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: 8396250
• 负责人: Gina M Kavanaugh
• 金额: $ 4.92万
• 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/8396250
• 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. PUBLIC HEALTH RELEVANCE: The cellular response to replication stress is complex and poorly understood. The goal of the aims proposed in this grant is to identify and characterize novel replication stress proteins using a whole genome siRNA library approach. Insights into the mechanistic function of these novel proteins will lead to greater understanding of the replication stress response and its role in maintaining genomic integrity to prevent cancer.

描述（由申请人提供）：DNA的正确复制对于细胞分裂时遗传信息的传递至关重要。由于内在和外在DNA损伤而发生的DNA密码的改变可引起基因突变，从而导致蛋白质功能和表达的改变。这可能最终导致疾病状态，如癌症。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，并且需要许多细胞途径的协调，包括细胞周期检查点、复制叉和复制体稳定化、复制叉重新激发的预防和重新启动， DNA损伤修复和DNA复制的重新启动。由于这些途径的复杂性，对这些反应的理解是一个持续的过程，在这个过程中，新的参与者会定期被发现。我们相信一些与复制应激反应相关的新蛋白质还有待鉴定。因此，在目标1中，我们提出使用全基因组siRNA方法以鉴定在复制应激反应中起作用的蛋白质。利用胸苷类似物掺入和复制应激诱导剂羟基脲的免疫荧光测定已被设计用于揭示复制应激修复和复制重启所必需的蛋白质。该测定利用384孔板格式和免疫荧光数据的高通量分析，以快速识别感兴趣的蛋白质。一旦目标蛋白得到验证，它们将在目标2中进一步表征，以将它们分类并优先考虑它们在复制应激反应中的功能。分类和优先级排序将基于生物信息学分析和实验评价，以确定对羟基脲和复制应激的敏感性、蛋白质的亚细胞定位、对DNA复制和复制叉动力学的影响，以及确定蛋白质是否参与ATR依赖性或非依赖性途径。这些分析将允许鉴定在复制应激反应中起作用的高优先级蛋白质。进一步的研究将集中在确定这些蛋白质在复制应激过程中发挥作用的机制上。提出的目标将允许识别新的复制应激反应蛋白，这将加深我们对DNA复制和修复以及这些途径如何有助于基因组稳定性的理解。对在基因组稳定性中起作用的蛋白质和机制的更大理解将最终导致癌症诊断和治疗的改进发展。 公共卫生相关性：细胞对复制应激的反应是复杂的，人们对其了解甚少。在这项资助中提出的目标的目标是使用全基因组siRNA文库方法鉴定和表征新的复制应激蛋白。深入了解这些新蛋白质的机制功能将有助于更好地理解复制应激反应及其在维持基因组完整性以预防癌症中的作用。