Effects of methylation on the activities of restriction enzymes

Many DNA molecules contain methylated bases. When restriction enzyme recognition sites are methylated, DNA cleavage may be blocked depending on the restriction enzyme and the type of methylation. Most strains of E. coli have two site-specific DNA methylases (methyltransferases), Dam methylase (G6mATC) and Dcm methylase (C5mCA/TGG). As a result, DNA molecules prepared from Dam⁺ Dcm⁺ E. coli strains are methylated at GATC and CCAGG/CCTGG sequences. Additionally, some mammalian DNA molecules are methylated by CG methylase (5mCG).

The table below lists restriction enzymes whose activities are affected by Dam methylase, Dcm methylase or CG methylase-mediated methylation. Sequences recognized by each methylase are also provided. Please refer to the footnotes below the table for additional details.

A–C, D–K, M–Z

Restriction enzyme	Recognition sequence¹	Methylase influencing restriction cleavage efficiency and its recognition sequence	Cleavage sites blocked by methylation²	Sequences not affected by methylation (From reported literature, etc.)	Sequences affected by methylation (From reported literature, etc.)
AatII	GACGTC	CG methylase (^5mCG)	GA^5mCGTC		GA^5mCGTC GACGT^5mC
AccI	GTMKAC	CG methylase (^5mCG)	GTMKA^5mCG GT^5mCGAC		GTMK^6mAC GTMKA^5mC³ GT^5mCGAC⁴
AccII	CGCG	CG methylase (^5mCG)	^5mCG^5mCG		^5mCGCG
AccIII	TCCGGA	dam methylase (G^6mATC)	TCCGG^6mATC	T^5mCCGGA TC^5mCGGA³	TCCGG^6mA
AfaI	GTAC	CG methylase (^5mCG)	GTA^5mCG		GTA^5mC^3,4
AflII	CTTAAG				^5mCTTAAG CTTA^6mAG
AluI	AGCT				^6mAGCT AG^4mCT AG^5mCT AG^5hmCT
Aor13HI	TCCGGA	CG methylase (^5mCG)	TC^5mCGGA		TC^5mCGGA
Aor51HI	AGCGCT	CG methylase (^5mCG)	AG^5mCGCT		AG^5mCGCT⁴
ApaI	GGGCCC	dcm methylase (C^5mCWGG) CG methylase (^5mCG)	GGGCC^5mCWGG GGGCC^5mCG		GGG^5mCCC GGGCC^5mC
ApaLI	GTGCAC	CG methylase (^5mCG)	GTGCA^5mCG	GTG^5mCAC GTGC^6mAC	GTGCA^5mC
AvaI	CYCGRG	CG methylase (^5mCG)	CY^5mCGRG	C^5mCCGGG	^5mCYCGRG CY^5mCGRG CTCG^6mAG³
AvaII	GGWCC	dcm methylase (C^5mCWGG) CG methylase (^5mCG)	GGWC^5mCWGG GGWC^5mCG	GGWC^4mC*3	GGW^5mCC GGWC^5mC GGW^5hmC5hmC
BalI	TGGCCA	dcm methylase (C^5mCWGG)	TGGC^5mCAGG		TGG^5mCCA TGGC^5mCA
BamHI	GGATCC			GGATC^4mC GGATC^5mC GG^6mATCC GG6^6mATC^5mC	GGAT^4mCC GGAT^5mCC GGAT^5hmC^5hmC
BanII	GRGCYC			GRGCY^5mC	GRG^5mCYC
BcnI	CCSGG			^5mCCSGG C^5mCSGG	C^4mCSGG
BglI	GCCN5GGC	CG methylase (^5mCG)	GCCN5GG^5mCG	GC^5mCN5GGC	G^5mCCN5GGC GCCN5GG^5mC GC^4mCN5GGC
BglII	AGATCT			AG^6mATCT	AGAT^5mCT AGAT^5hmCT
BlnI	CCTAGG
BmeT110I	CYCGRG			CC^5mCGRG CTCG^6mAG AG^5mCTCGGG ^5mCCCGGG
BmgT120I	GGNCC	dcm methylase (C^5mCWGG) CG methylase (^5mCG)	GGNC^5mCWGG GGNC^5mCG		GG^5mCCC GGK^5mCC
Bpu1102I	GCTNAGC
BspT104I	TTCGAA	CG methylase (^5mCG)	TT^5mCGAA		TT^5mCGAA
BspT107I	GGYRCC
Bsp1286I	GDGCHC			GDGCH^5mC	GDG^5mCHC
Bsp1407I	TGTACA
BssHII	GCGCGC	CG methylase (^5mCG)	G^5mCG^5mCGC		G^5mCGCGC
BstPI	GGTNACC			GGTNAC^5mC⁴
BstXI	CCAN6TGG			CCAN₄C^5mCTGG⁴	^5mCCAN₆TGG
Bst1107I	GTATAC	CG methylase (^5mCG)	GTATA^5mCG		GTATA^5mC
Cfr10I	RCCGGY	CG methylase (^5mCG)	RC^5mCGGY		R^5mCCGGY RC^5mCGGY⁴
Cfr13I	GGNCC	dcm methylase (C^5mCWGG) CG methylase (^5mCG)	GGNC^5mCWGG GGNC^5mCG		GGN^5mCC GGNC^5mC^{3, 4}
ClaI	ATCGAT	dam methylase (G^6mATC) CG methylase (^5mCG)	G^6mATCGAT ATCG^6mATC AT^5mCGAT		^6mATCGAT ATCG^6mAT AT^5mCGAT
CpoI	CGGWCCG	CG methylase (^5mCG)	^5mCGGWC^5mCG		CGGW^5mCCG⁴ CGGWC^5mCG⁴
A–C, D–K, M–Z
Restriction enzyme	Recognition sequence¹	Methylase influencing restriction cleavage efficiency and its recognition sequence	Cleavage sites blocked by methylation²	Sequences not affected by methylation (From reported literature, etc.)	Sequences affected by methylation (From reported literature, etc.)
DraI	TTTAAA			TTTA^6mAA
EaeI	YGGCCR	dcm methylase (C^5mCWGG) CG methylase (^5mCG)	YGGC^5mCAGG YGGC^5mCG		YGG^5mCCR YGGC^5mCR
EcoO65I	GGTNACC			GGTNAC^5mCAN⁴
EcoO109I	RGGNCCY	dcm methylase (C^5mCWGG)	RGGNC^5mCTGG	RGGNC^5mCY
EcoRI	GAATTC	CG methylase (^5mCG)	GAATT^5mCG	GAATT^5hmC GAA^5hmU^5hmUC	G^6mAATTC GA^6mATTC GAATT^5mC³
EcoRV	GATATC			GATAT^5mCGATAT^5hmC	G^6mATATC GAT^6mATC
EcoT14I	CCWWGG
EcoT22I	ATGCAT				ATG^5mCAT ATGC6mAT
Eco52I	CGGCCG	CG methylase (^5mCG)	^5mCGGC^5mCG		^5mCGGCCG4 CGGC^5mCG4 CGG^5mCCG*4
Eco81I	CCTNAGG
FbaI	TGATCA	dam methylase (G^6mATC)	TG^6mATCA		TG^6mATCA
FokI	GGATG (CATCC)			CAT^5mCC CATC^4mC CATC^5mC*3	C^6mATCC GG^6mATG
FseI	GGCCGGCC	CG methylase (^5mCG)	GGC^5mCGGCC		GG^5mCCGGCC GG^5mCCGG^5mCC GGC^5mCGGCC
HaeII	RGCGCY	CG methylase (^5mCG)	RG^5mCGCY		RG^5mCGCY RGCG^5mCY³ RG^5hmCG^5hmCY
HaeIII	GGCC			GGC^5mC	GG^5mCC³ GG^5hmC^5hmC
HapII	CCGG	CG methylase (^5mCG)	C^5mCGG		C^5mCGG
HhaI	GCGC	CG methylase (^5mCG)	G^5mCGC		G^5mCGC GCG^5mC G^5hmCG^5hmC
HincII	GTYRAC			GTYRA^5mC GT^5mCGAC⁴	GTYR^6mAC GTYRA^5hmC
HindIII	AAGCTT			A^6mAGCTT AAGC^5hmU^5hmU	^6mAAGCTT AAG^5mCTT AAG^5hmCTT
HinfI	GANTC			GANT^5mC³	G^6mANTC GANT^5hmC
Hin1I	GRCGYC	CG methylase (^5mCG)	GR^5mCGYC	GRCGY^5mC^3,4	GR^5mCGYC
HpaI	GTTAAC			GTTAA^5mC	GTTA^6mAC GTTAA^5hmC G^5hmU^5hmUAAC
KpnI	GGTACC			GGTA^5mCC GGTA^5mC^5mC GGTAC^5mC	GGT^6mACC GGTAC^4mC
A–C, D–K, M–Z
Restriction enzyme	Recognition sequence¹	Methylase influencing restriction cleavage efficiency and its recognition sequence	Cleavage sites blocked by methylation²	Sequences not affected by methylation (from reported literature, etc.)	Sequences affected by methylation (From reported literature, etc.)
MboI	GATC	dam methylase (G^6mATC)	G^6mATC	GAT^4mC GAT^5mC GA^5hmUC	G^6mATC GAT^5hmC
MboII	GAAGA (TCTTC)	dam methylase (G^6mATC)	GAAG^6mATC	T^5mCTT^5mC³ G^6mAAGA	GA^6mAGA GAAG^6mA
MflI	RGATCY	dam methylase (G^6mATC)	RG^6mATCY	^6mAGATCY³	RG^6mATCY RGAT^4mCY RGAT^5mCY
MluI	ACGCGT	CG methylase (^5mCG)	A^5mCG^5mCGT	^6mACGCGT	A^5mCGCGT
MspI	CCGG			^4mCCGG C^4mCGGC ^5mCGG³	^5mCCGG ^5hmC^5hmCGG
MunI	CAATTG				CA^6mATTG
MvaI	CCWGG			^5mCCWGGC^5mCWGG	^4mCCWGG ^5mC^5mCWGG C^4mCWGG CC^6mAGG
NaeI	GCCGGC	CG methylase (^5mCG)	GC^5mCGGC GCCGG^5mCG		G^5mCCGGC GC^5mCGGC GCCGG^5mC
NcoI	CCATGG			CC^6mATGG	^4mCCATGG ^5mCCATGG
NdeI	CATATG			^5mCATATG
NheI	GCTAGC	CG methylase (^5mCG)	GCTAG^5mCG		GCTAG^5mC
NotI	GCGGCCGC	CG methylase (^5mCG)	G^5mCGGC^5mCGC	GCGGCCG^5mC	GCGG^5mCCGC GCGGC^5mCGC
NruI	TCGCGA	dam methylase (G^6mATC) CG methylase (^5mCG)	TCGCG^6mATC T^5mCG^5mCGA	TCG^5mCGA	GCGG^5mCCGC GCGGC^5mCGC
NsbI	TGCGCA	CG methylase (^5mCG)	TG^5mCGCA	TGCGC^6mA	TG^5mCGCA
PmaCI	CACGTG	CG methylase (^5mCG)	CA^5mCGTG		CA^5mCGTG⁴
PshAI	GACN₄GTC	CG methylase (^5mCG)	GA^5mCGN₃GTC	GACN₄GT^5mC⁴	GA^5mCN₄GTC⁴
PshBI	ATTAAT
Psp1406I	AACGTT	CG methylase (^5mCG)	AA^5mCGTT		AA^5mCGTT
PstI	CTGCAG				^5mCTGCAG CTGC^6mAG C^5hmUGCAG
PvuI	CGATCG	CG methylase (^5mCG)	^5mCGAT^5mCG	CG^6mATCG	CGAT^4mCG CGAT^5mCG
PvuII	CAGCTG				CAG^4mCTG CAG^5mCTG
SacI	GAGCTC			G^6mAGCTC GAGCT^5mC	GAG^5mCTC
SacII	CCGCGG	CG methylase (^5mCG)	C^5mCG^5mCGG		^5mCCGCGG C^5mCGCGG
SalI	GTCGAC	CG methylase (^5mCG)	GT^5mCGAC	GTCGA^5mC	GT^5mCGAC GTCG^6mAC
Sau3AI	GATC	CG methylase (^5mCG)	GAT^5mCG	G^6mATC GA^5hmUC	GAT^4mC GAT^5mC³ GAT^5hmC
ScaI	AGTACT			AGTA^5mCT
SfiI	GGCCN₅GGCC	dcm methylase (C^5mCWGG) CG methylase (^5mCG)	GGC^5mCWGGN₂GGCC GGCCN₅GGC^5mCWGG GGCCN₅GGC^5mCG	GG^5mCCN₅GG^5mCC	GG^4mCCN₅GGCC GGC^5mCN₅GGCC GGCCN₅GGC^5mC
SmaI	CCCGGG	CG methylase (^5mCG)	CC^5mCGGG	C^5mCCGGG	^4mCCCGGG ^5mCCCGGG C^4mCCGGG CC^4mCGGG CC^5mCGGG³
SmiI	ATTTAAAT
SnaBI	TACGTA	CG methylase (^5mCG)	TA^5mCGTA		T^6mACGT^6mA TA^5mCGTA
SpeI	ACTAGT				^6mACTAGT A^5mCTAGT
SphI	GCATGC			GCATG^5mC G^5hmCATG^5hmC	GC^6mATGC
Sse8387I	CCTGCAGG			^5mCGCCTGCAGG^5mCG⁴	^5mCCTGCAGG⁴ C^5mCTGCAGG⁴ CCTG^5mCAGG⁴ CCTGC^6mAGG⁴
SspI	AATATT			^6mAATATT
StuI	AGGCCT	dcm methylase (C^5mCWGG)	AGGC^5mCTGG		AGG^5mCCT AGGC^4mCT AGGC^5mCT
TaqI	TCGA	dam methylase (G^6mATC)	TCG^6mATC	T^5mCGA	TCG^6mA T^5hmCGA
Tth111I	GACN₃GTC			GA^5mCN₃GT^5mC GACN₃GT^5mC⁴
Van91I	CCAN₅TGG	dcm methylase (C^5mCWGG)	C^5mCAGGN₃TGG		C^5mCAN₅TGG
VpaK11BI	GGWCC	dcm methylase (C^5mCWGG) CG methylase (^5mCG)	GGWC^5mCWGG GGWC^5mCG		GGWC^5mC
XbaI	TCTAGA	dam methylase (G^6mATC)	TCTAG^6mATC		T^5mCTAGA³ TCTAG^6mA T^5hmCTAGA
XhoI	CTCGAG			CT^5mCGAG^{3, 4}	^5mCTCGAG CTCG^6mAG
XspI	CTAG				^5mCTAG

1.	M: A or C
	K: G or T
	N: A or C or G or T
	R: A or G
	Y: C or T
	W: A or T
	S: C or G H: A or C or T
	H: A or C or T
	D: A or G or T
	^4mC: N4-methyl cytosine
	^5mC: C5-methyl cytosine
	^5hmC: C5-hydroxymethyl cytosine
	^6mA: N6-methyl adenine

2.	In case of CG methyalse, the partial digestion may be obeserved.

3.	Acc I: Nicks slowly the unmethylated strand of hemi-methylated GTMKA^5mC.
	Acc III: cleaves TC^5mCGGA slowly (1/75).
	Afa I: cleaves hemi-methylated GTA^5mC, but cannot cut bi-methylated GTA^5mC.
	Ava I: Nicks the unmethylated strand of hemi-methylated CTCG^5mAG slowly.
	Ava II: cleaves GGWC^4mC slowly.
	Bal I: In case of overlapping with dcm site (TGGC^5mCAGG), the digestion speed becomes very slow (less than 1/50).
	Bgl I: While cleaves hemi-methylated GC^5mCN5GGC, GC^4mCN5GGC and GCCN5GG^5mC slowly, can scarcely cleave bi-methylated M. Hae III-Bgl I site.
	Cfr13 I: cannot cleave hemi-methylated GGNC^5mC.
	Cla I: cleaves hemi-methylated AT^5mCGAT slowly.
	EcoR I: While cleaves hemi-methylated GAATT^5mC, cannot cleave bi-methylated GAATT^5mC.
	Fok I: cleaves CATC^5mC slowly (1/2 to 1/4).
	Hae II: cleaves RGCG^5mCY very slowly.
	Hae III: Nicks the unmethylated strand of hemi-methylated GG^5mCC.
	Hin1 I: cleaves GRCGY^5mC very slowly.
	Hind III: cleaves hemi-methylated AAG^5mCTT slowly.
	Hinf I: cleaves GANT^5mC slowly (1/10).
	Kpn I: influenced by hemi-methylated GGTA^5mCC and GGTAC^5mC, and digests hemi-methylated GGTA^5mC^5mC slowly.
	Mbo II: It has been reported that this cannot digest hemi-methylated T^5mCTT^5mC.
	Mfl I: cleaves ^6mAGATCY slowly.
	Msp I: cleaves GGC^5mCGG very slowly.
	Mva I: Nicks hemi-methylated CC^6mAGG and the unmethylated strand of hemi-methylated ^4mCCWGG.
	Pst I: cleaves hemi-methylated ^5mCTG^5mCAG slowly.
	Pvu II: cleaves hemi-methylated ^5mCAG^5mCTG slowly.
	Sal I: cleaves hemi-methylated GT^5mCGAC slowly.
	Sau3A I: Nicks the unmethylated strand of hemi-methylated GAT^5mC. It has been reported that the digestion speed of^5mAGATC becomes slow.
	Sma I: Nicks the unmethylated strand of hemi-methylated CC^5mCGGG.
	Spe I: cleaves hemi-methylated A^5mCTAGT slowly.
	Xba I: cleaves hemi-methylated T^5mCTAGA, but its digestion speed becomes very slow (less than 1/100).
	Xho I: cleaves CT^5mCGAG very slowly.
	Hemi-methylated DNA denotes DNA of which one strand out of two is methylated, and ( ) shows the relative speed.
4.	Takara Bio, unpublished observations.

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