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

描述（由申请人提供）：RAD 51 AP 1（AP 1相关蛋白1）是一种广泛表达的脊椎动物特异性新型RAD 51相互作用和DNA结合蛋白，在许多肿瘤类型中上调。没有直接证据表明RAD 51 AP 1与同源重组DNA修复（HRR）有关，其功能仍然是个谜。利用RNAi，我们现在已经积累了大量的证据，表明RAD 51 AP 1是HRR所必需的。此外，我们的细胞生物学敲除调查和生化研究，由合作者进行，表明RAD 51 AP 1功能的重组介质的下游，无论是在突触或突触后，两个知之甚少的步骤内的HRR途径。HRR对于S期自发双链断裂（DSB）以及S/G2期辐射和化学诱导的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不可或缺的。HRR在限制突变和癌症中是重要的，但也是肿瘤和预防性治疗的靶点。重要的是，HRR在brca 2细胞中受损，而BRCA 2蛋白，如RAD 51 AP 1，直接与RAD 51相互作用。本研究的主要目的是：1）验证我们的假设，即RAD 51 AP 1在HRR的中间阶段发挥作用（即重组介体的下游）（目标1和2）; 2）确定在稳定耗尽RAD 51 AP 1的人细胞中和在从DT 40细胞敲除rad 51 ap 1的人细胞中是否缺乏RAD 51 AP 1，反映或加剧其他HRR突变体的表型（例如brca 2）（目的1和2）; 3）测试HRR是否需要RAD 51 AP 1通过其C-末端结构域（CTD）与人RAD 51的相互作用（目的2&3）; 4）确定RAD 51 AP 1的其他区域是否对其RAD 51相互作用及其在HRR中的作用重要，包括NUCKS保守的区域（核，激酶底物），RAD 51 AP 1蛋白（目的3&4）;和5）测试NUCKS和RAD 51 AP 1的功能是否重叠（目的4）。为了实现这些目标，将采取以下方法：将使用慢病毒shRNA系统来广泛耗尽几种人类细胞系中的RAD 51 AP 1，将测试其RAD 51和RAD 54焦点形成。此外，还将评估姐妹染色单体交换和诱变水平。将开发和表征模型DT 40鸡系统中的rad 51 ap 1敲除，并将其与RAD 51 AP 1耗尽的人细胞一起使用，以测试RAD 51 AP 1-RAD 51相互作用和RAD 51 AP 1-CTD的生物学意义。NUCKS是一种缺乏RAD 51 AP 1-CTD基序的RAD 51 AP 1蛋白，将测试其在DNA修复中的作用，并测试RAD 51 AP 1中与NUCKS保守的区域，以确定它们是否对RAD 51 AP 1的正常功能很重要。将使用人细胞双杂交系统来检查RAD 51相互作用并测试其他RAD 51 AP 1相互作用。我们提出的实验应该有助于确定RAD 51 AP 1在HRR中的确切作用，这是更好地理解细胞对化疗和放疗反应的先决条件。

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.