Two intertwined methylation activities of the MmeI restriction-modification class-IIS system from Methylophilus methylotrophus.
The class-IIS restriction endonuclease, R.MmeI, was isolated from Methylophilus methylotrophus. It was originally described as a monomeric enzyme, with the native Mr 105000+/-7000, which did not cleave DNA efficiently [Boyd et al. (1986) Nucleic Acids Res. 14, 5255-5274; Tucholski et al. (1995) Gene 157, 87-92]. However, it was discovered that R.MmeI endonucleolytic activity is enhanced by S-adenosyl-l-methionine (AdoMet) and sinefungin, an analogue of AdoMet. Surprisingly, the purified R.MmeI endonuclease was found to have a second enzymatic activity, namely methylation of the adenine residue to N6-methyladenine in the top strand of the MmeI-recognition sequence, 5'-TCCR*AC-3' (*A=meA. The R.MmeI methylating activity requires AdoMet and is increased in the presence of several divalent cations, 20-fold by Mg2+ or Ca2+, and less by Mn2+, Zn2+ and Co2+; however, methylation is inhibited entirely by sinefungin, at concentrations above 9microM. The latter observation shows that the enhancing effect of AdoMet or sinefungin on the DNA cleavage was not related to the process of DNA methylation. Furthermore, a second component of the MmeI restriction-modification system, a M.MmeI methyltransferase, was isolated and purified. The M.MmeI protein was found to have an Mr of 48000+/-2000 (under denaturing conditions) and to methylate both adenine residues (*A) in the MmeI-recognition sequence 5'-TCCR*AC-3'/3'-*AGGYTG-5'. Methylation of the top strand does not inhibit the DNA cleavage by R.MmeI, whereas methylation of both DNA strands blocks the cleavage process.
Tucholski
J
J
Department of Biotechnology, Faculty of Biotechnology, University of Gdańsk and Medical University of Gdańsk, ul. Kladki 24, 80-822, Gdańsk, Poland.
Zmijewski
J W
JW
Podhajska
A J
AJ
D000241:Adenosine / Q000031:analogs & derivatives / Q000378:metabolism; D019175:DNA Methylation; D004269:DNA, Bacterial / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D006088:Gram-Negative Aerobic Bacteria / Q000201:enzymology* / Q000235:genetics; D008745:Methylation; D008780:Methyltransferases / Q000302:isolation & purification / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000031:analogs & derivatives / Q000378:metabolism
D004269:DNA, Bacterial; D012436:S-Adenosylmethionine; D008780:Methyltransferases; C049760:endodeoxyribonuclease MmeI; D015252:Deoxyribonucleases, Type II Site-Specific; D000241:Adenosine; C006235:sinefungin