ANALYSIS OF HRAD17: A DNA DAMAGE CHECKPOINT PROTEIN

HRAD17 的分析：DNA 损伤检查点蛋白

• 批准号: 6294291
• 负责人: LAURA A LINDSEY-BOLTZ
• 金额: $ 3.48万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-03-01 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6294291
• 关键词: DNA damage; DNA repair; HeLa cells; adenosine triphosphate; affinity chromatography; autoradiography; binding sites; biological signal transduction; cell cycle; cell cycle proteins; crosslink; hydrolysis; intermolecular interaction; molecular assembly /self assembly; molecular site; protein binding; protein purification; protein structure function; site directed mutagenesis; western blottings

Cell cycle checkpoints and DNA repair mechanisms help to maintain a cell's genomic integrity. Inactivation of genes required for these processes has been linked to syndromes causing predisposition to cancer. The DNA damage checkpoint involves the detection of damaged DNA and the generation of a signal that delays the cell cycle in order to allow time for the damaged DNA to be repaired. Proteins required for the DNA damage checkpoint have been identified primarily through genetic screens in yeast, and the human homologs have recently been cloned. Despite these momentous advances, the DNA damage checkpoint remains biochemically ill-defined. In particular, it is not known how the DNA damage is sensed. Six proteins in fission yeast have been implicated in sensing damage and activating the checkpoint kinases. It is not known whether these proteins directly interact with the DNA lesion or indirectly by interacting with repair complexes, or stalled transcription or replication complexes. One of these proteins, Rad17, has sequence homology to replication factor C (RFC) subunits, and has been shown to associate with RFC subunits in yeast. Rad17 hydrolyzes ATP in the process of loading PCNA onto DNA during replication and repair. RAD17 may perform a similar function to load checkpoint proteins onto DNA. The main goal of this proposal is to determine how hRad17 functions in the DNA damage checkpoint. This will be accomplished by: 1) determining if hRad17 binds and/or hydrolyzes ATP; 2) identifying proteins that interact with hRad17; and 3) determining if hRad17 associates with specific DNA structures, repair complexes, or repair intermediates. The results from these experiments will further our understanding of the role of hRad17 in the DNA damage checkpoint and may provide the groundwork for new cancer treatments.

细胞周期检查点和DNA修复机制有助于维持细胞的基因组完整性。这些过程所需的基因失活与综合征有关，导致癌症易感。 DNA损伤检查点涉及检测受损的DNA和延迟细胞周期的信号的产生，以便时间修复受损的DNA。 DNA损伤检查点所需的蛋白质主要是通过酵母中的遗传筛选来鉴定的，并且最近克隆了人类同源物。尽管有这些重要的进步，但DNA损伤检查点仍然在生化上定义不明。特别是，尚不清楚如何感觉到DNA损伤。裂变酵母中的六种蛋白质与感测损伤和激活检查点激酶有关。尚不清楚这些蛋白是通过与修复复合物或停滞转录或复制复合物相互作用而直接与DNA病变相互作用。这些蛋白质之一Rad17具有与复制因子C（RFC）亚基的序列同源性，并已证明与酵母中的RFC亚基相关联。在复制和修复过程中，RAD17在将PCNA加载到DNA的过程中水解ATP。 RAD17可能会执行与将检查点蛋白上载到DNA上的相似函数。该提案的主要目标是确定HRAD17在DNA损伤检查点中的功能。这将通过：1）确定HRAD17是否结合和/或水解ATP； 2）鉴定与HRAD17相互作用的蛋白质； 3）确定HRAD17是否与特定的DNA结构相关，修复复合物或修复中间体。这些实验的结果将进一步理解HRAD17在DNA损伤检查点的作用，并可能为新的癌症治疗提供基础。