Functions of WRN in Response to DNA Double-Strand Breaks

WRN 响应 DNA 双链断裂的功能

• 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
• 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

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）：沃纳综合征（WS）是一种常染色体隐性遗传病，以年龄相关症状的早期发病为特征。WS患者患罕见非上皮性癌症的风险也增加，尤其是间质瘤，如肉瘤。从WS细胞分离的成纤维细胞在培养中过早衰老，并显示出增加的染色体畸变。WRN是在WS中突变的蛋白，在RecQ家族蛋白中是独特的，在单个多肽中具有外切酶活性和32至52个解旋酶。越来越多的证据表明，WRN通过参与各种DNA损伤修复途径有助于维持基因组完整性，并在端粒维持中发挥作用。然而，WRN在DNA修复，特别是DNA双链断裂（DSB）修复中的作用机制尚不清楚。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）中都起作用。最近，有证据表明，WRN被PIKK3激酶家族招募到DNA损伤位点并被磷酸化，以响应DNA DSB。在本文中，我们将在体内验证WRN被招募到DNA双链断裂（DSB）的假设，以确定WRN磷酸化的功能，以及它在响应DNA损伤的NHEJ或HR过程中的参与。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)验证DNA双链断裂时，WRN被DNA- pk磷酸化，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）途径中发挥作用的假设。本研究的完成将有助于了解WRN在DNA损伤响应中的功能，并有助于进一步阐明WRN在癌症中的作用。公共卫生相关性：沃纳综合征（WS）是一种常染色体隐性遗传病，其特征是年龄相关症状的早期发作。WS患者患罕见的非上皮性癌症的风险也增加，尤其是间充质肿瘤，如肉瘤。wsn是WS中有缺陷的蛋白，参与基因组完整性的维持和DNA损伤的修复。为了了解WRN在DNA损伤应答中的功能，我们提出(i)确定WRN在体内被招募到DNA损伤位点的机制，（ii）找到负责WRN磷酸化的激酶，（iii）确定WRN磷酸化在通过非同源末端连接和/或同源重组介导的DNA双链断裂修复过程中的生物学意义。这些实验结果将进一步阐明WRN在基因组维持、癌症和DNA修复中的作用机制。