| 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.
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