Regulatory networks in DNA damage checkpoint response

描述(申请人提供)：细胞对DNA损伤的反应由检查点途径控制，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家族蛋白的调节机制。ATR蛋白激酶与伴侣蛋白ATrip相互作用，以ATR-ATrip复合体的形式发挥作用。在芽殖酵母中，Mec1和Ddc2分别对应于ATR和Atrip。受损的DNA必须由DNA修饰酶处理，才能进行适当的DNA修复。单链DNA的产生是早期损伤处理的关键步骤之一。在DNA损伤处产生的单链DNA被复制蛋白A(RPA)覆盖，复制蛋白A介导多种DNA修复途径。ATR-Trip/Mec1-Ddc2复合体与RPA覆盖的单链DNA相互作用，并在DNA损伤部位聚集。在萌芽酵母中，Mec1在DNA损伤部位磷酸化Rad9检查点介体。磷酸化的Rad9与Rad53激酶相互作用，Rad9-Rad53相互作用增加Rad53激酶的活性。激活的Rad53进一步使目标蛋白磷酸化，并将检查点信号传递到下游。因此，目前的研究已经为Mec1如何启动磷酸化级联反应提供了一个清晰的轮廓。然而，监管机制还没有完全被理解。本方案中的实验将旨在揭示Mec1是如何在DNA损伤部位被激活的(目标1)，以及磷酸酶如何抵消Mec1-Rad53磷酸化级联反应(目标2)。端粒与激活Mec1检查点通路的DNA断裂是不同的。这些实验还将旨在确定端粒如何抑制Mec1检查点途径(目标3)。正确的检查点激活失败会导致染色体不稳定，这可能会导致人类癌症的发展。对检查站控制的更好理解应该会导致更好的癌症治疗和预防。 与公共健康相关：细胞对DNA损伤的反应由检查点途径控制，从酵母到人类都高度保守。检查点激活的失败被认为是染色体不稳定的主要原因，而染色体不稳定会导致高等真核生物中的癌症。更好地理解DNA损伤检查点的调控机制使我们能够开发更好的癌症治疗和预防方法。

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.