Nucleic Acids Research, Vol 26, Issue 19 4409-4412, Copyright © 1998 by Oxford University Press
M Nishioka, S Fujiwara, M Takagi and T Imanaka
Two intein endonucleases, denoted PI- Pko I and PI- Pko II, in the DNA
polymerase gene of the hyperthermophilic archaeon Pyrococcus kodakaraensis
KOD1 were expressed in Escherichia coli and the recombinant endonucleases
were characterized. Both endonucleases were thermostable and cleaved their
intein-less DNA sequences leaving four base 3'-hydroxyl overhangs. PI-Pko I
exhibited 22 times higher specific activity than PI-Pko II and the activity
of PI-Pko II was enhanced at higher potassium ion concentrations (1 M).
Recognition sequences were also determined using synthetic oligonucleotides
inserted into plasmid pUC19. It was shown that DNA sequences of 19 and 16
bp are needed for cleavage by PI-Pko I and PI-Pko II, respectively. PI-Pko
II could cleave the downstream junction region between intein-encoding and
mature DNA polymerase regions and cleavage by PI-Pko II could be detected
even when chromosomal DNA of P.kodakaraensis KOD1 was used as substrate.
Therefore, it is suggested that these endonucleases are switching
endonucleases whose function lies in the rearrangement of chromosomal DNA.
ARTICLES
Characterization of two intein homing endonucleases encoded in the DNA polymerase gene of Pyrococcus kodakaraensis strain KOD1
Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
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