Regulatory networks in DNA damage checkpoint response

描述（由申请人提供）：细胞对DNA损伤的反应是由检查点途径控制的，从酵母到人类都是高度保守的。该项目的长期目标是明确控制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。在出芽酵母中，Mec1和Ddc2分别对应ATR和ATRIP。受损的DNA必须经过DNA修饰酶的处理才能进行适当的DNA修复。单链DNA （ssDNA）的生成是早期损伤处理的关键步骤之一。DNA损伤处产生的ssDNA被复制蛋白A （RPA）覆盖，RPA介导多种DNA修复途径。ATR-ATRIP/Mec1-Ddc2复合物与rpa覆盖的ssDNA相互作用，并在DNA损伤部位积累。在出芽酵母中，Mec1在DNA损伤位点磷酸化Rad9检查点介质。磷酸化的Rad9与Rad53激酶相互作用，Rad9-Rad53相互作用增加了Rad53激酶的活性。激活的Rad53进一步磷酸化靶蛋白，并将检查点信号传递给下游。因此，目前的研究为Mec1如何启动磷酸化级联提供了一个清晰的轮廓。然而，监管机制尚未完全了解。本提案中的实验旨在揭示Mec1如何在DNA损伤位点被激活（aim 1），以及磷酸酶如何抵消Mec1- Rad53磷酸化级联（aim 2）。端粒不同于激活Mec1检查点通路的DNA断裂。实验还将旨在确定端粒如何抑制Mec1检查点途径（aim 3）。检查点正常激活失败会导致染色体不稳定，从而可能导致人类癌症的发生。更好地了解检查点控制将有助于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. PUBLIC HEALTH RELEVANCE: The cellular responses to DNA damage are controlled by checkpoint pathways, which are highly conserved from yeast to human. The failure of the checkpoint activation has been implicated as a major cause of chromosomal instability, which leads to cancer in higher eukaryotes. A better understanding of the regulatory mechanism for DNA damage checkpoint enables us to develop better treatment and prevention of cancer.