Roles of ATM and ATR Kinases in DNA Damage Responses

描述(由申请人提供)： 细胞周期检查点是确保有序的信号转导途径 细胞周期的进程，以及增殖细胞不会试图 复制或分离受损的染色体。最近的研究准确地指出了两个 近端的P1 3-激酶相关蛋白家族的成员ATM和ATR G1、S和G2检查点通路中的信号元件。新兴的观点 自动柜员机功能是诱导适当检查点所必需的 对电离辐射诱导的DNA双链断裂的反应，而ATR 在紫外光诱导的DNA损伤反应中起主导作用， 以及由药物或内在错误引起的DNA复制应激 与DNA新陈代谢有关。尽管取得了这一进展，但这些机制 ATR的监管和功能仍不清楚，这在很大程度上是由于 由于缺乏遗传上易处理的ATR缺陷的细胞模型系统。 同样，尽管我们的初步研究发现了两个重要的 作为ATM/ATR底物的基因组监测蛋白BRCA1和hRad17 DNA损伤细胞中的激酶，这些酶的功能后果 磷酸化事件尚未定义。该项目将解决这些问题 通过实施以下具体目标解决关键问题：(1) 完成了一种新型ATR-/-细胞系的产生，并对其进行了表征 这些细胞中的检查点信号缺陷，以及(2)确定 ATR介导的BRCA1基因组维持功能的磷酸化， 通过使用两种新描述的BRCA1的功能分析，(3) 确定ATR/ATM依赖的磷酸化对 HRad17的检查点信令功能，以及(4)识别和功能 在暴露的细胞中表征用于ATR和ATM的新型含SIT-Q底物 对基因毒性压力的反应。

DESCRIPTION (provided by applicant): Cell-cycle checkpoints are signal transduction pathways that ensure orderly progression of the cell cycle, and that proliferating cells do not attempt to replicate or segregate damaged chromosomes. Recent studies have pinpointed two members of the P1 3-kinase related kinase family, ATM and ATR, as proximal signaling elements in the G1, S, and G2 checkpoint pathways. The emerging view is that ATM function is required for the induction of appropriate checkpoint responses to ionizing radiation-induced DNA double-strand breaks, while ATR plays dominant roles in the responses to ultraviolet light induced DNA damage, as well as to DNA replicative stress induced by drugs or intrinsic errors associated with DNA metabolism. In spite of this progress, the mechanisms underlying the regulation and function of ATR remain unclear, due in large part to the lack of a genetically tractable, ATR-deficient cellular model system. Similarly, although our preliminary studies have identified two important genome surveillance proteins, BRCA1 and hRad17, as substrates for the ATM/ATR kinases in DNA-damaged cells, the functional consequences of these phosphorylation events have not been defined. This project will address these critical issues through implementation of the following specific aims: (1) To complete the generation of a novel ATR-/- cell line, and to characterize the checkpoint signaling defects in these cells, and (2) To determine the impact of ATR-mediated phosphorylation on the genome maintenance functions of BRCA1, through the use of two newly described functional assays for BRCA1, (3) To define the regulatory effects of ATR/ATM-dependent phosphorylation on the checkpoint signaling functions of hRad17, and (4) To identify and functionally characterize novel SIT-Q-containing substrates for ATR and ATM in cells exposed to genotoxic stress.