Regulatory networks in DNA damage checkpoint response

描述（由申请人提供）：对DNA损伤的细胞反应由检查点途径控制，该途径从酵母到人类高度保守。该项目的长期目标是确定控制DNA损伤检查点的ATR家族蛋白的调控机制。ATR蛋白激酶与伴侣蛋白ATRIP相互作用，并以ATR-ATRIP复合物的形式起作用。在芽殖酵母中，Mec 1和Ddc 2分别对应于ATR和ATRIP。受损的DNA必须通过DNA修饰酶进行处理，以进行适当的DNA修复。单链DNA（single-strandDNA，ssDNA）的产生是损伤早期处理的关键步骤之一。在DNA损伤处产生的ssDNA被复制蛋白A（RPA）覆盖，其介导各种DNA修复途径。ATR-ATRIP/Mec 1-Ddc 2复合物与RPA覆盖的ssDNA相互作用，并在DNA损伤位点积累。在芽殖酵母中，Mec 1在DNA损伤位点磷酸化Rad 9检查点介质。磷酸化的Rad 9与Rad 53激酶相互作用，并且Rad 9-Rad 53相互作用增加Rad 53激酶活性。激活的Rad 53进一步磷酸化靶蛋白，并将检查点信号传递到下游。因此，目前的研究提供了一个清晰的轮廓Mec 1如何启动磷酸化级联反应。然而，监管机制尚未得到充分理解。该提案中的实验旨在揭示Mec 1如何在DNA损伤位点被激活（Aim 1）以及磷酸酶如何抵消Mec 1-Rad 53磷酸化级联反应（Aim 2）。端粒与激活Mec 1检查点途径的DNA断裂不同。这些实验还旨在确定端粒如何抑制Mec 1检查点途径（Aim 3）。检查点激活失败会导致染色体不稳定，这可能导致人类癌症的发展。更好地理解检查点控制应该导致更好地治疗和预防癌症。

DESCRIPTION (provided by applicant): The cellular responses to DNA damage are controlled by checkpoint pathways, which are highly conserved from yeast to human. The long-term goal of this project is to define the regulatory mechanism of the ATR family protein that controls the DNA damage checkpoint. The ATR protein kinase interacts with a partner protein, ATRIP, and acts in the form of the ATR-ATRIP complex. In budding yeast, Mec1 and Ddc2 correspond to ATR and ATRIP, respectively. Damaged DNAs have to be processed by DNA modification enzymes for proper DNA repair. Generation of single-strand DNA (ssDNA) is one of key steps at the early damage processing. Generated ssDNA at DNA lesions are covered with replication protein A (RPA), which mediates various DNA repair pathways. The ATR-ATRIP/Mec1-Ddc2 complex interacts with RPA-covered ssDNA and accumulates at sites of DNA damage. In budding yeast, Mec1 phosphorylates the Rad9 checkpoint mediator at sites of DNA damage. Phosphorylated Rad9 interacts with the Rad53 kinase, and the Rad9-Rad53 interaction increases Rad53 kinase activity. Activated Rad53 further phosphorylates the target proteins and relays checkpoint signals to the downstream. Current studies thus have provided a clear outline of how Mec1 initiates the phosphorylation cascade. However, the regulatory mechanism has not been fully understood yet. The experiments in this proposal will aim to uncover how Mec1 is activated at sites of DNA damage (Aim 1) and how phosphatases counteract the Mec1- Rad53 phosphorylation cascade (Aim 2). Telomeres are distinguished from DNA breaks that activate the Mec1 checkpoint pathway. The experiments will also aim to define how telomeres inhibit the Mec1 checkpoint pathway (Aim 3). Failure of proper checkpoint activation causes chromosome instability, which may result in cancer development in human. A better understanding of the checkpoint control should lead to better treatment and prevention of cancer.