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

描述（由申请人提供）：RAD51AP1 （AP1 - Associated Protein 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，在许多肿瘤类型中上调。没有直接证据表明RAD51AP1与同源重组DNA修复（homologous recombinational DNA repair， HRR）有关，其功能也一直是个谜。使用RNAi，我们现在已经积累了大量的证据来表明RAD51AP1是HRR所必需的。此外，我们的细胞生物学敲低研究和由合作者进行的生化研究表明，RAD51AP1在突触或突触后的重组介质下游起作用，这是HRR途径中两个鲜为人知的步骤。HRR对于S期自发双链断裂（dsb）和S/G2期辐射和化学诱导的dsb的修复至关重要，对于维持基因组稳定性和重新启动停滞的复制分叉是必不可少的。HRR在限制突变和癌症方面具有重要意义，同时也是肿瘤和预防治疗的靶点。重要的是，HRR在brca2细胞中受损，brca2蛋白，如RAD51AP1，直接与RAD51相互作用。这项关于人类RAD51AP1的研究有五个主要目标：1)验证我们的假设，即RAD51AP1在HRR的中间阶段起作用（即重组介质的下游）（目的1和2）；2)确定RAD51AP1的缺乏是否反映或加剧了其他HRR突变体（如brca2）的表型，无论是在RAD51AP1稳定耗尽的人类细胞中，还是在DT40细胞中剔除RAD51AP1的细胞中（目的1和2）；3)测试HRR是否需要RAD51AP1通过其c端结构域（CTD）与人类RAD51相互作用（目的2和3）；4)确定RAD51AP1的其他区域是否对其RAD51-相互作用及其在HRR中的作用很重要，包括与NUCKS（核，激酶底物），RAD51AP1类似物保守的区域（目标3和4）；5)测试NUCKS和RAD51AP1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系统在几种人类细胞系中广泛消耗RAD51AP1，并测试RAD51和RAD54的病灶形成。此外，姊妹染色单体交换和突变的水平将被评估。我们将在模型DT40鸡系统中开发rad51ap1基因敲除并对其进行表征，并将其与rad51ap1缺失的人类细胞一起用于测试rad51ap1 - rad51相互作用和rad51ap1 - ctd的生物学意义。缺少RAD51AP1- ctd基序的RAD51AP1类似物NUCKS将被测试其在DNA修复中的作用，并且RAD51AP1中与NUCKS保守的区域将被测试以确定它们是否对RAD51AP1的正常功能重要。人类细胞双杂交系统将用于检查RAD51相互作用和测试其他RAD51AP1相互作用。我们提出的实验应该有助于确定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.