Restriction endonuclease cleavage of 5-methyl-deoxycytosine hemimethylated DNA at high enzyme-to-substrate ratios.
We have investigated the ability of a large number of restriction enzymes to digest non-canonically hemimethylated DNA at high enzyme-to-substrate ratios. A single-stranded unmethylated phagemid was used as a template to complete synthesis of the second strand using 5-methyl-dCTP to substitute for all the deoxycytosine residues. A fragment of this double-stranded hemimethylated DNA which contains the multiple cloning site region was used as a substrate. For all the enzymes tested, at least some degree of protection from digestion is observed. Sites completely protected from digestion by their cognate enzymes are SalI, BstXI, SacI, SacII, SmaI, SstI, XhoI, PstI, HinfI, BamHI and AccI. Sites partially protected from digestion by their cognate enzymes are XbaI, HindIII, KpnI, SpeI, ClaI, EcoRI and PvuII. Knowledge of the sensitivity of commonly used restriction enzymes to hemimethylated substrates is useful for several applications, which will be discussed.
Nelson
P S
PS
Laboratory of Molecular Oncology, National Cancer Institute, Frederick, MD 21702-1201.
Papas
T S
TS
Schweinfest
C W
CW
D044503:5-Methylcytosine / Q000031:analogs & derivatives; D001483:Base Sequence; D003596:Cytosine / Q000031:analogs & derivatives* / Q000378:metabolism; D004247:DNA / Q000138:chemical synthesis / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D008745:Methylation; D008969:Molecular Sequence Data; D013379:Substrate Specificity
C065701:5-methyldeoxycytosine; D044503:5-Methylcytosine; D003596:Cytosine; D004247:DNA; D015252:Deoxyribonucleases, Type II Site-Specific