| Accurate duplication of parental DNA is a fundamental biological process,conserved in function across all life forms.The eukaryote GINS,Cdc45,and MCM proteins form an essential complex that moves with the DNA replication fork.The GINS protein complex has also been reported to associate with DNA polymerase.In Archaea,the third domain of life,DNA polymerase D(PoID)is essential for DNA replication,and the genes encoding PolDs exist only in the genomes of Archaea.The archaeal GAN(GINS-associated nuclease)is believed to be a homologue of the eukaryotic Cdc45.In this study,we found that the Thermococcus sp.4557 DP1(small subunit of PolD)interacted with GINS 15 in vitro,and the 3'-5'exonuclease activity of DP1 was inhibited by GINS15.We also demonstrated that the GAN,GINS15,and DP1 proteins interact to form a complex adapting a GAN-GINS15-DP1 order.The results of this study imply that the complex constitutes a core of the DNA replisome in archaea.In addition to basic theoretical research,we also made a technical attempt to develop DNA replicase resources.DNA polymerases also play central roles in modern molecular biology and biotechnology.It is significant to research and develop DNA polymerase products for all DNA polymerase application fields.We researched and developed two new products from hyperthermophilic microorganisms,Palaeococcus pacificus DNA polymerase(D09)and Thermococcus sp.4557 DNA polymerase(D18).Taking Thermus aquaticus DNA polymerase(D17)as a research and development case,we tried to achieve a high yield of purified D17 from 1 L to 100 L industrial-scale cell culture,finally formulating a standard procedure on large-scale protein expression and purification.As a result,we obtained four DNA polymerase products.In terms of yield and purity,we reached the industrialization standard.Among them,D13 DNA polymerase was from D17 mutation by deleting 5'-3'nucleic acid exonuclease domain of D17,suitable for fluorescence quantitative PCR.At the same time,the activity of purified DNA polymerase products was tested,including a large number of gene amplification from different sources,and different research institutes participating in products testing.PCR tests result show that the four DNA polymerases can compete with foreign brands in terms of elongation efficiency,fidelity and activity persistence.The best known DNA polymerase-based biotechnology application is PCR.However,this technique is often plagued with its low specificity and sensitivity.Great efforts have been made to improve PCR efficiency and specificity.In this research,we introduced a new approach of Hot Start PCR utilizing a modified Escherichia coli Exonuclease ?(EcoExo?M),by substituting residues in the DNA binding pocket and the activity center.Results showed that specificity and amplification of tested PCR reactions were significantly promoted by the addition of EcoExo?M.To explain EcoExo?M how to improve PCR at room temperature,we proposed the mechanism.In this paper,DNA polymerase and other proteins,involved in the process of DNA replication and repair,were studied experimentally with the combination of theory and application,and significant data were obtained.On the one hand,the mechanism of DNA replication in archaea was further understood.On the other hand,DNA polymerase from superthermophilic archaea from deep-sea hydrothermal vents was applied and developed,which promote the competitiveness of relevant enterprises in China. |
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