The Werner syndrome protein in CPT-induced DNA damage

CPT 诱导的 DNA 损伤中的沃纳综合征蛋白

• 批准号: 7069615
• 负责人: LUCIO COMAI
• 金额: $ 31.19万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-06-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7069615
• 关键词: DNA damage; DNA repair; DNA replication; DNA topoisomerases; SDS polyacrylamide gel electrophoresis; Werner' s syndrome; camptothecin; cell line; chromatin; cytogenetics; developmental genetics; enzyme activity; exonuclease; gene expression; helicase; human genetic material tag; human tissue; laboratory rabbit; mutant; nucleic acid sequence; protein kinase; protein structure function; ribosomal DNA; ribosomal RNA; tissue /cell culture

DESCRIPTION (provided by applicant): Werner syndrome (WS) is an autosomal recessive disorder leading to premature onset aging and aging-related diseases including cancer and atherosclerosis. WS results from the loss of function of the WRN gene. The WRN gene encodes a RecQ helicase protein with a unique exonuclease activity (WRN) whose cellular function is poorly understood. Cells from WS patients demonstrate premature senescence and sensitivity to DNA damaging agents such as camptothecin (CPT). Importantly, we have shown that WRN binds to Ku70/80, a heterodimeric complex known to play a critical role in the repair of DNA damage. This observation strongly supports the idea that WRN functions in a DNA damage response pathway. We therefore hypothesize that WRN is required for S-phase checkpoint activation or is directly involved in the repair of DNA lesions following exposure of cells to CPT. Specifically, in Aim I we will test whether loss of WRN leads to defective S-phase checkpoint controls in CPT-treated cells. Experiments proposed in Aim 2 will study the dynamics of the recruitment of WRN and its associated factors to chromatin in response to CPT-induced DNA damage. In Aim 3, we will test the hypothesis that loss of WRN results in genetic instability at the rDNA locus leading to aberrant ribosomal RNAs biosynthesis upon DNA damage. In Aim 4, biochemical insights into these processes will be obtained by studying the response to CPT-induced DNA damage of cells expressing mutant WRN proteins deficient in exonuclease or helicase activity, or lacking conserved structural domains. Taken together, these experiments should provide important mechanistic insights into the process of human aging.

描述（由申请人提供）：沃纳综合征（WS）是一种常染色体隐性遗传疾病，导致过早衰老和衰老相关疾病，包括癌症和动脉粥样硬化。 WS 是由 WRN 基因功能丧失引起的。 WRN 基因编码具有独特核酸外切酶活性 (WRN) 的 RecQ 解旋酶蛋白，但对其细胞功能知之甚少。 WS 患者的细胞表现出过早衰老和对喜树碱 (CPT) 等 DNA 损伤剂的敏感性。 重要的是，我们已经证明 WRN 与 Ku70/80 结合，这是一种已知在 DNA 损伤修复中发挥关键作用的异二聚体复合物。这一观察有力地支持了 WRN 在 DNA 损伤反应途径中发挥作用的观点。因此，我们假设 WRN 是 S 期检查点激活所必需的，或者直接参与细胞暴露于 CPT 后 DNA 损伤的修复。具体来说，在目标 I 中，我们将测试 WRN 丢失是否会导致 CPT 处理的细胞中 S 期检查点控制缺陷。目标 2 中提出的实验将研究响应 CPT 诱导的 DNA 损伤而招募 WRN 及其相关因子到染色质的动态。在目标 3 中，我们将检验以下假设：WRN 丢失会导致 rDNA 位点遗传不稳定，导致 DNA 损伤时核糖体 RNA 生物合成异常。在目标 4 中，通过研究表达缺乏核酸外切酶或解旋酶活性或缺乏保守结构域的突变 WRN 蛋白的细胞对 CPT 诱导的 DNA 损伤的反应，可以获得对这些过程的生化见解。总而言之，这些实验应该为人类衰老过程提供重要的机制见解。