FUNCTION OF THE HUMAN DNA-ACTIVATED PROTEIN KINASE

真核细胞有几种监测DNA或DNA 结构，其中一些可能对维持基因组完整性至关重要 包括检测DNA链断裂并激活G1细胞的基因 循环检查点。 p53依赖性G1检查点机制的缺失 导致基因组不稳定， 肿瘤的 检测DNA链断裂并激活细胞周期的酶 检查点仍有待确定，但一种DNA信号酶， 参与其中的是DNA-PK。 DNA-PK是一种中等丰度的，核， 丝氨酸/苏氨酸蛋白激酶在体外被具有切口的DNA激活， 缺口、断裂或单链到双链的转变。 最近的研究 强烈表明，DNA-PK是位点特异性V（D）J所必需的 重组和用于修复DNA双链的至少一种途径 休息. DNA-PK磷酸化多种核DNA结合蛋白， 包括p53蛋白，控制转录，DNA复制， 重组和修复。 因此，DNA-PK还可以调节其他方面 包括细胞周期的进展和 细胞对DNA链断裂的反应 DNA-PK活性很容易在人细胞提取物中通过以下方法测量： 一种高度特异性的基于肽的测定，但DNA-PK活性的状态 在体内不能被监测。 这项建议的主要目的是 开发用于监测组织中DNA-PK活性的非放射性方法 培养细胞，因此，以确定的情况和因素， 调节DNA-PK活性。 特别感兴趣的是DNA-PK是否是 由正常核过程激活（或抑制），包括 转录和DNA复制，作为细胞周期状态的函数， 对各种DNA损伤的反应 我们也将决定 DNA-PK（包括p53）的特异性体外底物是否 在体内通过DNA-PK磷酸化。 许多用于癌症治疗的药物 是导致DNA链断裂的DNA损伤诱导剂。 如果DNA-PK 参与细胞对DNA损伤的反应或调节细胞 周期进展，然后开发可以调节其活性的药物， 可能会带来更好的癌症治疗。

Eukaryotic cells have several mechanisms for monitoring DNA or DNA structures, some of which may be critical for maintaining genome integrity including one that detects DNA strand breaks and activates the G1 cell cycle checkpoints. Loss of the p53-dependent G1 checkpoint mechanism leads to genome instability and an enhanced probability of developing tumors. The enzymes that detect DNA strand breaks and activate cell cycle checkpoints remain to be identified, but one DNA signaling enzyme that may be involved is DNA-PK. DNA-PK is a moderately abundant, nuclear, serine/threonine protein kinase activated in vitro by DNAs with nicks, gaps, breaks, or single-to-double strand transitions. Recent studies strongly suggest that DNA-PK is required for site specific V(D)J recombination and for at least one pathway for repairing DNA double-strand breaks. DNA-PK phosphorylates a variety of nuclear, DNA-binding proteins, including the p53 protein, that control transcription, DNA replication, recombination, and repair. Thus, DNA-PK also may regulate other aspects of DNA metabolism including progression through the cell cycle and the cellular responses to DNA strand breaks. DNA-PK activity is easily measured in extracts of human cells by means of a highly specific peptide based assay, but the status of DNA-PK's activity in vivo cannot be monitored. The major aims of this proposal are to develop non-radioactive methods for monitoring DNA-PK activity in tissue culture cells, and hence, to identify the circumstances and factors that regulate DNA-PK activity. Of specific interest is whether DNA-PK is activated (or inhibited) by normal nuclear processes including transcription and DNA replication, as a function of cell cycle status and in response to various kinds of DNA damage. Also we will determine whether specific in vitro substrates of DNA-PK, including p53, are phosphorylated by DNA-PK in vivo. Many agents used in cancer therapies are DNA-damage-inducing agents that create DNA strand breaks. If DNA-PK is involved in the cellular response to DNA damage or in regulating cell cycle progression, then developing drugs that can modulate its activity may lead to better cancer therapies.