FUNCTION OF THE HUMAN DNA-ACTIVATED PROTEIN KINASE

真核细胞有多种监测DNA或DNA的机制 结构，其中一些可能是维持基因组完整性的关键 包括检测DNA链断裂并激活G1细胞的一个 循环检查站。P53依赖的G1检查点机制的丢失 导致基因组不稳定，并增加发育的可能性 肿瘤。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信号酶可能 参与其中的是DNA-PK。DNA-PK是一种中等丰富的，有核的， 含NICS的DNA体外激活的丝氨酸/苏氨酸蛋白激酶， 间隙、断裂或单链到双链的转变。最新研究 强烈提示DNA-PK是特异的V(D)J所必需的 重组和至少一条修复DNA双链的途径 休息一下。DNA-PK可使多种核DNA结合蛋白磷酸化， 包括控制转录、DNA复制的P53蛋白， 重组和修复。因此，DNA-PK还可能调节其他方面 DNA新陈代谢的过程，包括细胞周期和 细胞对DNA链断裂的反应。 DNA-PK活性很容易通过以下方法在人体细胞提取液中测量 一种基于高度特异性多肽的分析，但DNA-PK的活性状况 在体内不能被监测。这项建议的主要目的是 建立监测组织中DNA-PK活性的非放射性方法 培养细胞，并因此确定环境和因素 调节DNA-PK活性。特别令人感兴趣的是DNA-PK是否 由正常的核过程激活(或抑制)，包括 转录和DNA复制，作为细胞周期状态和 以应对各种DNA损伤。此外，我们还将确定 DNA-PK的特定体外底物，包括P53，是否 在体内被DNA-PK磷酸化。癌症治疗中使用的许多药物 是造成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 循环进行，然后开发可以调节其活动的药物 可能会带来更好的癌症疗法。

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.