【作者】 万力；

【导师】 黄俊；

【作者基本信息】 浙江大学 ， 细胞生物学， 2015， 博士

【摘要】 为了应对DNA损伤威胁,真核生物进化出了一系列的DNA损伤应答反应机制(DNA damage response, DDR).DDR是一个由许多不同的信号通路所组成的复杂的信号网络,负责在整个细胞周期内应对各种内源性和外源性的损伤威胁,这些信号通路包括：DNA修复、细胞周期检验点、转录调控和细胞凋亡等。DNA双链断裂是细胞内所有损伤类型中最为严重的损伤之一,为了应对这种威胁性的损伤类型,机体演化出了两种主要修复机制：非同源末端连接(non-homologous end-joining, NHEJ)和同源重组修复(homologous recombination, HR).HR修复方式在维持基因组的稳定性方面发挥了关键的作用,这一修复通路中的任一关键蛋白发生突变都会导致肿瘤以及其他严重疾病的发生。布鲁姆综合症是一类临床表现为发育缺陷、免疫缺陷、基因组高度不稳定以及高癌症诱发率的疾病,其诱因是RecQ家族成员之一的BLM基因发生了突变。BLM(Bloom’s syndrome protein),即布鲁姆综合症蛋白,它可以与HR信号通路精密协作,在修复过程中促进DNA的精准合成,高效地完成双链断裂的修复。然而,BLM与HR在机体内究竟是怎样精准协作的分子机制尚不清楚。在本论文的第一个课题中,我们定义了一个新的蛋白SPIDR (scaffolding protein involved in DNA repair, SPIDR),并证明了它具有连接BLM和HR修复信号通路的桥梁功能。SIPDR可以分别与BLM和RAD51相互作用,以形成一个在机体内发挥重要功能的BLM/RAD51复合体。用RNA干扰的方法降低内源性的SPIDR的表达之后,BLM和RAD51不能有效定位到DNA损伤位点,并使姐妹染色单体交换(sister-chromatid exchanges, SCE)的水平明显升高,而SCE水平过高是布鲁姆综合症的表型之一。此外,降低内源性的SPIDR的表达,也会导致基因组的不稳定、同时增加了细胞对损伤诱导剂的敏感性。所以我们猜想,SPIDR作为一个脚架蛋白,通过调节BLM与RAD51的协作关系,一方面促进了HR修复的发生,另一方面也阻止或降低了不必要的甚至是具有毒性的代谢产物的生成。ATR (ataxia telangiectasia-mutated and Rad3-related kinase)激酶是PIKK家族的成员之一,在DDR的细胞周期检验点信号通路中发挥了关键作用。当DNA损伤发生之后,ATR-ATRIP(ATR-interacting protein)复合物会被单链DNA结合蛋白——-RPA (replication protein A)复合物招募到损伤位点,这对于ATR的激活是至关重要的。PSO4复合物是由PSO4/PRP19/SNEV、CDC5L、PLRG1和SPF27所组成的异源四聚体复合物,之前的研究报道了其在mRNA前体的剪切方面的重要作用。最近的报道指出,PSO4复合物可能在DDR中也扮演了重要的角色,但其具体的分子机制尚不明确。在本论文的第二个课题中,我们发现BCAS2通过与RPA相互作用的方式使PSO4复合物被招募到了损伤位点。降低内源性的BCAS2或者PSO4的表达水平,都会影响ATRIP在损伤位点的聚集、CHK1的激活和RPA2的磷酸化水平。之后,我们进一步证明,无论是BCAS2与RPA1结合作用的缺陷,还是PSO4作为泛素E3连接酶活性的失去,都会使ATRIP在损伤位点的聚集、CHK1的激活状况和RPA2的磷酸化水平受到明显影响。所以我们的课题证明了,在面对DNA损伤压力的情况下,PSO4复合物通过与RPA结合,继而介导了由ATR调控的细胞周期检验点信号通路,最终参与到了DNA损伤应答反应之中。更多还原

【Abstract】 To counteract DNA damage, eukaryotic cells have evolved the DNA damage response(DDR). DDR represents a complex network of multiple signaling pathways involving DNA repair, cell cycle checkpoints, transcriptional programs, and apoptosis, through which cells maintain genome stability following exposure to various endogenous or exogenous DNA-damaging agents.Of the different types of DNA damage that arise in cells, DNA double-strand break(DSB) is one of the most cytotoxic forms of DNA damage. Two major pathways for DSB repair have been defined:non-homologous end-joining (NHEJ) and homologous recombination (HR). HR repair plays a vital role in the maintenance of genomic integrity, and mutations in key components in the HR repair pathway are often associated with cancers and other human diseases. Bloom’s syndrome, characterized by growth deficiency, immunodeficiency, genome instability, and a high predisposition to cancer, is caused by mutation of the RecQ family gene BLM. BLM, the Bloom’s syndrome protein, is crucial for collaborating with HR machinery to promote efficient DNA repair and prevent DSB repair by less precise pathways. However, exactly how such coordination occurs in vivo is not clearly defined. Here, we identified a protein termed SPIDR (scaffolding protein involved in DNA repair) as the link between BLM and the HR machinery. SPIDR independently interacts with BLM and RAD51and promotes the formation of a BLM/RAD51-containing complex of biological importance. Consistent with its role as a scaffolding protein for the assembly of BLM and RAD51foci, depletion of SPIDR by RNA-mediated interference methods results in the high levels of sister-chromatid exchanges (SCE), the hallmark of Bloom’s syndrome cells. Moreover, SPIDR depletion leads to genome instability and causes hypersensitivity to DNA-damaging agents. We propose that, through providing a scaffold for the cooperation of BLM and RAD51in a multifunctional DNA-processing complex, SPIDR not only promotes the efficiency of HR, but also optimises the HR pathway and prevents unnecessary and potentially deleterious product in repair processes.The ataxia telangiectasia-mutated and Rad3-related (ATR) kinase, a member of PIKK family, is a master checkpoint regulator of DDR. Upon DNA damage, the ATR-ATRIP (ATR-interacting protein) complex is recruited to DNA damage sites by RPA-ssDNA(replication protein A-single-stranded DNA), which is thought to play a crucial role in ATR activation. PSO4complex, containing PSO4/PRP19/SNEV, CDC5L, PLRG1, and SPF27, has a well-defined role in pre-mRNA splicing from yeast to humans. Recently, studies have described that the PSO4complex had a role in DDR, However, the specific role for the PSO4complex in DDR remains enigmatic. We showed that BCAS2subunit of the PSO4complex directly interacts with RPA1and is recruited to DNA damage sites in an RPA-dependent manner. Depletion of BCAS2or PSO4results in defective recruitment of ATRIP to DNA damage sites and compromises CHK1activation and RPA2phosphorylation. Moreover, we demonstrated that both the RPA1-binding ability of BCAS2and the E3ligase activity of PSO4are required for efficient accumulation of ATRIP at DNA damage sites and the subsequent CHK1activation and RPA2phosphorylation. Our results demonstrated that PSO4complex associates with RPA and regulates ATR-mediated cell-cycle checkpoint in response to genotoxic agents.更多还原