FUNCTION OF THE HUMAN DNA-ACTIVATED PROTEIN KINASE

人类 DNA 激活蛋白激酶的功能

• 批准号: 2415326
• 负责人: CARL W ANDERSON
• 金额: $ 18.18万
• 依托单位: ASSOCIATED UNIV-BROOKHAVEN NATL LAB
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-05-01 至 1998-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2415326
• 关键词: DNA; DNA binding protein; DNA damage; DNA replication; chemical fingerprinting; electrofocusing; enzyme activity; enzyme inhibitors; enzyme mechanism; enzyme substrate; genetic transcription; immunoprecipitation; laboratory rabbit; method development; molecular cloning; phosphorylation; posttranslational modifications; protein kinase; synchronous cell division; tissue /cell culture; transfection; transfection /expression vector; western blottings

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.

真核细胞有几种监测DNA或DNA的机制