Characterization of two intein homing endonucleases encoded in the DNA polymerase gene of Pyrococcus kodakaraensis strain KOD1

Abstract

Two intein endonucleases, denoted PI-PkoI and PI-PkoII 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 inteinless DNA sequences leaving four base 3'-hydroxyl overhangs. PI-PkoI exhibited 22 times higher specific activity than PI-PkoII and the activity of PI-PkoII 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-PkoI and PI-PkoII, respectively. PI-PkoII could cleave the downstream junction region between intein-encoding and mature DNA polymerase regions and cleavage by PI-PkoII 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.