Exogenous AdoMet and its analogue sinefungin differentially influence DNA cleavage by R.EcoP15I--usefulness in SAGE.
While it has been demonstrated that AdoMet is required for DNA cleavage by Type III restriction enzymes, here we show that in the presence of exogenous AdoMet, the head-to-head oriented recognition sites are cleaved only on a supercoiled DNA. On a linear DNA, exogenous AdoMet strongly drives methylation while inhibiting cleavage reaction. Strikingly, AdoMet analogue sinefungin results in cleavage at all recognition sites irrespective of the topology of DNA. The cleavage reaction in the presence of sinefungin is ATP dependent. The site of cleavage is comparable with that in the presence of AdoMet. The use of EcoP15I restriction in presence of sinefungin as an improved tool for serial analysis of gene expression is discussed.
Raghavendra
Nidhanapati K
NK
Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India.
Rao
Desirazu N
DN
D000241:Adenosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D004247:DNA / Q000378:metabolism*; D004270:DNA, Circular / Q000378:metabolism; D004278:DNA, Superhelical / Q000378:metabolism*; D015263:Deoxyribonucleases, Type III Site-Specific / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020869:Gene Expression Profiling / Q000379:methods; D012097:Repressor Proteins / Q000494:pharmacology; D012436:S-Adenosylmethionine / Q000494:pharmacology*; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000037:antagonists & inhibitors / Q000378:metabolism*
D004270:DNA, Circular; D004278:DNA, Superhelical; D012097:Repressor Proteins; D012436:S-Adenosylmethionine; D004247:DNA; C089712:DNA modification methylase EcoP15I; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); C023451:endodeoxyribonuclease EcoP15I; D015263:Deoxyribonucleases, Type III Site-Specific; D000241:Adenosine; C006235:sinefungin