Dam, Dcm and CpG Methylases
DNA methyltransferases (MTases), which transfer a methyl group from S-adenosylmethionine to either adenine or cytosine residues, are found in a wide variety of prokaryotes and eukaryotes. Methylation needs to be considered when digesting DNA with restriction endonucleases because cleavage can be blocked or impaired when a particular base in the recognition site is methylated.
In prokaryotes, MTases have most often been identified as elements of restriction/modification systems that act to protect host DNA from cleavage by the corresponding restriction endonuclease. Most laboratory strains of E. coli contain three site-specific DNA methylases. The methylase encoded by the dam gene (Dam methylase) transfers a methyl group to the N6 position of the adenine residues in the sequence GATC (1,2). The Dcm methylase, encoded by the dcm gene, methylates the internal cytosine residues in the sequences CCAGG and CCTGG (1,3) at the C5 position. The EcoK I methylase, M. EcoK I, modifies adenine residues in the sequences AAC(N6)GTGC and GCAC(N6)GTT. Some or all of the sites for a restriction endonuclease may be resistant to cleavage when isolated from strains expressing the Dam or Dcm methylases if the methylase recognition site overlaps the endonuclease recognition site. For example, plasmid DNA isolated from Dam+ E. coli is completely resistant to cleavage by Mbo I, which cleaves at GATC sites.
Not all DNA isolated from E. coli is methylated to the same extent. While pBR322 DNA is fully modified (and is therefore completely resistant to Mbo I digestion), only about 50% of l DNA Dam sites are methylated, presumably because the methylase does not have the opportunity to methylate the DNA fully before it is packaged into the phage head. As a result, enzymes blocked by Dam or Dcm modification will yield partial digestion patterns with l DNA.
CpG MTases, found in higher eukaryotes (e.g., Dnmt1), transfer a methyl group to the C5 position of cytosine residues. Patterns of CpG methylation are heritable, tissue specific, and correlate with gene expression. Consequently CpG methylation has been postulated to play a role in differentiation and gene expression (4). The effects of CpG methylation are mainly of concern when digesting eukaryotic genomic DNA. It should be noted that CpG methylation patterns are not retained once the DNA is cloned into a bacterial host.
The table below summarizes methylation sensitivity for NEB restriction enzymes, indicating whether or not cleavage is blocked or impaired by Dam, Dcm or CpG methylation if or when it overlaps each recognition site. This table should be viewed as a guide to the behavior of the enzymes listed rather than an absolute indicator. Consult REBASE <http://rebase.neb.com/rebase/>, the restriction enzyme database, for more detailed information and specific examples upon which these guidelines are based.
References
1. Marinus, M.G. and Morris, N.R. (1973) J.
Bacteriol. 114, 1143–1150.
2. Geier, G.E.and Modrich, P. (1979) J.
Biol. Chem. 254, 1408–1413.
3. May, M.S. and Hattman, S.(1975) J.
Bacteriol. 123, 768–770.
4.
Siegfried, Z. and Cedar, H. (1997) Curr.
Biol. 7, r305–307.
| Legend | |
|---|---|
 |
Not Sensitive |
 |
Blocked |
 |
Blocked by Overlapping |
 |
Blocked by Some Combinations of Overlapping |
 |
Impaired |
 |
Impaired by Overlapping |
 |
Impaired by Some Combinations of Overlapping |
| A | B | C | D | E | F | H | I | K | M | N | P | R | S | T | X | Z |
| Enzyme | Sequence | Dam | Dcm | CpG |
|---|---|---|---|---|
| Aat II | GACGTC | |
|
|
| Acc I | GTMKAC | |
|
|
| Acc65 I | GGTACC | |
|
|
| Aci I | CCGC | |
|
|
| Acl I | AACGTT | |
|
|
| Acu I | CTGAAG(16/14) | |
|
|
| Afe I | AGCGCT | |
|
|
| Afl II | CTTAAG | |
|
|
| Afl III | ACRYGT | |
|
|
| Age I | ACCGGT | |
|
|
| Ahd I | GACNNNNNGTC | |
|
|
| Ale I | CACNNNNGTG | |
|
|
| Alu I | AGCT | |
|
|
| Alw I | GGATC(4/5) | |
|
|
| AlwN I | CAGNNNCTG | |
|
|
| Apa I | GGGCCC | |
|
|
| ApaL I | GTGCAC | |
|
|
| Apo I | RAATTY | |
|
|
| Asc I | GGCGCGCC | |
|
|
| Ase I | ATTAAT | |
|
|
| AsiS I | GCGATCGC | |
|
|
| Ava I | CYCGRG | |
|
|
| Ava II | GGWCC | |
|
|
| Avr II | CCTAGG | |
|
|
| Bae I | (10/15)AC(N4)GTAYC(12/7) | |
|
|
| BamH I | GGATCC | |
|
|
| Ban I | GGYRCC | |
|
|
| Ban II | GRGCYC | |
|
|
| Bbs I | GAAGAC(2/6) | |
|
|
| Bbv I | GCAGC(8/12) | |
|
|
| BbvC I | CCTCAGC | |
|
|
| Bcc I | CCATC(4/5) | |
|
|
| BceA I | ACGGC(12/14) | |
|
|
| Bcg I | (10/12)CGA(N6)TGC(12/10) | |
|
|
| BciV I | GTATCC(6/5) | |
|
|
| Bcl I | TGATCA | |
|
|
| Bfa I | CTAG | |
|
|
| BfrB I | ATGCAT | |
|
|
| BfuA I | ACCTGC(4/8) | |
|
|
| BfuC I | GATC | |
|
|
| Bgl I | GCCNNNNNGGC | |
|
|
| Bgl II | AGATCT | |
|
|
| Blp I | GCTNAGC | |
|
|
| Bme1580 I | GKGCMC | |
|
|
| BmgB I | CACGTC | |
|
|
| Bmr I | ACTGGG(5/4) | |
|
|
| Bmt I | GCTAGC | |
|
|
| Bpm I | CTGGAG(16/14) | |
|
|
| Bpu10 I | CCTNAGC | |
|
|
| BpuE I | CTTGAG(16/14) | |
|
|
| Bsa I | GGTCTC(1/5) | |
|
|
| BsaA I | YACGTR | |
|
|
| BsaB I | GATNNNNATC | |
|
|
| BsaH I | GRCGYC | |
|
|
| BsaJ I | CCNNGG | |
|
|
| BsaW I | WCCGGW | |
|
|
| BsaX I | (9/12)AC(N5)CTCC(10/7) | |
|
|
| BseR I | GAGGAG(10/8) | |
|
|
| BseY I | CCCAGC | |
|
|
| Bsg I | GTGCAG(16/14) | |
|
|
| BsiE I | CGRYCG | |
|
|
| BsiHKA I | GWGCWC | |
|
|
| BsiW I | CGTACG | |
|
|
| Bsl I | CCNNNNNNNGG | |
|
|
| Bsm I | GAATGCN | |
|
|
| BsmA I | GTCTC(1/5) | |
|
|
| BsmB I | CGTCTC(1/5) | |
|
|
| BsmF I | GGGAC(10/14) | |
|
|
| BsoB I | CYCGRG | |
|
|
| Bsp1286 I | GDGCHC | |
|
|
| BspCN I | CTCAG | |
|
|
| BspD I | ATCGAT | |
|
|
| BspE I | TCCGGA | |
|
|
| BspH I | TCATGA | |
|
|
| BspM I | ACCTGC(4/8) | |
|
|
| Bsr I | ACTGGN | |
|
|
| BsrB I | CCGCTC | |
|
|
| BsrD I | GCAATGNN | |
|
|
| BsrF I | RCCGGY | |
|
|
| BsrG I | TGTACA | |
|
|
| BssH II | GCGCGC | |
|
|
| BssK I | CCNGG | |
|
|
| BssS I | CACGAG | |
|
|
| BstAP I | GCANNNNNTGC | |
|
|
| BstB I | TTCGAA | |
|
|
| BstE II | GGTNACC | |
|
|
| BstF5 I | GGATGNN | |
|
|
| BstN I | CCWGG | |
|
|
| BstU I | CGCG | |
|
|
| BstX I | CCANNNNNNTGG | |
|
|
| BstY I | RGATCY | |
|
|
| BstZ17 I | GTATAC | |
|
|
| Bsu36 I | CCTNAGG | |
|
|
| Btg I | CCRYGG | |
|
|
| BtgZ I | GCGATG(10/14) | |
|
|
| Bts I | GCAGTGNN | |
|
|
| Cac8 I | GCNNGC | |
|
|
| Cla I | ATCGAT | |
|
|
| CviA II | CATG | |
|
|
| Dde I | CTNAG | |
|
|
| Dpn II | GATC | |
|
|
| Dra I | TTTAAA | |
|
|
| Dra III | CACNNNGTG | |
|
|
| Drd I | GACNNNNNNGTC | |
|
|
| Eae I | YGGCCR | |
|
|
| Eag I | CGGCCG | |
|
|
| Ear I | CTCTTC(1/4) | |
|
|
| Eci I | GGCGGA(11/9) | |
|
|
| EcoN I | CCTNNNNNAGG | |
|
|
| EcoO109 I | RGGNCCY | |
|
|
| EcoP15 I | CAGCAG(25/27) | |
|
|
| EcoR I | GAATTC | |
|
|
| EcoR V | GATATC | |
|
|
| Fat I | CATG | |
|
|
| Fau I | CCCGC(4/6) | |
|
|
| Fnu4H I | GCNGC | |
|
|
| Fok I | GGATG(9/13) | |
|
|
| Fse I | GGCCGGCC | |
|
|
| Fsp I | TGCGCA | |
|
|
| Hae II | RGCGCY | |
|
|
| Hae III | GGCC | |
|
|
| Hga I | GACGC(5/10) | |
|
|
| Hha I | GCGC | |
|
|
| Hinc II | GTYRAC | |
|
|
| Hind III | AAGCTT | |
|
|
| Hinf I | GANTC | |
|
|
| HinP1 I | GCGC | |
|
|
| Hpa I | GTTAAC | |
|
|
| Hpa II | CCGG | |
|
|
| Hph I | GGTGA(8/7) | |
|
|
| Hpy188 I | TCNGA | |
|
|
| Hpy188 III | TCNNGA | |
|
|
| Hpy99 I | CGWCG | |
|
|
| HpyCH4 III | ACNGT | |
|
|
| HpyCH4 IV | ACGT | |
|
|
| HpyCH4 V | TGCA | |
|
|
| Kas I | GGCGCC | |
|
|
| Kpn I | GGTACC | |
|
|
| Mbo I | GATC | |
|
|
| Mbo II | GAAGA(8/7) | |
|
|
| Mfe I | CAATTG | |
|
|
| Mlu I | ACGCGT | |
|
|
| Mly I | GAGTC(5/5) | |
|
|
| Mme I | TCCRAC(20/18) | |
|
|
| Mnl I | CCTC(7/6) | |
|
|
| Msc I | TGGCCA | |
|
|
| Mse I | TTAA | |
|
|
| Msl I | CAYNNNNRTG | |
|
|
| Msp I | CCGG | |
|
|
| MspA1 I | CMGCKG | |
|
|
| Mwo I | GCNNNNNNNGC | |
|
|
| N.Alw I | GGATCNNNN | |
|
|
| Nae I | GCCGGC | |
|
|
| Nar I | GGCGCC | |
|
|
| Nci I | CCSGG | |
|
|
| Nco I | CCATGG | |
|
|
| Nde I | CATATG | |
|
|
| NgoM IV | GCCGGC | |
|
|
| Nhe I | GCTAGC | |
|
|
| Nla III | CATG | |
|
|
| Nla IV | GGNNCC | |
|
|
| Not I | GCGGCCGC | |
|
|
| Nru I | TCGCGA | |
|
|
| Nsi I | ATGCAT | |
|
|
| Nsp I | RCATGY | |
|
|
| Pac I | TTAATTAA | |
|
|
| PaeR7 I | CTCGAG | |
|
|
| Pci I | ACATGT | |
|
|
| PflF I | GACNNNGTC | |
|
|
| PflM I | CCANNNNNTGG | |
|
|
| Pho I | GGCC | |
|
|
| Ple I | GAGTC(4/5) | |
|
|
| Pme I | GTTTAAAC | |
|
|
| Pml I | CACGTG | |
|
|
| PpuM I | RGGWCCY | |
|
|
| PshA I | GACNNNNGTC | |
|
|
| Psi I | TTATAA | |
|
|
| PspG I | CCWGG | |
|
|
| PspOM I | GGGCCC | |
|
|
| Pst I | CTGCAG | |
|
|
| Pvu I | CGATCG | |
|
|
| Pvu II | CAGCTG | |
|
|
| Rsa I | GTAC | |
|
|
| Rsr II | CGGWCCG | |
|
|
| Sac I | GAGCTC | |
|
|
| Sac II | CCGCGG | |
|
|
| Sal I | GTCGAC | |
|
|
| Sap I | GCTCTTC(1/4) | |
|
|
| Sau3A I | GATC | |
|
|
| Sau96 I | GGNCC | |
|
|
| Sbf I | CCTGCAGG | |
|
|
| Sca I | AGTACT | |
|
|
| ScrF I | CCNGG | |
|
|
| SexA I | ACCWGGT | |
|
|
| SfaN I | GCATC(5/9) | |
|
|
| Sfc I | CTRYAG | |
|
|
| Sfi I | GGCCNNNNNGGCC | |
|
|
| Sfo I | GGCGCC | |
|
|
| SgrA I | CRCCGGYG | |
|
|
| Sma I | CCCGGG | |
|
|
| Sml I | CTYRAG | |
|
|
| SnaB I | TACGTA | |
|
|
| Spe I | ACTAGT | |
|
|
| Sph I | GCATGC | |
|
|
| Ssp I | AATATT | |
|
|
| Stu I | AGGCCT | |
|
|
| Sty I | CCWWGG | |
|
|
| StyD4 I | CCNGG | |
|
|
| Swa I | ATTTAAAT | |
|
|
| Taq I | TCGA | |
|
|
| Tfi I | GAWTC | |
|
|
| Tli I | CTCGAG | |
|
|
| Tse I | GCWGC | |
|
|
| Tsp45 I | GTSAC | |
|
|
| Tsp509 I | AATT | |
|
|
| TspR I | NNCASTGNN | |
|
|
| Tth111 I | GACNNNGTC | |
|
|
| Xba I | TCTAGA | |