【作者】 卢淑红；

【导师】 申玉龙；

【作者基本信息】 山东大学 ， 微生物学， 2008， 硕士

【摘要】 生存在自然环境中的所有生物都会不可避免地面临各种不利于其生长的危险,例如来自于生活环境中的各种有害化学物质以及生物自身体内的有害代谢产物等等,这些因素都会对生物的基因组DNA造成损伤,而生物体内拥有完整的一套DNA修复系统,维持其基因组稳定。值得注意的是超嗜热古菌比起一般生物生活环境更加恶劣,但是仍然能活跃生存在那种极端环境中,其体内必定含有更优越的DNA损伤修复机制。此外,由于古菌DNA代谢中的各种因子与细菌相比更相似于真核生物,所以探索古菌DNA代谢奥秘,无疑也会为复杂的真核生物研究提供可靠的帮助。滑动夹(sliding clamp)参与DNA复制,修复以及细胞周期调控等多种DNA代谢途径,是各种生物维持生存必要的蛋白因子,可以作为一种平台与DNA代谢中的多种蛋白因子相互作用,例如DNA复制聚合酶,连接酶等。细菌中的滑动夹为DNA复制聚合酶的一个亚基称β-夹子(β-clamp),真核生物和古菌中称这一蛋白为PCNA(proliferating cell nuclear antigen)。不同生物的滑动夹虽然在序列上并不是保守的,但是结构上都是一种相似的环状复合物,中间具有一DNA通过的孔道,预示着它们发挥功能作用的方式可能是相似的。例如,几种滑动夹都需要一种辅助蛋白的参与作用,即滑动夹装载蛋白(clamp loader),细菌中为γ-复合物,而真核生物和细菌中为RFC(replication factor C,RFC)。然而,滑动夹复合物在三种生物域中也具有多样性,例如,他们寡聚状态也并不保守,细菌中的β-夹子为同源二聚体,真核生物及广古菌中PCNA为同源三聚体,而泉古菌中为异源三聚体。由于在古菌DNA修复系统中,PCNA是不可缺少的因子,因此,研究古菌的这一蛋白对于了解其特有的DNA修复机制十分有意义。本文主要介绍了对超嗜热古菌Sulfolobus tokodaii三个PCNA亚基间的相互作用和一些生化性质的初步研究。主要利用基因重组技术获得了来自s.tokodaii三个PCNA亚基的重组蛋白,首先利用His-pull down,酵母双杂交以及分子筛层析等蛋白互作方法,检测到体内,体外PCNA单亚基均不能形成同源聚体,PCNA1和PCNA3可形成二聚体,PCNA1,PCNA2和PCNA3形成三聚体。很有意思的是我们发现PCNA2和PCNA3也可形成三聚体,其中的成分有两种可能,即PCNA223和PCNA323。接下来我们设计实验验证其成分,主要是利用His-pull down方法,鉴定出了一种新的PCNA三聚体,其成分为PCNA323。然后结合Pull down和分子筛层析两种方法将PUNA123和PCNA323两种三聚复合体分别纯化出来,并分别检测它们对参与DNA修复的几个蛋白因子活性的影响。发现两种复合体均以相同程度抑制解旋酶StoHjm以及连接酶StoLigase活性,PCNA323较强于PCNA123刺激核酸内切酶StoHjc的活性。本实验所获得的实验数据与其他报道相比较分析,发现古菌中的PCNA亚基间相互作用关系具有一定的多样性,即使是同属泉古菌的S.solfataricus或Aeropyrum pernix中的同源PCNA都存在着差别。关于本文中鉴定出的PCNA两种三聚体复合体的具体功能,以及相互之间差异还需要进一步的实验分析.另外,有文章报道在S.solfataricus中PCNA可以与重组修复过程中的核酸酶Hjc(Holliday junction cleavage)具有相互作用,pyrococcus.furiosus中PCNA与Hjm具有相互作用。同时,我们发现S.tokodaii中StoHjc可以与同样参与重组修复途径的StoHjm解旋酶(Holliday junction migration)具有相互作用,根据以上信息,我们推测PCNA,StoHjc和StoHjm可能会形成一种三聚体行使某种生理功能,于是着手实验验证这种猜测。在本论文的后一部分,我们介绍了有关StoHjm和StoHjc的工作,通过分子筛层析,His-pull down和酵母双杂交证明了StoHjm可以与StoHjc之间存在相互作用,并且StoHjc抑制StoHjm的解旋酶活性。目前,虽然没有检测到StoHjm和核酸内切切酶StoHjc与PCNA之间具有相互作用,但是我们会优化实验条件继续研究。另外,为了了解StoHjm与StoHjc相互作用的分子机制,我们计划利用缺失突变方法进一步探索。本文初步研究了S.tokodaii中PCNA,以及Hjm和Hjc这几个蛋白的生化性质及其相互之间的关系,为深入了解S.tokodaii的修复机制提供了依据,并为进一步研究奠定了基础。更多还原

【Abstract】 Organisms in the environments always encounter many barriers to survival, for examples, DNA damages caused by chemical agents from environments and by the byproducts of normal metabolism. So, there exist various pathways in organisms living actively in the environments to overcome these damages and stabilize genomes. Especially, hyperthermophilic archaea can flourish in habitats of extreme temperatures, without obvious difference in genetic mutation frequency from organisms living in mesophilic environments. This fact indicates the existence of a unique and effective DNA repair system in thermophilic archaea. Intriguingly, genome sequence comparison has revealed that archaeal information processing processes (DNA replication, transcription, and translation) are far more closely related to those in eukarya than bacteria, although metabolic and cell division proteins in archaea resemble those of bacteria. Therefore, it is very helpful to investigate protein factors involving in the DNA metabolism in archaea for the research on eukarya with more complicated systems and dealing with many diseases in human.Sliding clamps, called proliferating cell nuclear antigen (PCNA) in Archaea or Eukarya andβ-clamp in bacteria, play an essential role in many DNA metabolic processes, including cell cycle control, DNA replication, and repair in all organisms. This protein may function as a moving platform on which enzymes implicated in genetic information processes are exchanged. All sliding clamps from prokaryotes and eukaryotes form similar planar ring structures with a central channel that is of sufficient width to encircle duplex DNA, which suggests all sliding clamps may share the similar molecular mechanism in function. For example, architecture and mechanism of clamps and clamp loaders (γ-complex in E. coli, RFC in archaea and eukarya) are conserved across the three domains of life. Even though all sliding clamps share several similar features, individual clamps from different domains of life exist in low level of sequence identity and different Oligomeric states. Specifically, twoβ-clamp protomers in E. coli dimerize to form a ring, and PCNA is a homotrimeric ring in Eukarya and Euryarchaeota, while there exist heterotrimeric complexes in Crenarchaeota. Given that diverse PCNAs are key factors in DNA replication and repair system, studies of PCNA could provide much information for the further exploration on the specific repair mechanism in archaea. Here, in the main part of this study, we described the biochemical properties of the three PCNAs from Sulfolobus tokodaii strain 7, a hyperthermophilic archaeon belonging to Crearchaeota. We cloned genes encoding PCNA1, PCNA2, PCNA3, Hjm, Hjc and Ligase from S. tokodaii, overexpressed the recombinant proteins in E. coli, and purified these proteins uisng Ni-NTA or HiTrap Q column in vitro.About the relationship among three subunits of PCNAs, some interesting results were obtained by methords of gel filtration and yeast two-hybrid. Firstly, it was found that none of the PCNAs homo-multimerize and PCNA1 and PCNA3 can interact with each other, but PCNA1 and PCNA2 can not. Secondly, we identified for the first time a novel trimeric PCNA complex (PCNA323) composed of one PCNA2 and two PCNA3 probably similar to the ring complex (PCNA123) formed by PCNA1, PCNA2 and PCNA3.In order to compare difference in function of the PCNA323 and PCNA123 complex, we purified them by combination of His-pulldown with gel filtration methods. Then, we added the two complexes into the reaction systems of different enzymes including StoHjm, StoLigase and StoHjc respectively. The results indicated that both complexes inhabited the unwinding activity of Hjm and Ligase in vitro. What is more, PCNA323 stimulated the cleavage activity of Hjc more strongly than PCNA123. Further experiment is needed to determine the detailed difference or similarity between the two complexes.Additionally, it has been reported that PCNA can stimulate the cleavage activity of Hjc (Holliday junction cleavage) involving in recombination repair pathway in S. solfataricus, and PCNA can interact physically with Hjm in Pyrococcus. furiosus. Furthermore, our initial result on StoHjm showed that Hjm also physically interacts with StoHjc in S. tokodaii. Based on these results, we predicted that PCNA, StoHjm and StoHjc may form a heterotrimeric complex implicating in the same repair pathway. In the second part of this thesis, results on the interaction between StoHjm and StoHjc were presented. We described that StoHjm physically interacts with StoHjc in S. tokodaii by using His-pull down, gel filtration and yeast two-hybrid, and StoHjc inhibits unwinding activity of StoHjm. Although the interaction between PCNA and StoHjc or StoHjm could not been detected, interaction may be ditected by optimizing experiment protocol or analyzing the three proteins all together. Additionally, to clarify the molecular mechanism about StoHjm interacting with StoHjc, we plan to construct a series of deletion mutants of StoHjm and test the relationship of these mutants and StoHjc.In conclusion, we described the biochemical properties and physical interactions of several proteins related to DNA metabolism in S. tokodaii, including PCNA, Hjm, and Hjc. Our results may facilitate further understanding of the specific DNA repair system in archaea.更多还原