Gene Structure and Properties of DNA Polymerase Isolated from Thermococcus fumicolans
Joel QUERELLOU*a and Marie-Anne CAMBONb
a IFREMER, Centre de Brest, Laboratoire de Biotechnologie des Microorganismes Hydrothermaux, BP 70, 29280 Plouzane, France
b IFREMER, Centre de Brest, Laboratoire de Caracterisation des Microorganismes Marins, BP 70, 29280 Plouzane, France
The DNA polymerase genes of eight new strains of Thermococcales isolated from various deep-sea vent environments were either cloned or amplified by PCR in order to compare their gene structure and the frequency of intervening sequences (IVS) coding for inteins. Among these new strains, Thermococcus fumicolans displays an original structure with two IVS inserted in motifs A and C of the DNA polymerase gene. The entire gene including IVS was subcloned into expression vectors and appropriate E. coli strains. Co-expression of DNA polymerase and inteins was obtained. The recombinant DNA polymerase of T. fumicolans (Tfu) was purified to homogeneity and its properties for PCR use were investigated. Thermostability was studied for both polymerase and proofreading activities. Polymerase activity is characterized by a half life of 2 hours at 100℃ but the proofreading activity is less thermostable with 2 hours at 95℃. Moreover, the Tfu proofreading activity displays a lower sensitivity to dNTP concentration than Tli polymerase (from Thermococcus litoralis). The strong proofreading activity can be modulated by use of various buffers, but makes necessary to protect oligonucleotides in 3', againt hydrolysis in PCR.
