Mechanisms And Application Of DNA Polymerases From Thermophilic Archaea Thermococcus Sp.4557

DNA replication is a key process of transferring genetic information between generations of all kind of life. Archaea, the third domain of life possess the DNA replication enzymes similar but simpler than that of Eukayote. This feature made archaea an ideal organism to study complex DNA replication mechanisms.More than 20 DNA replication proteins from archaea were previously studied, including DNA polymerases as a key factor that directly synthesis nascent strand. There are two families of DNA polymerase in the Euriarchaea. The B family (PolB) is a single subunit possessing 5'-3'synthesis activity and 3'-5'exo-nuclease activity serving as proof reading to guarantee the fidelity. The widely known Pfu and KOD from the market belongs to PolB family. PolD the other DNA polymerase was uniquely identified from the Euriarchaea. Most PolD is consisted of a large subunit containing polymerase activity and a small subunit containing 3'-5'exo-nuclease. It was reported that PolD but not PolB is essential in Thermococcus kodakarensis, which indicated the possibility that PolD is the DNA replicative polymerase.In this thesis, we performed researches on the PolB and PolD DNA polymerase using 4557. The application of 4557-PolB in PCR amplification of ALV gene fragment was invested in the study of ALV. The complex containing PolD, GINS, and GAN was firstly studied in detail. Several technique including gel-filtration and pull-down was used to reveal the regulation mechanism of PolD complex.