Cloning, purification, and characterization of a new DNA polymerase from a hyperthermophilic archaeon, Thermococcus sp. NA1.
Genomic analysis of Thermococcus sp. NA revealed the presence of a 3,927-base-pair (bp) family B-type DNA polymerase gene, TNA1_pol. TNA1_pol, without its intein, was overexpressed in Escherichia coli, purified using metal affinity chromatography, and characterized. TNA1_pol activity was optimal at pH 7.5 and 75 degrees C. TNA1_pol was highly thermostable, with a half-life of 3.5 h at 100 degrees C and 12.5 h at 95 degrees C. Polymerase chain reaction parameters of TNA1_pol such as error-rate, processivity, and extension rate were measured in comparison with rTaq, Pfu, and KOD DNA polymerases. TNA1_pol averaged one incorrect bp every 4.45 kilobases (kb), and had a processivity of 150 nucleotides (nt) and an extension rate of 60 bases/s. Thus, TNA1_pol has a much faster elongation rate than Pfu DNA polymerase with 7-fold higher fidelity than that of rTaq.
Kim
Yun Jae
YJ
Korea Ocean Research and Development Institute, Ansan RPO. Box 29, Seoul 425-600, Korea.
Lee
Hyun Sook
HS
Bae
Seung Seob
SS
Jeon
Jeong Ho
JH
Lim
Jae Kyu
JK
Cho
Yona
Y
Nam
Ki Hoon
KH
Kang
Sung Gyun
SG
Kim
Sang-Jin
SJ
Kwon
Suk-Tae
ST
Lee
Jung-Hyun
JH
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001105:Archaea / Q000201:enzymology*; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004259:DNA-Directed DNA Polymerase / Q000032:analysis / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D006207:Half-Life; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013696:Temperature; D019712:Thermococcus / Q000201:enzymology* / Q000235:genetics
D019843:Archaeal Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D004259:DNA-Directed DNA Polymerase