Characterization of intein homing endonuclease encoded in the DNA polymerase gene of Thermococcus marinus.
The DNA polymerase gene of Thermococcus marinus (Tma) contains an intein inserted at the pol-b site that possesses a 1611-bp ORF encoding a 537-amino acid residue. The LAGLIDADG motif, often found in site-specific DNA endonucleases, was detected within the amino acid sequence of the intein. The intein endonuclease, denoted as PI-Tma, was purified as a naturally spliced product from the expression of the complete DNA polymerase gene in Escherichia coli. PI-Tma cleaved intein-less DNA sequences, leaving four-base-long, 3'-hydroxyl overhangs with 5'-phosphate. Nonpalindromic recognition sequences 19 bp long were also identified using partially complementary oligonucleotide pair sequences inserted into the plasmid pET-22b(+). Cleavage by PI-Tma was optimal when present in 50mM glycine-NaOH (pH 10.5), 150mM KCl and 12mM MgCl(2) at 70 degrees C.
Bae
Heejin
H
Department of Genetic Engineering, Sungkyunkwan University, Suwon, Korea.
Kim
Kee Pum
KP
Song
Jung Min
JM
Kim
Jun-Hwan
JH
Yang
Joo-Sung
JS
Kwon
Suk-Tae
ST
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004720:Endonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D047668:Inteins; D008969:Molecular Sequence Data; D010802:Phylogeny; D019154:Protein Splicing; D019712:Thermococcus / Q000737:chemistry / Q000145:classification / Q000201:enzymology* / Q000235:genetics
D019843:Archaeal Proteins; D004259:DNA-Directed DNA Polymerase; D004720:Endonucleases