组完整性方面起着核心作用，使细胞有更多时间修复染色体双链断裂（dsb）或消除无法完成修复的细胞。在出芽酵母中，单个DSB的产生足以激活Mec1 （ATR）和Tel1 （ATM）检查点蛋白激酶，这两种蛋白激酶都修饰DSB周围的染色质，并触发一系列磷酸化，导致细胞周期进程在后期之前被阻止。本研究研究了检查点反应的三个主要方面：检查点的激活和维持，通过g-H2AX和g-H2B的形成对染色质的修饰，以及DDR与纺锤体组装检查点的协同相互作用，该检查点负责延长阻滞状态。在第一个Aim中，将通过研究Mec1上磷酸化位点的新发现的关闭检查点所需的突变来研究如何维持Mec1依赖性G2/M细胞周期阻滞；因此，在没有DSB修复的情况下，细胞不能适应并在12-15小时后恢复有丝分裂。将研究Mec1如何监测DNA损伤的存在，重点是通过外切酶切除DSB末端的5‘到3’和维持检查点之间的关系。先前的研究结果表明，在DSB修复后，细胞恢复细胞周期进程的能力取决于细胞被阻滞的时间，这可能与检查点信号的强度有关。检查点信号的强度将通过人为地捆绑Mec1的Ddc1和Ddc2 （ATRIP）激活子来增强反应来检测，以引发独立于DSB的反应。第二个目标将集中于组蛋白H2B (g-H2B) c端Mec1和tel1依赖性磷酸化，这类似于已被充分研究的g- H2AX修饰，但似乎在DNA损伤信号传导和修复中具有不同的作用。第三个目标将关注DNA损伤检查点和纺锤体组装检查点之间的协同作用，跟踪我们的发现，删除Mad2缩短了野生型细胞中的检查点阻滞，抑制了适应缺陷突变的永久阻滞，这种抑制可以通过删除遭受未修复的DSB的染色体上的着丝粒来模拟。

DESCRIPTION (provided by applicant): The induction and maintenance of the DNA damage response (DDR) plays a central role in protecting genome integrity, allowing cells more time to repair chromosomal double-strand breaks (DSBs) or to eliminate cells that fail to accomplish repair. In budding yeast, creation of a single DSB is sufficient to activate the Mec1 (ATR) and Tel1 (ATM) checkpoint protein kinases that both modify chromatin around the DSB and trigger a cascade of phosphorylations that result in the arrest of cell cycle progression prior to anaphase. This proposal investigates three major aspects of the checkpoint response: the activation and maintenance of the checkpoint, the modification of chromatin by formation of g-H2AX and - as we have discovered - g-H2B, and a synergistic interaction of the DDR with the Spindle Assembly Checkpoint that is responsible for the prolongation of the arrested state. In the first Aim, how Mec1-dependent G2/M cell cycle arrest is maintained will be investigated by studying newly-discovered mutations of phosphorylation sites on Mec1 that are required to turn the checkpoint off; consequently cells fail to adapt and resume mitosis after 12-15 h when there is no DSB repair. How Mec1 monitors the presence of DNA damage will be examined, focusing on the relation between 5' to 3' resection of the DSB ends by exonucleases and maintenance of the checkpoint. Previous results suggest that the ability of cells to resume cell cycle progression after a DSB is repaired depends on how long cells have been arrested, which may correlate with the strength of the checkpoint signal. The strength of checkpoint signaling will be examined by augmenting the response by artificially tethering the Ddc1 and Ddc2 (ATRIP) activators of Mec1 to elicit a response independent of a DSB. A second Aim will focus on the Mec1- and Tel1-dependent phosphorylation of the C-terminus of histone H2B (g-H2B) that resembles the well-studied g- H2AX modification but appears to have separate roles in DNA damage signaling and repair. A third Aim will focus on the synergy between the DNA damage checkpoint and the Spindle Assembly Checkpoint, following up our findings that deleting Mad2 shortens checkpoint arrest in wild type cells and suppresses the permanent arrest of adaptation-defective mutations and that this suppression can be mimicked by deleting the centromere on the chromosome suffering an unrepaired DSB.