Functions of WRN in Response to DNA Double-Strand Breaks

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，特征是早期出现与年龄相关的症状。WS患者患罕见的非上皮性癌的风险也增加，尤其是间充质肿瘤，如肉瘤。从WS细胞分离的成纤维细胞在培养过程中过早衰老，并表现出更多的染色体畸变率。WRN是在WS中突变的蛋白，在具有外切酶活性的RecQ家族蛋白中是唯一的，在单个多肽中具有32-52个解旋酶。越来越多的证据表明，WRN通过参与各种DNA损伤修复途径来维持基因组的完整性，并在端粒维持中发挥作用。然而，WRN在DNA修复，特别是在DNA双链断裂(DSB)修复中的作用机制仍不清楚。体外和间接证据表明，WRN可能在同源重组(HR)和非同源末端连接(NHEJ)中发挥作用。最近有证据表明，WRN被募集到DNA损伤部位，并被PIKK3激酶家族磷酸化，以响应DNA DSB。在这个方案中，我们将检验WRN被招募到体内DNA双链断裂(DSB)的假设，以确定WRN的磷酸化功能，以及它参与NHEJ或HR响应DNA损伤的过程。我们的具体目标是：(1)确定WRN被招募到DNA双链断裂位点的机制；(2)检验WRN在DNA双链断裂反应中被DNA-PK磷酸化以及WRN的磷酸化状态调节其在DNA损伤部位的功能的假设；以及(3)验证WRN在DNA双链修复的非同源末端连接(NHEJ)和/或同源重组(HR)途径中发挥作用的假设。完成这项拟议的研究将有助于理解WRN在应对DNA损伤方面的功能，并有助于进一步阐明WRN在癌症中的作用。

DESCRIPTION (provided by applicant): Werner Syndrome (WS) is an autosomal recessive condition characterized by an early onset of age-related symptoms. WS patients also experience an increased risk of rare non-epithelial cancers, especially mesenchymalneoplasms such as sarcomas. Fibroblasts isolated from WS cells senesce prematurely in culture and display increased chromosomal aberrations. WRN, the protein mutated in WS, is unique among the RecQ family proteins possesses on exonuclease activity and 32 to 52 helicase in a single polypeptide. There is accumulating evidence suggesting that WRN contributes to the maintenance of genomic integrity through its involvement in various DNA damage repair pathways and plays a role in telomere maintenance. However, the mechanism by which WRN functions in DNA repair, especially in DNA double-strand break (DSB) repair is still elusive. In vitro and indirect evidence leads to the conclusion that WRN may play a role both in homologous recombination (HR) as well as nonhomologous end joining (NHEJ). Recently, evidence showed that WRN is recruited to DNA damage sites and phosphorylated by PIKK3 kinase family in response to DNA DSB. In this proposal, we will test the hypothesis that WRN is recruited to DNA double-strand breaks (DSB) in vivo, to determine the function of WRN phosphorylation, and its involvement in the process of NHEJ or HR in response to DNA damage. Our specific aims are: (1) To determine the mechanism by which WRN is recruited to the sites of DNA double-strand breaks; (2) To test the hypothesis that WRN is phosphorylated by DNA-PK in response to DNA double-strand breaks and the phosphorylation status of WRN modulates its functions at DNA damage sites; and (3) To verify the hypothesis that WRN plays a role in nonhomologous end joining (NHEJ) and/or homologous recombination (HR) pathways of DNA double-strand break repair. Accomplishment of the proposed research would lead to the understanding of WRN's function in response to DNA damage and help further elucidate the role of WRN in cancer.