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  • 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 orImpaired 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