Functions of WRN in Response to DNA Double-Strand Breaks

WRN 响应 DNA 双链断裂的功能

基本信息

批准号：
7652143
负责人：
DAVID J CHEN
金额：
$ 32.58万
依托单位：
UT SOUTHWESTERN MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-04-01 至 2014-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7652143
关键词：
Affect Alanine Aspartic Acid Biochemical Biological Biological Assay Cell Line Cells Chromatin Chromosome abnormality DNA DNA Binding DNA Damage DNA Double Strand Break DNA Repair DNA-PKcs DNA-dependent protein kinase Dominant-Negative Mutation Double Strand Break Repair Epithelial Event Exonuclease Family Family member Fibroblasts Genome Genomics Human In Vitro Interruption Kinetics Knockout Mice Laboratories Lasers Lead Life Maintenance Malignant Neoplasms Measures Mediating Mesenchymal Cell Neoplasm Mus Mutate Mutation Nonhomologous DNA End Joining Pathway interactions Patients Phosphorylation Phosphorylation Site Phosphotransferases Play Process Protein Family Recruitment Activity Reporter Research Risk Roentgen Rays Role Site Study Section Symptoms System Telomere Maintenance Testing Werner Syndrome XRCC5 gene age related base cellular imaging early onset endodeoxyribonuclease SceI experience helicase homologous recombination human WRN protein in vivo kinase inhibitor mutant polypeptide protein protein interaction public health relevance repaired research study response sarcoma spatial relationship

项目摘要

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. PUBLIC HEALTH RELEVANCE: 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 mesenchymal neoplasms such as sarcomas. WRN, the protein defective in WS is involved in maintenance of genomic integrity and DNA damage repair. To understand the function of WRN in DNA damage response, we proposed to (i) determine the mechanism by which WRN is recruited to the sites of DNA damage in vivo, (ii) find out kinases that are responsible for WRN phosphorylation and (iii) determine the biological significance of WRN phosphorylation in the process of DNA double strand break repair mediated through non- homologous end joining and/or homologous recombination. The results obtained with these experiments will further elucidate the mechanism by which WRN contributes in genome maintenance, cancer and DNA repair.

描述（由申请人提供）：Werner综合征（WS）是一种常染色体隐性疾病，其特征是与年龄相关的症状早期发作。 WS患者还经历了罕见的非上皮癌的风险，尤其是肉瘤等间质癌的风险。从WS细胞中分离出来的成纤维细胞在培养物中过早地衰老，并显示出增加的染色体畸变。 WRN是WS中突变的蛋白质，在RECQ家族蛋白中具有外丝酶活性的独特，单个多肽中的核酸酶活性和32至52个旋转酶。有积极的证据表明，WRN通过参与各种DNA损伤修复途径的基因组完整性有助于维持基因组完整性，并在端粒维持中起作用。但是，WRN在DNA修复中起作用的机制，尤其是在DNA双链断裂（DSB）修复中仍然难以捉摸。在体外和间接证据中得出的结论是，WRN可能在同源重组（HR）以及非同源结局（NHEJ）中发挥作用。最近，有证据表明，将WRN募集到DNA损伤位点，并由Pikk3激酶家族磷酸化，以响应DNA DSB。在此提案中，我们将检验以下假设：在体内募集WRN到DNA双链断裂（DSB），以确定WRN磷酸化的功能及其参与NHEJ或HR的参与，以响应DNA损伤。我们的具体目的是：（1）确定将WRN募集到DNA双链断裂部位的机制；（2）为了测试响应DNA双链断裂的DNA-PK磷酸化的假设，WRN的磷酸化状态调节其在DNA损伤位点的功能；（3）验证WRN在DNA双链断裂修复的非同源末端连接（NHEJ）和/或同源重组（HR）途径中起作用的假设。拟议研究的完成将导致对DNA损伤的响应WRN功能的理解，并有助于进一步阐明WRN在癌症中的作用。公共卫生相关性：Werner综合征（WS）是一种常染色体隐性疾病，其特征是与年龄相关的症状早期发作。 WS患者还经历了罕见的非上皮癌的风险，尤其是间质肿瘤，例如肉瘤。 WR，WS中的蛋白质缺陷参与了基因组完整性和DNA损伤修复的维持。为了了解WRN在DNA损伤反应中的功能，我们建议（i）确定将WRN募集到体内DNA损伤部位的机制，（ii）找出导致WRN磷酸化和（iii）的激酶确定通过非同源末端连接和/或同源重组介导的DNA双链断修复过程中WRN磷酸化的生物学意义。这些实验获得的结果将进一步阐明WRN在基因组维持，癌症和DNA修复方面贡献的机制。