Identification and Characterization of Novel Replication Stress Response Proteins

新型复制应激反应蛋白的鉴定和表征

• 批准号: 8565648
• 负责人: Gina M Kavanaugh
• 金额: $ 4.85万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8565648
• 关键词: Affect; Bioinformatics; Biological Assay; Biological Preservation; Biological Process; Cancer Diagnostics; Categories; Cell Cycle Arrest; Cell Cycle Checkpoint; Cell Cycle Progression; Cells; Classification; Code; Complex; DNA; DNA Damage; DNA Repair; DNA Repair Pathway; DNA biosynthesis; DNA lesion; Data; Development; Disease; Evaluation; Fellowship; Foundations; Gene Mutation; Genes; Genetic; Genetic Screening; Genome; Genome Stability; Genomics; Goals; Grant; Health; Heat shock proteins; Human; Immunofluorescence Immunologic; Knowledge; Lead; Libraries; Maintenance; Malignant Neoplasms; Metabolism; Molecular Target; Mutation; Organism; Pathway interactions; Play; Prevention; Process; Proteins; Replication Initiation; Research Personnel; Role; Small Interfering RNA; Stress; TOPBP1 Gene; TREX1 gene; Therapeutic; Thymidine; Training; analog; base; biological adaptation to stress; carcinogenesis; career; design; genome wide association study; genome-wide; high throughput analysis; hydroxyurea; improved; insight; interest; novel; prevent; protein expression; protein function; repaired; research study; response; stress protein

DESCRIPTION (provided by applicant): Proper replication of the DNA is critical for passing on of the genetic information when a cell divides. Alterations to the DNA code, which occur as a result of both intrinsic and extrinsic DNA damage, can cause gene mutations leading to alterations in protein function and expression. This may ultimately lead to disease states such as cancer. Therefore, the cellular response to replication stress is important for maintaining the genomic integrity and health of an organism. The replication stress response is complex and requires the coordination of a number of cellular pathways, including cell cycle checkpoint, replication fork and replisome stabilization, prevention and restart of replication fork re-firing, DNA damage repair, and restart of DNA replication. Due to the intricacy of these pathways, understanding of these responses is an ongoing process in which new players are being discovered regularly. We believe a number of novel proteins associated with the replication stress response are yet to be identified. Consequently, in Aim 1 we propose to use a whole genome siRNA approach in order to identify proteins that play a role in replication stress responses. An immunofluorescence assay utilizing thymidine analog incorporation and the replication stress inducer hydroxyurea has been designed to uncover proteins that are necessary for replication stress repair and replication restart. This assay takes advantage of the 384-well plate format and high-throughput analysis of immunofluorescent data in order to quickly identify proteins of interest. Once proteins of interest are validated, they will be furthe characterized in Aim 2 to classify them and prioritize their function in the replication stress response. Classification and prioritization will be based upon bioinformatics analyses and experimental evaluation to determine sensitivity to hydroxyurea and replication stress, subcellular localization of the protein, effect on DNA replication and replication fork dynamics, as well as determination of whether the protein is involved in an ATR-dependent or -independent pathway. These analyses will allow for identification of high- priority proteins playing roles in the replication stress response. Further studies will focus on determining the mechanisms by which these proteins function during replication stress. The proposed aims will allow for identification of novel replication stress response proteins that will deepen our understanding of DNA replication and repair and how these pathways contribute to genomic stability. A greater appreciation for the proteins and mechanisms that function in genomic stability will ultimately lead to improved developments in cancer diagnostics and therapeutics.

描述（由申请人提供）：当细胞分裂时，DNA的适当复制对于遗传信息的传递至关重要。DNA密码的改变，是由于内在和外在的DNA损伤而发生的，可以引起基因突变，导致蛋白质功能和表达的改变。这可能最终导致癌症等疾病。因此，细胞对复制应激的反应对于维持生物体的基因组完整性和健康是重要的。复制应激反应是复杂的，需要许多细胞途径的协调，包括细胞周期检查点、复制叉和复制体的稳定、复制叉重新点燃的预防和重启。