SIVA1调节跨损伤DNA合成的分子机制研究

发布时间：2018-03-27 08:38

本文选题：基因组稳定性　切入点：跨损伤DNA合成　出处：《浙江大学》2016年博士论文

【摘要】：细胞的基因组DNA持续受到内源性因素(如代谢产生的活性氧等)及外源性物质(如阳光中的紫外线,化学药物及电离辐射等)的攻击,造成不同类型的DNA损伤。这些损伤阻碍了细胞的复制和转录过程,如果不能得到及时、精确的修复,将导致基因突变、染色体畸变、细胞老化或凋亡。因此,细胞启用一系列高效的监控和修复途径来检测、修复不同类型的DNA损伤,从而维持基因组稳定性。跨损伤DNA合成(translesion DNA synthesis, TLS)是从酵母到哺乳动物都高度保守的DNA损伤耐受机制,由TLS聚合酶取代高保真的DNA聚合酶,对受损伤的DNA进行复制。DNA受到损伤后往往结构发生异变,异变的DNA结构阻碍了DNA聚合酶的复制进程,导致复制叉停滞,对处在复制期的细胞产生了巨大的威胁。TLS聚合酶的催化中心更灵活,能适应不同类型的异变的DNA模板,取代高保真性的DNA聚合酶重新启动暂停的复制叉,完成DNA的复制,这对于维持遭受DNA损伤的细胞的存活至关重要。TLS聚合酶主要由Y家族的DNA聚合酶构成,包括Polη、Polη、Polκ和REV1。研究表明每种TLS聚合酶倾向于复制不同类型的损伤模板,从损伤模板中提取正确的遗传信息。环境中广泛存在的紫外线照射常常导致DNA中相邻的嘧啶碱基发生交联,形成环丁烷嘧啶二聚体(Cyclobutane pyrimidine dimers, CPDs).胸腺嘧啶二聚体(T-T dimer)是最常见的CPDs, Po1η能以T-T dimer为模板进行复制,正确地插入A·A碱基。缺失有功能的Polη易患着色性干皮病(XP-V), XP-V患者的细胞对UV照射非常敏感,患者在青少年时期就容易得皮肤癌。调节跨损伤DNA合成途径的关键事件是PCNA的单泛素化修饰。目前大量的研究表明当细胞遭受DNA损伤或遇到复制压力时,泛素结合酶RAD6及泛素连接酶RAD18组成的复合物特异地对底物PCNA的第164位赖氨酸进行单泛素化修饰。单泛素化的PCNA与Y家族的TLS聚合酶亲和力升高,主要是通过Y家族聚合酶的PCNA结合结构域和泛素结合结构域来实现的。TLS聚合酶取代常规的复制聚合酶对受损的DNA进行复制,重新启动停滞的复制叉。目前RAD6-RAD18复合物如何精确地识别PCNA并对其进行单泛素化修饰的分子机制并不清楚。我们利用串联亲和纯化的方法研究PCNA的相互作用蛋白,发现SIVAI在调节PCNA的单泛素化修饰过程中发挥着重要的功能。我们的实验结果表明SIVAI通过PIP box结构域持续地与PCNA相互作用。细胞敲减SIVAI后,UV损伤诱导的PCNA的单泛素化水平降低,TLS聚合酶Polη的招募明显减少,细胞对UV损伤的敏感度和突变率升高。我们证明SIVAI与RAD18直接相互作用,作为接头蛋白帮助RAD18识别PCNA,从而促进RAD18对PCNA的单泛素化修饰,保证细胞能快速地启动跨损伤DNA合成,高效地对受损伤的DNA进行复制。因此,我们的实验创新性地发现E3连接酶RAD18识别其底物PCNA需要接头蛋白SIVAI,为UV损伤诱导的PCNA单泛素化修饰的调节机制提供了新的见解,填补了跨损伤DNA合成途径中遗留的一个重要空缺。
[Abstract]:The genome of DNA cells continued to be endogenous factors (such as metabolism of active oxygen) and exogenous substances (such as UV rays, chemicals and ionizing radiation) attacks, causing DNA damage of different types. These injuries hampered the replication and transcription processes in the cell, if they can not get timely and accurate repair that will lead to gene mutation, chromosome aberration, cell senescence or apoptosis. Therefore, cells enable a series of effective monitoring and repair pathways to detect DNA damage repair of different types, in order to maintain genomic stability. Translesion DNA synthesis (translesion DNA, synthesis, TLS) is DNA damage tolerance mechanisms are highly conserved from yeast to mammals and by TLS polymerase to replace the high fidelity DNA polymerase, often on structure damage DNA replication damage.DNA mutation, DNA mutation blocked DNA poly structure The copy process synthase, leading to stalled replication forks, in replicating cells produced a huge threat to.TLS polymerase catalytic center is more flexible and can adapt to different types of mutation of the DNA template, instead of high fidelity DNA polymerase to initiate replication fork suspension, complete DNA replication, which is to maintain suffer the damage of DNA cell survival is mainly composed of Y.TLS polymerase family DNA polymerase, including Pol, Pol, Pol, K and REV1. research shows that each template damage TLS polymerase tended to replicate the different types of extraction of genetic information correctly from the damage mode of the board. The environment is often lead to ultraviolet irradiation pyrimidine bases in DNA adjacent crosslink formation of cyclobutane pyrimidine dimers (Cyclobutane pyrimidine two dimers, CPDs). Two thymine dimer (T-T dimer) is the most common CPDs, Po1 to T-T di. Mer as the template for replication, correctly inserted into the A A base. The lack of functional Pol is susceptible to xeroderma pigmentosum (XP-V) patients, XP-V cells are very sensitive to UV irradiation, patients easily at a young age to skin cancer. Key events regulate translesion DNA synthesis pathway is monoubiquitinated modification of PCNA. Many studies have shown that when the cells were subjected to DNA damage or replication encountered pressure, complex ubiquitin conjugating enzyme RAD6 and the ubiquitin ligase RAD18 composed of specific substrate PCNA 164th lysine of monoubiquitin. Increased ubiquitination of PCNA and Y family TLS polymerase affinity, mainly the family of Y polymerase PCNA binding domain and ubiquitin binding domain to achieve.TLS polymerase of conventional polymerase substituted replication of damaged DNA replication, restart the stalled replication forks. At present RAD6-RAD18 complexes How to accurately identify PCNA and its molecular mechanism of monoubiquitin is not clear. We use a series of interacting protein affinity purification method of PCNA, found that SIVAI played an important role in the process of modification of single ubiquitin mediated PCNA. Our experimental results show that the SIVAI through the PIP box domain continuously the interaction with PCNA cells. Knockdown of SIVAI, ubiquitination of UV damage induced by PCNA level decreased, the recruitment of TLS polymerase Pol is significantly reduced, the sensitivity of cells to UV damage and mutation rate increased. We prove that SIVAI and RAD18 interact directly, as adapter proteins help identify RAD18 PCNA, so as to promote RAD18 monoubiquitin to PCNA, to ensure the cell can quickly start translesion DNA synthesis and replication of damaged DNA efficiently. Therefore, our experiment innovatively found that E3 is connected with the enzyme RAD 18, recognition of its substrate PCNA requires a joint protein SIVAI, which provides new insights into the regulatory mechanism of UV damage induced PCNA ubiquitination modification, and fills an important gap left in the cross injury DNA synthesis pathway.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q75

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