Determining the role of RAD51AP1: a new gene in DNA repair and genomic stability

确定 RAD51AP1 的作用：DNA 修复和基因组稳定性中的新基因

• 批准号: 7584220
• 负责人: DAVID SCHILD
• 金额: $ 38.29万
• 依托单位: UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-03-07 至 2012-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7584220
• 关键词: Antibodies; BRCA2 Protein; Biochemical; Biological; Biological Assay; Biological Models; Biological Testing; C-terminal; Cell Cycle; Cell Line; Cells; Chickens; Chromatin; Chromosome Pairing; DNA Damage; DNA Repair; DNA Repair Gene; DNA Repair Pathway; DNA repair protein; DNA-Binding Proteins; Defect; Dominant-Negative Mutation; Exposure to; Frequencies; Genes; Genetic Recombination; Genome Stability; Goals; Human; Human Cell Line; Induced Mutation; Investigation; Knock-out; Letters; Link; Malignant Neoplasms; Mediator of activation protein; Modeling; Mutagenesis; Nuclear; Pathway interactions; Phase; Phenotype; Phosphotransferases; Preventive; Proteins; Publishing; RNA Interference; Radiation; Radiation therapy; Roentgen Rays; Role; Sequence Homology; Sister Chromatid Exchange; Staging; System; TK1 gene; Testing; Time; Transcription Factor AP-1; Work; base; carcinogenesis; chemotherapy; environmental mutagens; in vivo; meetings; mutant; novel; paralogous gene; prevent; public health relevance; repaired; research study; response; small hairpin RNA; tool; tumor; yeast two hybrid system

DESCRIPTION (provided by applicant): RAD51AP1 (AP1 - Associated Protein 1) is a widely expressed, vertebrate-specific, novel RAD51-interacting and DNA-binding protein that is up-regulated in many tumor types. No direct evidence linked RAD51AP1 to homologous recombinational DNA repair (HRR), and its function has remained mysterious. Using RNAi, we have now accumulated a large body of evidence to show that RAD51AP1 is required for HRR. In addition, both our cell biological knockdown investigations and biochemical studies, conducted by a collaborator, suggest that RAD51AP1 functions downstream of the recombinational mediators either in synapsis or post-synapsis, two poorly understood steps within the HRR pathway. HRR is critical for the repair of spontaneous double-strand breaks (DSBs) in S-phase and radiation- and chemically-induced DSBs in S/G2, and is indispensible for maintaining genomic stability and restarting stalled replication forks. HRR is important in limiting mutagenesis and cancer, but also is a target in both tumor and preventive therapy. Importantly, HRR is impaired in brca2 cells and the BRCA2 protein, like RAD51AP1, directly interacts with RAD51. This proposal on human RAD51AP1 has five main goals: 1) to test our hypothesis that RAD51AP1 functions at an intermediate stage in HRR (i.e. downstream of the recombinational mediators) (Aims 1 & 2); 2) to determine if lack of RAD51AP1, both in human cells stably depleted for RAD51AP1 and in a rad51ap1 knockout from DT40 cells, mirrors or exacerbates the phenotype of other HRR mutants (e.g. brca2) (Aims 1 & 2); 3) to test if the interaction of RAD51AP1, through its C-terminal domain (CTD), with human RAD51 is required for HRR (Aims 2 & 3); 4) to determine if other regions of RAD51AP1 are important for its RAD51- interaction and its role in HRR, including regions conserved with NUCKS (Nuclear, Kinase Substrate), a RAD51AP1 paralog (Aims 3 & 4); and 5) to test if the functions of NUCKS and RAD51AP1 overlap (Aim 4). To meet these goals the following approaches will be taken: a lentiviral shRNA system will be used to extensively deplete RAD51AP1 in several human cell lines, which will be tested for RAD51 and RAD54 foci formation. In addition, the levels of sister chromatid exchanges and mutagenesis will be assessed. A rad51ap1 knockout in the model DT40 chicken system will be developed and characterized, and used with RAD51AP1- depleted human cells to test for the biological significance of the RAD51AP1-RAD51 interaction and of the RAD51AP1-CTD. NUCKS, a RAD51AP1 paralog that lacks the RAD51AP1-CTD motif, will be tested for its role in DNA repair, and regions in RAD51AP1 that are conserved with NUCKS will be tested to determine if they are important for the proper function of RAD51AP1. A human cell two-hybrid system will be used to examine the RAD51 interaction and to test for other RAD51AP1 interactions. Our proposed experiments should help establish the exact role of RAD51AP1 in HRR, a prerequisite for better understanding the cellular responses to chemo- and radiotherapy.

描述（由申请人提供）：RAD51AP1（AP1-相关蛋白1）是一种广泛表达的，脊椎动物特异性的，新型的Rad51相互作用和DNA结合蛋白，在许多肿瘤类型中被上调。没有直接证据将RAD51AP1与同源重组DNA修复（HRR）联系起来，其功能仍然是神秘的。使用RNAi，我们现在积累了大量证据，以表明HRR需要RAD51AP1。此外，我们的细胞生物敲低研究和由合作者进行的生化研究都表明，在突触或同伴后重组介体下游的RAD51AP1在HRR途径中的两步骤中的步骤都不足。 HRR对于在S/G2中的S期和辐射和化学诱导的DSB中自发双链断裂（DSB）的修复至关重要，并且对于维持基因组稳定性和重新启动停滞的复制叉是必不可少的。 HRR在限制诱变和癌症方面很重要，但在肿瘤和预防疗法中也是一个靶标。重要的是，HRR在BRCA2细胞中受损，而BRCA2蛋白（如RAD51AP1）直接与RAD51相互作用。关于人Rad51AP1的这一建议具有五个主要目标：1）测试我们的假设，即Rad51AP1在HRR的中间阶段起作用（即重组介体的下游）（AIMS 1＆2）； 2）确定是否缺乏RAD51AP1，在人类细胞中是否稳定地耗尽了RAD51AP1的耗尽以及在DT40细胞的Rad51AP1敲除中，镜子或加剧了其他HRR突变体的表型（例如BRCA2）（例如，AIMS 1＆2）； 3）要测试HRR是否需要Rad51ap1通过其C末端结构域（CTD）的相互作用（AIMS 2＆3）； 4）确定RAD51AP1的其他区域对于其RAD51-相互作用及其在HRR中的作用是否重要，包括与Nuck（核，激酶底物）保守的区域，RAD51AP1旁系同源物（AIMS 3＆4）； 5）测试Nucks和Rad51AP1的功能是否重叠（AIM 4）。为了满足这些目标，将采取以下方法：慢病毒shRNA系统将用于在几种人类细胞系中广泛耗尽rad51ap1，这将对RAD51和RAD54 FOCI形成进行测试。另外，将评估姐妹染色单体交换和诱变水平。 DT40鸡系统中的RAD51AP1敲除将开发和表征，并与Rad51AP1耗尽的人类细胞一起使用，以测试RAD51AP1-RAD51相互作用和RAD51AP1-CTD的生物学意义。 Nucks是一种缺乏RAD51AP1-CTD基序的RAD51AP1旁系同源物，将测试其在DNA修复中的作用，而Rad51ap1中与Nucks保守的区域将经过测试，以确定它们对于RAD51AP1的正常功能是否重要。人类细胞两杂交系统将用于检查RAD51相互作用并测试其他RAD51AP1相互作用。我们提出的实验应有助于确定RAD51AP1在HRR中的确切作用，这是更好地理解细胞对化学和放射疗法的细胞反应的先决条件。