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，导致早发、衰老和与衰老相关的疾病，包括癌症和动脉粥样硬化。WS是由于WRN基因功能丧失所致。WRN基因编码一种具有独特核酸外切酶活性(WRN)的RecQ解旋酶蛋白，其细胞功能尚不清楚。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，Ku70/80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体。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阶段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。具体地说，在Aim I中，我们将测试WRN的丢失是否会导致CPT处理的细胞中S期检查点控制的缺陷。目标2中提出的实验将研究WRN及其相关因素在CPT诱导的DNA损伤反应中对染色质的招募动力学。在目标3中，我们将检验这样的假设，即WRN的丢失导致rDNA基因座的遗传不稳定，从而导致DNA损伤时核糖体RNA的异常生物合成。在目标4中，通过研究表达缺乏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，将获得对这些过程的生化见解。综上所述，这些实验应该会为人类衰老过程提供重要的机械学见解。

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.