【作者】 刘长东；

【导师】 朱广；

【作者基本信息】 华东师范大学 ， 无线电物理， 2011， 博士

【摘要】 本论文包含两部分：1)在真核细胞中,DNA的复制受到严格的控制,以保证在每个细胞周期中DNA准确的复制一次。DNA复制的起始阶段是复制过程中关键的一步,该过程受复制前复合物(pre-replication complex, pre-RC)的严格控制。微小染色体维持蛋白(minichromsome maintenance, MCM)复合物是pre-RC的重要组分,该复合物是由同一家族结构相类似的6个亚单位(Mcm2-Mcm7)组成,目前被认为是DNA复制过程中的解链酶,并在复制叉的延伸过程中起关键作用。在细胞周期的M晚期和G1早期,Mcm2-7复合物依赖于另一个pre-RC的重要亚单位Cdc10依赖性转录因子(Cdc10 dependent transcriptl, Cdt1)而结合到染色质上面。Mcm6是Mcm2-7复合物中的一个亚单位,由位于蛋白中部高度保守的MCM结构域和C-末端尚未被研究的结构域构成。本文研究表明,高度保守的Mcm6 C-末端能够与Cdtl相互作用,并将与Cdtl相互作用的区域称为Cdtl结合结构域(Cdt1 binding domain, CBD),并通过高分辨率核磁共振方法解析出其空间结构。CBD的结构为典型的"winged-helix"折叠。"winged-helix"折叠通常参与蛋白质-核酸的相互作用,但本文的研究发现CBD同DNA没有直接相互作用,表明CBD特异性的参与蛋白质—蛋白质之间的相互作用。同时我们也发现了位于Cdtl上面与Mcm6相结合的区域(Mcm6 binding domain, MBD)。利用核磁共振滴定实验,我们确认出位于CBD上面直接与Cdtl相互作用的氨基酸,E757和L766,通过生物化学以及细胞学实验验证了这两个氨基酸在Mcm6-Cdtl相互结合中的关键作用。同时,通过高分辨率核磁共振方法解析了MBD的溶液结构为α螺旋,利用核磁共振滴定实验确定了MBD上与CBD的相互结合区域。通过13C-edited (F1), 13C/15N-filtered (F3)3DNOESY观测到了CBD与MBD分子间的NOE,解析出CBD-MBD复合物结构。确定了CBD与MBD结构域相互作用的区域和重要氨基酸,阐述了CBD与MBD通过HTH结构基元相互识别的特异性。我们认为,Mcm6通过其CBD与Cdtl的MBD相互作用可能引起Mcm2-7复合物与染色质相结合而完成pre-RC的装配,本文的研究工作为深入理解DNA的复制起始机理提供了重要结构依据。2) SWIRM结构域是在许多染色质修饰与重塑蛋白质分子中存在的一个非常保守的模块,比在如染色质修饰复合物中的LSDl和Ada2蛋白质分子中都存在有SWIRM结构域。本文通过NMR方法解析了LSDl中SWIRM结构域的溶液结构并与已经发表的包含有同样序列的晶体和NMR结构相比较发现,N-末端的十个氨基酸并未如已发表的结构一样形成α螺旋,而是呈现出无规卷曲结构。因此,将N末端序列延长5个氨基酸,通过2D 1H-15N-HSQC谱以及异核1H-15N NOE实验研究发现,这五个氨基酸对α螺旋的形成和稳定起到了重要作用。更多还原

【Abstract】 This thesis contains two parts:1) In all eukaryotic cells, initiation of genomic DNA replication is tightly controlled to ensure that DNA replication occurs only once in each cell cycle. DNA replication initiation is the critical step during the whole DNA replication process, initiation step is strictly controlled by the formation of pre-replication complex (pre-RC) which. is composed of several important protein complexes and proteins including the. minichromosome maintenance MCM complex. The MCM complex is composed of six evolutionally conserved proteins Mcm2-Mcm7, and it is believed to be the replicative helicase in eukaryotic species, and it plays essential roles in the initiation and elongation phases of DNA replication. During late M and early G1, the Mcm2-7 complex is loaded onto chromatin to form pre-RC in a Cdtl-dependent manner.Mcm6 is one of the six subunit of the Mcm2-7 complex, and it contains the highly conserved MCM domain and one uncharacterized C-terminal domain.In this study we demonstrate that the previously uncharacterized C-terminal domain of human Mcm6 is the Cdtl binding domain (CBD).With the use of high-resolution nuclear magnetic resonance (NMR) technique, we solved the 3D structure of CBD. The structure of CBD exhibits a typical "winged-helix" fold that is generally involved in protein-nucleic acid interaction. Nevertheless, the CBD failed to interact with DNA in our studies indicating that it is specific for protein-protein interaction. Also, we mapped the Mcm6 binding domain (MBD) of Cdtl. Based on NMR titration experiment and GST Pull-down assay we identified E757 and L766 on CBD are the critical residues for the interaction with Cdt1.The fundamental role of these two residues in Mcm6-Cdtl interaction was further confirmed with subcellular co-localization assay. Meanwhile, we solved the 3D structure of MBD which contains a a helix in solution and mapped the residues involved in binding with CBD by NMR. Based on the observed intermolecular NOEs in 13C-edited (F1), 13C/15N-filtered (F3)3D NOESY experiment, we have determined the structure of the CBD-MBD complex and mapped the key residues on the interface in solution. The structure of the CBD-MBD complex elucidates the specific recognization through the HTH motif between CBD and MBD. We speculate that Cdtl directs Mcm2-7 complex chromatin loading to accomplish pre-RC formation through the direct interaction between the MBD of Cdtl and CBD of Mcm6. In conclusion, our structural and functional studies in Mcm6-Cdtl interaction will definitely help to better understand the process of Mcm2-7 complex chromatin loading and pre-RC assembly which are the vital steps in eukaryotic DNA replication.2) The evolutionarily conserved SWIRM (Swi3p, Rsc8p and Moira) domain is a module found in many chromatin modification or remodeling proteins, such as the Ada2 and LSD1 subunits of chromatin modification complexes. In this study, we present the solution NMR structure of the SWIRM domain from LSD1 and compare with the published crystal and NMR structure which contains the same sequence. We found that the N-terminal 10 amino acids show a random coil but not a short helix in the published structure. The additional 5 amino acids were added on the N terminus. Based on the 2D 1H-15N HSQC spectrum and heteronuclear 1H-15N NOE data, the additional 5 amino acids are very important for the formation and stabilization of the N-terminal helix which is not observed in our structure.更多还原