New high fidelity polymerases from Thermococcus species.
Two DNA polymerase genes have been isolated from Thermococcus strains, Thermococcus zilligii from New Zealand, and the other, Thermococcus 'GT', a fast-growing strain isolated from the Galapagos trench. Both genes were isolated by genomic walking PCR, a technique that does not require expression of the gene product. Phylogenetic analysis of SSU rDNA showed that the two strains were not closely related, as confirmed by an examination of the DNA polymerase sequences. Inteinless versions of each gene were generated by overlap-extension PCR and transferred into plasmid expression vectors. The proteins were produced in an Escherichia coli strain with additional copies of tRNAs corresponding to rarely used codons and purified by standard chromatographic procedures. Both enzymes were able to support PCR, but the Thermococcus 'GT' polymerase required higher concentrations of template than the enzyme from T. zilligii. Both enzymes showed 3' to 5' exonuclease activity, which was abolished in the case of T. zilligii by mutating the aspartic acid at position 141 and the glutamic acid at position 143 to alanine. Both enzymes showed a significant increase in fidelity of replication compared to the family A Thermus aquaticus DNA polymerase, in agreement with other results reported for family B polymerases with proof-reading ability.
Griffiths
Kate
K
Department of Biological Sciences, Macquarie University, North Ryde, NSW 2109, Australia.
Nayak
Sunil
S
Park
Kyusung
K
Mandelman
David
D
Modrell
Brett
B
Lee
Jun
J
Ng
Bernie
B
Gibbs
Moreland D
MD
Bergquist
Peter L
PL
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D017931:DNA Primers; D019641:DNA, Archaeal / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000302:isolation & purification / Q000378:metabolism*; D005091:Exons; D010802:Phylogeny; D015183:Restriction Mapping; D019712:Thermococcus / Q000145:classification / Q000201:enzymology* / Q000235:genetics
D019843:Archaeal Proteins; D017931:DNA Primers; D019641:DNA, Archaeal; D004275:DNA, Ribosomal; D004259:DNA-Directed DNA Polymerase