Effects of CpG Methylation on Restriction Enzyme Cleavage

DNA methyltransferases (MTases) are found in a wide variety of prokaryotes and eukaryotes. In prokaryotes, MTases have most often been identified as elements of restriction/modification systems in which they act to protect host DNA from cleavage by the corresponding restriction endonuclease. CpG MTases found in higher eukaryotes(e.g., Dnmt1) are not involved in restriction and modification. Nonetheless, 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 (1). CpG methylase patterns can be approximated in vitro using the prokaryotic MTase, M.Sss I (NEB #M0226).

We have screened a wide variety of restriction endonucleases whose recognition sites can be modified by M.Sss I in order to determine if cleavage is blocked by this modification. Three broad classes of enzymes were identified: (i) Those whose cleavage was completely blocked by modification, (ii) those whose cleavage was not blocked by modification, and (iii) those whose cleavage was blocked or impaired when the canonical restriction site overlapped the CG dinucleotide. The results are summarized below.

DNA modified by M.Sss I is restricted by McrA, McrBC, and Mrr systems in E. coli (2,3). Accordingly, DNAs which have been treated with M.Sss I should be transformed into Mcr- Mrr- strains, such as those available without charge from New England Biolabs.

In vivo methylation by M.Sss I has proven useful in overcoming methylation-based restriction during transformation (4) and in mapping chromatin structure in vivo (5).

References:

  1. Siegfried, Z. and Cedar, H. (1997) Curr. Biol. 7, r305-307. PMID: 9115385
  2. Kelleher, J.E. and Raleigh, E. (1991) J. Bacteriol. 173, 5220-5223. PMID: 1830580
  3. Rees, P. et al. (1991) J. Bacteriol. 173, 5207-5219. PMID: 1650347
  4. Butler, C.A. and Gotschlich, E.C. (1991) J. Bacteriol. 173, 5793-5799. PMID: 1653220
  5. Kladde, M.P. and Simpson, R.T. (1996) Methods Enzymol. 274, 214-233. PMID: 8902807

Note: Restriction enzyme cleavage is blocked when the recognition sequence is methylated by the cognate methylase. Methylation at other bases can block cleavage, leave cleavage unaffected or slow the rate or extent of cleavage. The rate of cleavage may also be affected by the DNA sequence flanking the recognition site. As a result, cleavage may depend on reaction conditions and on the site being studied, and cleavage of a substrate with multiple sites may yield a mixture of complete and partial digestion products. The accompanying tables should be viewed as a guide to the behavior of the enzymes listed rather than an absolute indicator. Consult REBASE, the restriction enzyme database, for more detailed information and specific examples upon which these guidelines are based.

Footnotes:

  1. Cleavage rate slowed significantly (impaired) by methylation.
  2. Multiple overlaps required to block cleavage
Single Letter Code
Cleavage Blocked at All Sites
AatII GACGTC
AciI CCGC
AclI AACGTT
AfeI AGCGCT
AgeI ACCGGT
AscI GGCGCGCC
AsiSI GCGATCGC
AvaI CYCGRG
BceAI ACGGC
BmgBI CACGTC
BsaAI YACGTR
BsaHI GRCGYC
BsiEI CGRYCG
BsiWI CGTACG
BsmBI CGTCTC
BspDI ATCGAT
BspEI1 TCCGGA
BsrBI1 CCGCTC
BsrFI RCCGGY
BssHII GCGCGC
BstBI TTCGAA
BstUI CGCG
ClaI ATCGAT
EagI CGGCCG
FauI CCCGC
FseI GGCCGGCC
FspI TGCGCA
HaeII RGCGCY
HgaI GACGC
HhaI GCGC
HinP1I GCGC
HpaII CCGG
Hpy99I CGWCG
HpyCH4IV ACGT
KasI GGCGCC
MluI ACGCGT
NaeI GCCGGC
NarI GGCGCC
NgoMIV GCCGGC
NotI GCGGCCGC
NruI TCGCGA
PaeR7I1 CTCGAG
PmlI CACGTG
PvuI CGATCG
RsrII CGGWCCG
SacII CCGCGG
SalI GTCGAC
SfoI GGCGCC
SgrAI CRCCGGYG
SmaI CCCGGG
SnaBI TACGTA
TilI1 CTCGAG
XhoI1 CTCGAG
Cleavage Blocked or Impaired Only at Sites with Overlapping CG
AccI GTMKAC
Acc65I GGTACC
AhdI2 GAC(N5)GTC
ApaI GGGCCC
ApaLI GTGCAC
AvaII GGWCC
BaeI2 AC(N4)GTAYC
BanI2 GGYRCC
BcgI2 CGA(N6)TGC
BbvCI CCTCAGC
BfuAI ACCTGC
BglI2 GCC(N5)GGC
BsaI GGTCTC
BsaBI1 GAT(N4)ATC
BslI2 CC(N7)GG
BsmAI GTCTC
BsmFI GGGAC
BssKI CCNGG
BstAPI2 GCA(N5)TGC
BstZI GTATAC
Cac8I GCNNGC
DpnII GATC
DraIII CACNNNGTG
DrdI1 GAC(N6)GTC
EaeI YGGCCR
EarI CTCTTC
EciI2 GGCGGA
EcoRI2 GAATTC
EcoRV GATATC
Fnu4HI GCNGC
HincII1 GTYRAC
HinfI GANTC
HpaI2 GTTAAC
Hpy188III TCNNGC
MboI GATC
MspA1I CMGCKG
MwoI2 GC(N7)GC
NciI CCSGG
NheI GCTAGC
NlaIV GGNNCC
PleI2 GAGTC
PmeI GTTAAAC
PshAI2 GAC(N4)GTC
RsaI2 GTAC
SaII GTCGAC
Sau3AI GATC
Sau96I GGNCC
SfaNI2 GCATC
SfiI GGCC(N5)GGCC
TfiI2 GAWTC
TseI2 GCWGC
Cleavage Not Blocked at Sites with Overlapping CG
AflIII ACRYGT
AhdI GAC(N5)GTC
AlwI GGATC
BamHI GGATCC
BanII GRGCYC
BbsI GAAGAC
BbvI GCAGC
BciVI GTATCC
BlpI GCTNAGC
Bme1580I GKGCMC
BsaJI CCNNGG
BsaWI WCCGGW
BsaXI AC(N5)CTCC
BsgI GTGCAG
BsiHKAI GWGCWC
BsmI GAATGC
BsoBI CYCGRG
Bsp1286I GDGCHC
BspMI ACCTGC
BssSI CTCGTG
BstEII GGTNACC
BstYI RGATCY
BtgI CCRYGG
EcoO109I RGGNCCY
FokI GGATG
HaeIII GGCC
HphI GGTGA
Hpy188I TCNGA
HpyCH4III ACNGT
KpnI GGTACC
MlyI GAGTC
MnlI CCTC
MspI CCGG
PflFI GACNNNGTC
PspOMI GGGCCC
SacI GAGCTC
SapI GCTCTTC
SphI GCATGC
Taq αI TCGA
Tsp45I GTSAC
Tth111I GACNNNGTC
XmaI CCCGGG
XmnI GAA(N4)TTC