【作者】 刘伟；

【导师】 蔡刚；

【作者基本信息】 中国科学技术大学 ， 生物化学与分子生物学， 2019， 硕士

【摘要】 基因组不断受到来自内外环境的压力(如紫外线和电离辐射等)造成DNA损伤。细胞内DNA损伤触发细胞周期检查点激活,导致细胞周期进程延迟或阻滞,阻止细胞复制并且诱导细胞进行DNA损伤修复。Clamp 9-1-1复合体和clamp loader Rad24-RFC在细胞周期检查点激活方面起着重要作用。9-1-1复合体是由Rad9-Hus1-Rad1组成的环形异源三聚体复合物,其中Rad9/Ddc1的C端含有大量磷酸化的位点和蛋白相互作用位点,包括DNA损伤修复关键激酶ATR和TopBP1等。但是由于Rad9/Ddc1 C端结构柔性程度高,目前还没有9-1-1复合体的完整结构。解析9-1-1复合体在不同状态下的(包括DNA损伤条件下)完整结构,尤其是获得Rad9/Ddc1的结构变化能够更好地帮助我们理解9-1-1复合体如何参与DNA损伤响应。Rad24-RFC是一个异源五聚体,由最大的亚基Rad24和四个小亚基RFC2-5组成。在DNA损伤修复过程中,Rad24-RFC负责协助9-1-1复合体加载到DNA损伤位点,起到稳定DNA构象和激活DNA损伤检查点的作用。基于9-1-1复合体与增殖细胞核抗原(PCNA)在结构上的相似性,目前认为9-1-1复合体与Rad24-RFC间相互作用可能与PCNA与RFC间相互作用相似,但是目前并没有直接的证据表明9-1-1复合体与Rad24-RFC行使相似的机制。因此,获得9-1-1复合体与Rad24-RFC复合物的完整结构及其动态相互作用的结构信息将会极大地帮助我们理解clamp加载过程,但是目前还没有二者相互作用的结构信息,甚至连Rad24-RFC的结构信息也很少。本课题我们利用冷冻电镜技术解析了酿酒酵母中9-1-1复合体的完整结构,首次解析了Ddc1亚基的全长结构并发现Ddc1结构柔性受到精细调节。在正常生理状态下,9-1-1复合体具有多种构象,Ddc1 C-tail是高度动态的。我们获得了三种相对稳定的构象,包括Ddc1 C-tail位于环状区域的阻挡DNA结合的构象,Ddc1 C-tail远离环状区域的闭合构象以及环打开的开口构象。甲基磺酸甲酯(MMS)是一种DNA损伤诱导剂,能够造成细胞基因组发生断裂,为了深入研究,我们也获得了MMS诱导条件下的9-1-1复合体。在生化上,MMS诱导的9-1-1复合体的Ddc1亚基在电泳上迁移出多条条带,质谱结果显示均为Ddc1,考虑到Ddc1 C-tail包含众多磷酸化位点,这暗示在DNA损伤条件下Ddc1发生了不同程度的磷酸化。同时我们也解析了MMS诱导的9-1-1复合体的完整结构,与正常生理状态下的9-1-1复合体结构相比,MMS诱导的9-1-1复合体构象变得均一,Ddc1 C-tail主要位于环状区域外。基于生化和结构分析,Ddc1 C-tail的结构柔性很大可能与DNA损伤响应相关。Rad53作为DNA损伤响应的效应激酶,是DNA检查点激活的重要指示剂,我们的磷酸化实验发现,Ddc1 C-tail敲除将会导致G1/G2期Rad53的磷酸化完全丧失或降低,这一数据说明Ddcl C-tail在DNA损伤响应中发挥着重要作用。为了解析Rad24-RFC的结构,我们尝试了内源性表达和重组表达。在内源性纯化Rad24-RFC时,我们发现能够共纯化出Mec1/Ddc2(ATR/ATRIP),二维结构分析也发现存在二者复合体。在之前的研究中Rad24-RFC一直被认为作为clamp loader协助9-1-1复合体加载到DNA损伤位点,一旦完成,Rad24-RFC将会从DNA损伤处解离。我们的共纯化结果或许暗示了Rad24-RFC在体内能够与Mec1/Ddc2发生直接的相互作用。由于内源性纯化的Rad24-RFC浓度和均一度比较低,因此我们尝试重组表达获得Rad24-RFC样品。在高盐、高去垢剂的纯化条件下,我们获得了高浓度和高纯度的Rad24-RFC,同时我们也首次解析了Rad24-RFC的完整结构。在负染结构中,Rad24-RFC部分构象呈现出“U”型结构,中间含有一条深沟;部分构象呈现出“爪”型。参与DNA复制的clamp loader RFC复合体(RFC1-5)与Rad24-RFC共享了四个相同的小亚基RFC2-5,基于RFC复合体的晶体结构,我们确定了Rad24模块结构。同时我们将RFC1与Rad24的density进行单独比较,二者结构十分相似。结合PCNA与9-1-1复合体的结构相似性,Rad24-RFC与RFC复合体的结构相似或许暗示着这两对clamp-clamp loader的相互作用机制存在一定的相似。基于以上结构信息,我们也对9-1-1复合体与Rad24-RFC间相互作用进行了研究,我们发现,二者相互作用在体内受到DNA损伤应激调控。在体外我们也成功组装了Rad24-RFC/9-1-1超级复合物,这也为我们后续解析三维结构奠定了基础。更多还原

【Abstract】 The genome is constantly subject to stress from internal and external environments(such as ultraviolet light and ionizing radiation)causing DNA damage.Cellular DNA damage triggers cell cycle checkpoint activation,leading to a delay or arrest in cell cycle progression to prevent cell replication and inducing DNA damage repair.The clamp 9-1-1 complex and the clamp loader Rad24-RFC play an important role in cell cycle checkpoint activation.The 9-1-1 complex is a circular heterotrimeric complex composed of Rad9-Husl-Rad1,in which the C-terminus of Rad9/Ddcl contains a large number of pHospHorylation sites and protein interaction sites,including DNA damage repair key kinase ATR,TopBP1,etc.However,due to the flexibility of the Rad9/Ddcl C-terminal structure,there is currently no intact structure of the 9-1-1 complex.Obtaining the intact structure of the 9-1-1 complex under different conditions(including DNA damage conditions),especially determining the structural changes of Rad9/Ddc1,can better help us understand how the 9-1-1 complex is involved in DNA damage response.Rad24-RFC is a heteropentamer consisting of the largest subunit Rad24 and four small subunits RFC2-5.During DNA damage repair,Rad24-RFC is responsible for assisting the loading of the 9-1-1 complex into the DNA damage site,which serves to stabilize the DNA conformation and activate DNA damage checkpoints.Based on the structural similarities between the 9-1-1 complex and proliferating cell nuclear antigen(PCNA),it is currently believed that the interactions between the 9-1-1 complex and Rad24-RFC may be similar to the interactions between PCNA and RFC,but there is currently no direct evidence that the 9-1-1 complex performs a similar mechanism to Rad24-RFC.Therefore,obtaining the structural information of the intact structure of the 9-1-1 complex and the Rad24-RFC complex and its dynamic interaction will greatly help us understand the clamp loading process,but there is no structural information of the interactions between them,and even the structural information of the Rad24-RFC is rare.In this paper,the intact structure of the 9-1-1 complex in Saccharomyces cerevisiae was determined by cryo-electron microscopy(Cryo-EM).The full-length structure of the Ddc1 subunit was first analyzed and it was found that the structural flexibility of Ddc1 was finely regulated.Under normal pHysiological conditions,the 9-1-1 complex has multiple conformations and the Ddcl C-tail is highly dynamic.We obtained three relatively stable conformations,including the blocking conformation of the Ddcl C-tail in the ring region,the closed conformation of the Ddcl C-tail away from the ring region,and the open conformation of the ring opening.Methyl methanesulfonate(MMS)is a DNA damage inducer that can cause cell genome breakage.For further study,we also obtained the 9-1-1 complex under MMS induction conditions.Biochemically,the MMS-induced Ddcl subunit migrated multiple bands on the electropHoresis,and the mass spectrometry(MS)results showed that all were Ddc1.Considering that Ddc1 C-tail contains many pHospHorylation sites,it suggests that Ddcl undergoes different degrees of pHospHorylation under DNA damage conditions.At the same time,we also determined the intact structure of the MMS-induced 9-1-1 complex.Compared with the structure of the 9-1-1 complex under normal pHysiological conditions,the MMS-induced 9-1-1 complex conformation became homogeneous,Ddcl C-tail is mainly located outside the ring area.Based on biochemical and structural analysis,the structural flexibility of Ddcl C-tail is likely to be related to DNA damage response.Rad53,an effector kinase in response to DNA damage,is an important indicator of DNA checkpoint activation.Our pHospHorylation experiments revealed that Ddc1 C-tail knockout will result in complete loss or reduction of pHospHorylation of Rad53 in G1/G2 pHase.It suggests that Ddc1 C-tail plays an important role in DNA damage response.To determine the structure of Rad24-RFC,we tried endogenous expression and recombinant expression.When Rad24-RFC was endogenously purified,we found that Mecl/Ddc2(ATR/ATRIP)could be co-purified,and the Rad24-RFC/Mecl-Ddc2 complex was also found in the two-dimensional structure.In previous studies,Rad24-RFC has been considered as a clamp loader to assist the loading of the 9-1-1 complex into the DNA damage site.Once completed,Rad24-RFC will dissociate from the DNA damage.Our co-purification results may suggest that Rad24-RFC can interact directly with Mec1/Ddc2 in vivo.Due to the low concentration and purity of endogenously purified Rad24-RFC,we attempted recombinant expression to obtain Rad24-RFC samples.Under high salt and high detergent conditions,we obtained high concentration and high purity Rad24-RFC,and we also determined the complete structure of Rad24-RFC for the first time.In the negative-stained structure,the partial conformation of Rad24-RFC exhibits a "U"-shaped structure with a deep groove in the middle;the partial conformation exhibits the shape of a "claw.The clamp loader RFC complex(RFC1-5)involved in DNA replication shares four identical small subunits RFC2-5 with Rad24-RFC.Based on the crystal structure of the RFC complex,we determined the structure of the Rad24 module.At the same time,we compare the structure of RFC1 with the density of Rad24 and find that the structures are very similar.Combining the structural similarities between PCNA and the 9-1-1 complex,the similar structure of the Rad24-RFC to the RFC complex may suggest that the interaction mechanisms of the two pair of clamp-clamp loaders are similar.Based on the above structural information,we also studied the interactions between the 9-1-1 complex and Rad24-RPC,and we found that the interactions between the two are regulated by DNA damage pressure in vivo.We also successfully assembled the Rad24-RFC/9-1-1 supercomplex in vitro,which also laid the foundation for our subsequent acquisition of three-dimensional structure.更多还原