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Support >  Applications >  Restriction_Enzymes >  Methylation_Effects

Star Activity of Restriction Enzymes

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 (also known as 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). Information about restriction enzymes whose activities are affected by Dam methylase, Dcm methylase or CG methylase-mediated methylation are listed below. Sequences recognized by each methylase are also provided. Please refer to the footnotes below the table for additional details.

Restriction EnzymeRecognition
Sequence1
Methylase influencing restriction cleavage efficiency and its recognition sequence Cleavage sites blocked by methylation2 Reports confirmed in scientific literature
Sequences not blocked
by methylation
Sequences blocked
by methylation
Aat II GACGTC CG methylase (5mCG) GA5mCGTC   GA5mCGTC
GACGT5mC
Acc I GTMKAC CG methylase (5mCG)

GTMKA5mCG
GT5mCGAC

  GTMK6mAC
GTMKA5mC3
GT5mCGAC4
Acc II CGCG CG methylase (5mCG) 5mCG5mCG   5mCGCG
Acc III TCCGGA dam methylase (G6mATC) TCCGG6mATC T5mCCGGA
TC5mCGGA3
TCCGG6mA
Afa I GTAC CG methylase (5mCG) GTA5mCG   GTA5mC3,4
Afl II CTTAAG       5mCTTAAG
CTTA6mAG
Alu I AGCT      

6mAGCT
AG4mCT
AG5mCT
AG5hmCT

Aor 13H I TCCGGA CG methylase (5mCG) TC5mCGGA   TC5mCGGA
Aor 51H I AGCGCT CG methylase (5mCG) AG 5mCGCT   AG5mCGCT4
Apa I GGGCCC dcm methylase (C5mCWGG)
CG methylase (5mCG)
GGGCC5mCWGG
GGGCC5mCG
 

GGG5mCCC
GGGCC5mC

ApaL I GTGCAC CG methylase (5mCG) GTGCA5mCG GTG5mCAC
GTGC6mAC
GTGCA5mC
Ava I CYCGRG CG methylase (5mCG) CY5mCGRG C5mCCGGG 5mCYCGRG
CY5mCGRG
CTCG6mAG3

Ava II GGWCC dcm methylase (C5mCWGG)


CG methylase (5mCG)
GGWC5mCWGG
GGWC5mCG
GGWC4mC*3

GGW5mCC
GGWC5mC
GGW5hmC5hmC

Bal I TGGCCA dcm methylase (C5mCWGG) TGGC5mCAGG  

TGG5mCCA
TGGC5mCA

BamH I GGATCC    

GGATC4mC
GGATC5mC
GG6mATCC
GG66mATC5mC

GGAT4mCC
GGAT5mCC
GGAT5hmC5hmC

Ban II GRGCYC     GRGCY5mC

GRG5mCYC

Bcn I CCSGG    

5mCCSGG
C5mCSGG

C4mCSGG
Bgl I GCCN5GGC CG methylase (5mCG) GCCN5GG5mCG GC5mCN5GGC

G5mCCN5GGC
GCCN5GG5mC
GC4mCN5GGC

Bgl II AGATCT     AG6mATCT

AGAT5mCT
AGAT5hmCT

Bln I CCTAGG        
BmeT110 I CYCGRG     CC5mCGRG
CTCG6mAG
AG5mCTCGGG
5mCCCGGG
 
BmgT120 I GGNCC dcm methylase
(C5mCWGG)
CG methylase (5mCG)
GGNC5mCWGG
GGNC5mCG

GG5mCCC
GGK5mCC
Bpu1102 I GCTNAGC



BspT104 I TTCGAA CG methylase (5mCG) TT 5mCGAA
TT 5mCGAA
BspT107 I GGYRCC



Bsp1286 I GDGCHC

GDGCH 5mC GDG 5mCHC
Bsp1407 I TGTACA



BssH II GCGCGC CG methylase (5mCG) G 5mCG 5mCGC
G 5mCGCGC
BstP I GGTNACC

GGTNAC 5mC4
BstX I CCAN6TGG

CCAN4C5mCTGG4 5mCCAN6TGG
Bst1107 I GTATAC CG methylase (5mCG) GTATA5mCG
GTATA5mC
Cfr10 I RCCGGY CG methylase (5mCG) RC5mCGGY
R5mCCGGY
RC5mCGGY4
Cfr13 I GGNCC dcm methylase
(C5mCWGG)
CG methylase (5mCG)
GGNC5mCWGG
GGNC5mCG

GGN5mCC
GGNC5mC3, 4
Cla I ATCGAT dam methylase (G6mATC)
CG methylase (5mCG)
G6mATCGAT
ATCG6mATC
AT5mCGAT

6mATCGAT
ATCG6mAT
AT5mCGAT
Cpo I CGGWCCG CG methylase (5mCG) 5mCGGWC5mCG
CGGW5mCCG4
CGGWC5mCG4
Dra I TTTAAA

TTTA6mAA
Eae I YGGCCR dcm methylase
(C5mCWGG)
CG methylase (5mCG)
YGGC5mCAGG
YGGC5mCG

YGG5mCCR
YGGC5mCR
Eam1105 I GACN5GTC

GA5mCN5GTC4
GACN5GT5mC4

EcoO65 I GGTNACC

GGTNAC5mCAN4
EcoO109 I RGGNCCY dcm methylase
(C5mCWGG)
RGGNC5mCTGG RGGNC5mCY
EcoR I GAATTC CG methylase (5mCG) GAATT5mCG GAATT5hmC
GAA5hmU5hmUC
G6mAATTC
GA6mATTC
GAATT5mC3
EcoR V GATATC

GATAT5mCGATAT5hmC G6mATATC
GAT6mATC
EcoT14 I CCWWGG



EcoT22 I ATGCAT


ATG5mCAT
ATGC6mAT
Eco52 I CGGCCG CG methylase (5mCG) 5mCGGC5mCG
5mCGGCCG*4
CGGC5mCG*4
CGG5mCCG*4
Eco81 I CCTNAGG



Fba I TGATCA dam methylase (G6mATC) TG6mATCA
TG6mATCA
Fok I GGATG (CATCC)

CAT5mCC
CATC4mC
CATC5mC*3
C6mATCC
GG6mATG
Fse I GGCCGGCC CG methylase (5mCG) GGC5mCGGCC
GG5mCCGGCC
GG5mCCGG5mCC
GGC5mCGGCC
Hae II RGCGCY CG methylase (5mCG) RG5mCGCY
RG5mCGCY
RGCG5mCY3
RG5hmCG5hmCY
Hae III GGCC

GGC5mC GG5mCC3
GG5hmC5hmC
Hap II CCGG CG methylase (5mCG) C5mCGG
C5mCGG
Hha I GCGC CG methylase (5mCG) G5mCGC
G5mCGC
GCG5mC
G5hmCG5hmC
Hinc II GTYRAC

GTYRA5mC
GT5mCGAC4
GTYR6mAC
GTYRA5hmC
Hind III AAGCTT

A6mAGCTT
AAGC5hmU5hmU
6mAAGCTT AAG5mCTT
AAG5hmCTT
Hinf I GANTC

GANT5mC3 G6mANTC
GANT5hmC
Hin1 I GRCGYC CG methylase (5mCG) GR5mCGYC GRCGY5mC3,4 GR5mCGYC
Hpa I GTTAAC

GTTAA5mC GTTA6mAC
GTTAA5hmC
G5hmU5hmUAAC
Kpn I GGTACC

GGTA5mCC
GGTA5mC5mC
GGTAC5mC
GGT6mACC
GGTAC4mC
Mbo I GATC dam methylase (G6mATC) G6mATC GAT4mC
GAT5mC
GA5hmUC
G6mATC
GAT5hmC
Mbo II GAAGA
(TCTTC)
dam methylase (G6mATC) GAAG6mATC T5mCTT5mC3
G6mAAGA
GA6mAGA
GAAG6mA
Mfl I RGATCY dam methylase (G6mATC) RG6mATCY 6mAGATCY3 RG6mATCY
RGAT4mCY
RGAT5mCY
Mlu I ACGCGT CG methylase (5mCG) A5mCG5mCGT 6mACGCGT A5mCGCGT
Msp I CCGG

4mCCGG
C4mCGGC
5mCGG3
5mCCGG
5hmC5hmCGG
Mun I CAATTG


CA6mATTG
Mva I CCWGG

5mCCWGGC5mCWGG 4mCCWGG
5mC5mCWGG
C4mCWGG
CC6mAGG
Nae I GCCGGC CG methylase (5mCG) GC5mCGGC
GCCGG5mCG

G5mCCGGC
GC5mCGGC
GCCGG5mC
Nco I CCATGG

CC6mATGG 4mCCATGG
5mCCATGG
Nde I CATATG

5mCATATG
Nhe I GCTAGC CG methylase (5mCG) GCTAG5mCG
GCTAG5mC
Not I GCGGCCGC CG methylase (5mCG) G5mCGGC5mCGC GCGGCCG5mC GCGG5mCCGC
GCGGC5mCGC
Nru I TCGCGA dam methylase (G6mATC)
CG methylase (5mCG)
TCGCG6mATC
T5mCG5mCGA
TCG5mCGA GCGG5mCCGC
GCGGC5mCGC
Nsb I TGCGCA CG methylase (5mCG) TG5mCGCA TGCGC6mA TG5mCGCA
PmaC I CACGTG CG methylase (5mCG) CA5mCGTG
CA5mCGTG4
PshA I GACN4GTC CG methylase (5mCG) GA5mCGN3GTC GACN4GT5mC4 GA5mCN4GTC4
PshB I ATTAAT



Psp1406 I AACGTT CG methylase (5mCG) AA5mCGTT
AA5mCGTT
Pst I CTGCAG


5mCTGCAG
CTGC6mAG
C5hmUGCAG
Pvu I CGATCG CG methylase (5mCG) 5mCGAT5mCG CG6mATCG CGAT4mCG
CGAT5mCG
Pvu II CAGCTG


CAG4mCTG
CAG5mCTG
Sac I GAGCTC

G6mAGCTC
GAGCT5mC
GAG5mCTC
Sac II CCGCGG CG methylase (5mCG) C5mCG5mCGG
5mCCGCGG
C5mCGCGG
Sal I GTCGAC CG methylase (5mCG) GT5mCGAC GTCGA5mC GT5mCGAC
GTCG6mAC
Sau3A I GATC CG methylase (5mCG) GAT5mCG G6mATC
GA5hmUC
GAT4mC
GAT5mC3
GAT5hmC
Sca I AGTACT

AGTA5mCT
Sfi I GGCCN5GGCC dcm methylase
(C5mCWGG)
CG methylase (5mCG)
GGC5mCWGGN2GGCC
GGCCN5GGC5mCWGG
GGCCN5GGC5mCG
GG5mCCN5GG5mCC GG4mCCN5GGCC
GGC5mCN5GGCC
GGCCN5GGC5mC
Sma I CCCGGG CG methylase (5mCG) CC5mCGGG C5mCCGGG 4mCCCGGG
5mCCCGGG
C4mCCGGG
CC4mCGGG
CC5mCGGG3
Smi I ATTTAAAT



SnaB I TACGTA CG methylase (5mCG) TA5mCGTA
T6mACGT6mA
TA5mCGTA
Spe I ACTAGT


6mACTAGT
A5mCTAGT
Sph I GCATGC

GCATG5mC
G5hmCATG5hmC
GC6mATGC
Sse8387 I CCTGCAGG

5mCGCCTGCAGG5mCG4 5mCCTGCAGG4
C5mCTGCAGG4
CCTG5mCAGG4
CCTGC6mAGG4
Ssp I AATATT

6mAATATT
Stu I AGGCCT dcm methylase
(C5mCWGG)
AGGC5mCTGG
AGG5mCCT
AGGC4mCT
AGGC5mCT
Taq I TCGA dam methylase (G6mATC) TCG6mATC T5mCGA TCG6mA
T5hmCGA
Tth111 I GACN3GTC

GA5mCN3GT5mC
GACN3GT5mC4

Van91 I CCAN5TGG dcm methylase
(C5mCWGG)
C5mCAGGN3TGG
C5mCAN5TGG
VpaK11B I GGWCC dcm methylase
(C5mCWGG)
CG methylase (5mCG)
GGWC5mCWGG
GGWC5mCG

GGWC5mC
Xba I TCTAGA dam methylase (G6mATC) TCTAG6mATC
T5mCTAGA3
TCTAG6mA
T5hmCTAGA
Xho I CTCGAG

CT5mCGAG3, 4 5mCTCGAG
CTCG6mAG
Xsp I CTAG


5mCTAG

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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 GTMKA5mC.

Acc III : cleaves TC5mCGGA slowly (1/75).

Afa I : cleaves hemi-methylated GTA5mC, but cannot cut bi-methylated GTA5mC.

Ava I : Nicks the unmethylated strand of hemi-methylated CTCG5mAG slowly.

Ava II : cleaves GGWC4mC slowly.

Bal I : In case of overlapping with dcm site (TGGC5mCAGG), the digestion speed becomes very slow (less than 1/50).

Bgl I : While cleaves hemi-methylated GC5mCN5GGC, GC4mCN5GGC and GCCN5GG5mC slowly, can scarcely cleave bi-methylated M. Hae III-Bgl I site.

Cfr13 I : cannot cleave hemi-methylated GGNC5mC.

Cla I : cleaves hemi-methylated AT5mCGAT slowly.

EcoR I : While cleaves hemi-methylated GAATT5mC, cannot cleave bi-methylated GAATT5mC.

Fok I : cleaves CATC5mC slowly (1/2 to 1/4).

Hae II : cleaves RGCG5mCY very slowly.

Hae III : Nicks the unmethylated strand of hemi-methylated GG5mCC.

Hin1 I : cleaves GRCGY5mC very slowly.

Hind III : cleaves hemi-methylated AAG5mCTT slowly.

Hinf I : cleaves GANT5mC slowly (1/10).

Kpn I : It has been reported that this is influenced by hemi-methylated GGTA5mCC and GGTAC5mC, and digests hemi-methylated GGTA5mC5mC slowly.

Mbo II : It has been reported that this cannot digest hemi-methylated T5mCTT5mC.

Mfl I : cleaves 6mAGATCY slowly.

Msp I : cleaves GGC5mCGG very slowly.

Mva I : Nicks hemi-methylated CC6mAGG and the unmethylated strand of hemi-methylated 4mCCWGG.

Pst I : cleaves hemi-methylated 5mCTG5mCAG slowly.

Pvu II : cleaves hemi-methylated 5mCAG5mCTG slowly.

Sal I : cleaves hemi-methylated GT5mCGAC slowly.

Sau3A I : Nicks the unmethylated strand of hemi-methylated GAT5mC. It has been reported that the digestion speed of 5mAGATC becomes slow.

Sma I : Nicks the unmethylated strand of hemi-methylated CC5mCGGG.

Spe I : cleaves hemi-methylated A5mCTAGT slowly.

Xba I : cleaves hemi-methylated T5mCTAGA, but its digestion speed becomes very slow (less than 1/100).

Xho I : cleaves CT5mCGAG very slowly.

Hemi-methylated DNA denotes DNA of which one strand out of two is methylated, and ( ) shows the relative speed.
4 Takara, unpublished observations.

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