The Werner syndrome protein in CPT-induced DNA damage

描述（申请人提供）：沃纳综合征（WS）是一种常染色体隐性遗传疾病，可导致早发性衰老和衰老相关疾病，包括癌症和动脉粥样硬化。WS由WRN基因功能丧失引起。WRN基因编码RecQ解旋酶蛋白，其具有独特的外切核酸酶活性（WRN），其细胞功能知之甚少。来自WS患者的细胞表现出过早衰老和对DNA损伤剂如喜树碱（CPT）的敏感性。 重要的是，我们已经证明WRN与Ku 70/80结合，Ku 70/80是一种异二聚体复合物，已知在DNA损伤修复中起关键作用。这一观察结果有力地支持了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。具体而言，在目标I中，我们将测试WRN的丢失是否导致CPT处理的细胞中的S期检查点对照缺陷。目的2中提出的实验将研究响应CPT诱导的DNA损伤的WRN及其相关因子向染色质的募集的动态。在目标3中，我们将检验WRN的缺失导致rDNA位点的遗传不稳定性，从而导致DNA损伤后核糖体RNA生物合成异常的假设。在目标4中，将通过研究对CPT诱导的表达外切核酸酶或解旋酶活性缺陷或缺乏保守结构域的突变WRN蛋白的细胞的DNA损伤的反应来获得对这些过程的生物化学见解。总之，这些实验应该为人类衰老过程提供重要的机制见解。

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.