site-specific methylation: effect on dna modification

© 1991 Oxford University Press Nucleic Acids Research, Vol. 19, Supplement 2045

Site-specific methylation: effect on DNA modificationmethyltransferases and restriction endonucleases

Michael Nelson and Michael McClelland*California Institute of Biological Research, 11099 North Torrey Pines Road, La Jolla, CA 92037 USA

INTRODUCTION

We present in Table I an updated list of the sensitivities of over240 restriction endonucleases to the site-specific DNAmodifications m4C, "^C, iBnSC, and "^A, four modifications thatare common in DNA prokaryotes, eukaryotes, and their viruses(Mc2,Mc5,Mc8,Mcll,Ne3,Ne4).

Table II is a list of over 130 characterized DNAmethyltransferases. A detailed list of cloned restriction-modification genes has been made Wilson (Wi4).

Table III lists the sensitivities of over 20 Type II DNAmethyltransferases to m4C, "^C, ^ C , and m6A modification.Most DNA methyltransferases are sensitive to non-canonicalmodifications within their recognition sequences(Bu5,MclO,Ne3,Pc4), and this sensitivity may differ from thatof their restriction endonuclease partners.

Finally, several restriction endonuclease isoschizomers areknown to differ in their ability to cleave DNA which has beenmethylated. Table IV lists over 20 known isoschizomer pairsand one isomethylator pair, along with the modified recognitionsites at which they differ.

Effect of and ""^NG on restriction endonucleasesEnzymes that are not sensitive to site-specific methylation areparticularly useful for achieving complete digestion of methylatedDNA. For instance, endonucleases that are unaffected by '"'CGand '"'CNG are useful for digestion of plant DNA which isfrequently methylated at these positions. Endonucleases that areunaffected by these two cytosine modifications include: AccXR,AflR, AhaJR, Asel, AsplOOl AsuR, Bbul, Bed, BspHl, BspNl,BstER, BstNl, CviQI, Dpnl, Dral, EcoRV, HinCR, Hpal, Kpnl,MboR, Msel, Ndel, NdeR, Pad, Rsal, RspXl, Spel, Sphl, Sspl,Swal, Taql, TthYW. and Xmnl.

CpG sequences occur infrequently and are often methylatedin mammalian genomes (Mc9). Almost all the enzymes that couldgenerate large fragments of mammalian DNA are blocked bythis "^CpG modification at overlapping sites, including AatU,Apel, Avm, Bbe\, BmaDl, BssHR, BspMR, BstBl, Clal, Cspl,Csp45l, Eagl, EclXl, Eco47Ul, Fsel, Fspl, Kpri2l Mlul,Mh&mi, Mu9273II, Mrol, Noel, Nari, Notl, Nrul, Pfiil, Pndl,PpuAd, Pvul, RsrR, Sail, SalDl, Sbol3l, Sfil, Smal, SnaBl, Spll,Spol, Xhol and XorR (see Table I).

Only four enzymes suitable for pulsed field mapping ofeukaryotic chromosomes are known to cut "^CG-modifiedDNA: AccRl, AsuR, Cfr9\ and Xmal. It has been determinedthat Sfil is sensitive to "^C modification at the second cytosineof its recognition sequence, GGC^CNsGGCC. Sfil is therefore

sensitive to overlapping m5CG methylation atGGCm5CGN4GGCC sites in mammals and overlapping danmethylation at GGC^CWGGNNGGCC sequences in E. coli..

•"•C and "^C Cytosine modificationsIn some cases, a restriction enzyme may differ with to sensitivityto rcAQ and "^C at a particular sequence. For example, BstNland Mval cut "^C, but not "*€ modified CCWGG sequences.Kpnl cuts GGTAC^C but not GGTAC1"^. BstYl cuts RG-AT^CY but not RGATm4CY. Restriction enzymes we havetested for sensitivity to m4C include: Aatl, Afll, Alwl, AvaR,Banl, Bgll, Bstl, BstNl, BstYl, Dpnl, Fokl, Mbol, Mval, Nari,Neil, PflMl, Sau3A, and ScrFl.

Rate of cleavage at methylated restriction sitesm4C, "^C, ^ C , and "^A are bulky alkyl substitutions in themajor groove of B-form DNA. It is therefore not surprising thatsite-specific DNA methylation can interfere wim many sequence-specific DNA binding proteins (e.g. St2,Wa8) including bindingof restriction endonucleases and DNA methyltransferases. DNAmethylation may cause long-range perturbations of DNA minorand major grooves, and a range of rate effects are observed whenmodified substrates are used in restriction-modification reactions.Results can be summarized as follows.

(1) Canonical site-specific methylation always inhibits DNAcleavage by a restriction endonuclease. For example, M.BamHlmethylase modifies GGATm4CC; and BamYR endonucleasecannot cut this methylated sequence.

(2) In about one half of the cases tested, methylation at non-canonical sites inhibits the rate of duplex DNA cleavage at leastten-fold (Table I). However, in other cases non-canonicalmethylation has no effect on restriction cleavage. For example,BamYR cuts DNA which has been modified at GGATC1™^ orGGATC^C, but cannot cut DNA methylated at GGAT^CC.

(3) There are a few examples in which non-canonicalmethylation slows the rate of cleavage or permits nicking of onestrand of a hemi-methylated duplex. Examples of such rate effectsare presented in footnotes to Table I.

(4) Sometimes base modifications which lie outside arecognition sequence can influence the rate of DNA cleavage bya restriction enzyme. For example, Nari does not cut atoverlapping M.Mval-Narl GGCGCC^CCWGG sites (Nel);and HaeRl cannot cut certain GGCCT sites, where T aremodified thymine residues (Wil). Such methylation-induced'action at a distance' may be more common than has been

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2046 Nucleic Acids Research, Vol. 19, Supplement

previously appreciated. We have tested only a few enzymes forsensitivity to base modifications outside their canonicalrecognition sequences.

Effect of site-specific methylation on DNA methyltransferasesTwenty-three Type n methyltransferases have been tested forsensitivity to non-canonical DNA modifications, of which ninewere blocked (Me 10 and Table HI). As with restrictionendonucleases, rate effects are sometimes seen with DNAmethyltransferases at non-canonically modified sequences. Forexample, E. coli Dam methyltransferase is unaffected by G-AT1™^, but methylates GAT^C relatively slowly. Such data issummarized in Table HI and footnotes to Table I.

Methylase/endonuclease combinations can produce novelDNA cleavage specificitiesSeveral strategies involving combinations of modificationmethyltransferases and restriction endonucleases have been usedto generate rare or novel DNA cleavage sites.

For example, certain adenine methyltransferases may be usedin conjunction with the methylation-dependent restrictionendonuclease Dpnl to create cleavages at eight- to twelve-base-pair sequences (Mc6,Mcl2). MClal and Dpnl have been usedto cut the 2.8 million base pair Staphylococcus aureus genomeinto two pieces at the sequence ATCGATCGAT (Wei). Twelve-base-pair TCTAGATCTAGA MXbal/Dpnl sites in atransposon have been introduced into bacterial genomes andpermit cleavage one or more times depending on the number oftransposons integrated (Ha5).

Protection of a subset of restriction endonuclease cleavage sitesby methylation at overlapping methyltransferase/endonucleasetargets has been described (Hul ,K11 ,Ne6). This two-step 'cross-protection' strategy has produced over 60 new cleavagespecificities, and many more are possible (Ja2,Ka2,Kll,Ne6).Extremely specific DNA cleavages may result from certain'cross-protections.' For example, MFnuDWNotl cleavage hasbeen used to cut the 4.7 million base pair E. coli K12 genomeinto fourteen pieces (Qi2).

Methylases have been used to compete with endonucleases forrecognition sites in a method called methylase-limited partialdigestion. This method is particularly useful for performing partialdigests in agarose plugs for pulsed field gel electrophoresis(Ha6). Blocking a subset of DNA methyltransferase sites byoverlapping methylation (sequential double-methylation) canexpose a subset of restriction endonuclease sites for cleavage(Mc9,Ne3,Po3). For instance, MHpaU, M-BomHI, and BamHlhave been used in a sequential three-step methyltrans-ferase/methyltransferase/endonuclease reaction to achieveselective DNA cleavage at the ten base pair sequence, CCGG-ATCCGG (MclO).

Polypyrimidine oligonucleotides have been used in DNAtriplexes to selectively mask restriction-modification sites. Forexample, polyppyrimidine triplexes which overlap M.Taql siteshave been used to enable selective restriction cleavage (Ma7).

Finally, methods based on the sequential use of purified lacrepressor protein, DNA methyltransferases, and restrictionendonucleases have been used to achieve highly selective DNAcleavages (Ko2).

Methylation-dependent restriction systems in bacteriaE. coli K-12 contains at least three different methylation-dependent restriction systems which selectively restrict methylated

target sequences: mrr C^A), mcrA ("^CG), mcr B (R^C)(Br5,Dil,He3,Ral,Ra2). In vivo or in vitro modified DNA isinefficiently cloned into E. coli. For example, human DNA whichis extensively methylated at "^CpG is restricted by mcrA (Wo2).Appropriate non-restricting strains of £ coli (Go2,Krl, Ral,Ra2)should be chosen for efficient transformation and cloning ofmethylated DNA. Other species have such restriction systems(e.g. Ma2).

Engineered altered methylase specificitiesMany methylase gene have now been sequenced. Extensivehomologies between closely related enzymes (Wi3) or commonmotifs (Po5,Sm3) allow new specificities to be developed (e.g.Ba4,Tr4).

Data in electronic formThis paper is available as a text file on a 3.5' Macintosh diskette.The data can be supplied as a Microsoft Word, Macwrite or MS-DOS file. Please contact Michael McClelland at CIBR, phone619 535 5486, FAX 619 535 5472. There are tentative plansto provide the supplement issue on a CD-ROM.

ACKNOWLEDGEMENTS

This work is supported by grants from the National Institutesof Health and the U.S. Dept. of Energy. We gratefullyacknowledge the editorial assistance of Charlie Peterson, MikeBiros and Dotty Crosei.

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1324-1332.Ko6. Kosykh V. G., Bur'yanov Ya.I. and Bayev A.A.: Mol. Gen. Genet. Mc9.

178 (1980) 717-718.Ko7. Kosykh V.G., Glinskaite I., Bur'yanov Ya.I. and Bayev A.A.: Dokl. MclO.

Akad. Nauk SSSR 265 (1982) 727-730. Mcll.Ko8. Kosykh V.G.,SoloninA.S., Bur'yanov Ya.I. and Bayev A.A.: Bkxhim. Mcl2.

Biophys. Ada 655 (1981) 102-106.Krl. KretzP.L, Reid C.H., Greener A., J.M.Short (1989) Nucleic Acids Res. Mcl3.

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Lai. Labbe D., Holtke H.J. and Lau P.C.K.: Mol. Gen. Genet. 224 (1990)101-110. Na2.

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Schildkraut 1.: Gene 77 (1989) 1-10.Landry D.: (unpublished observations).Lange C , Noyer-Weidner M, Trautner T.A., Weinewr M., and Zahler

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J. Gen. Microbiol. 132 (1986) 3055-3059.Loenen W.A.M., Daniel A.S., Braymer H.D. and Murray N.E.: J. Mol.

Biol. 198 (1987) 159-170.Looney M.C., Moran L.S., Jack W.E., Feehery G.R., Brenner J.S.,

Slatko B.E. and Wilson G.G.: Gene 80 (1989) 193-208.Lui A.C.P., McBndge B.C., Vovis G.F. and Smith M.: Nucleic Acids

Res. 6 (1979) 1-15.Lunnen K.D., Barsomian J.M., Camp R.R., Card CO. , Chen S.Z.,

Croft R., Looney M.C., Meda M.M., Moran L.S., Nwankwo D.O.,Slatko B.E., Van Cott E.M. and Wilson G.G.: Gene 74 (1988) 25-32.

Lunnen K. (unpublished).Lunnen K.D., Morgan R.D., Timan C.J., Krzyclri J.A., Reeve J.N.

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81 (1984) 983-987.McClelland M., and Nelson M.: (unpublished results).McClelland M. and Nelson M.: Nucleic Acids Res. 13 (1985)

r201 -r207.McClelland M. and Nelson M.: Gene Amplification and Analysis,Vo\.

5 (1987) J. Chirikjian, Ed., Elsevier, NY, pp257-282.McClelland M. and Nelson M.: Gene 74 (1988) 169-176.McClelland M. and Nelson M.: Gene 74 (1988) 291-304.McClelland M., Nelson M. and Cantor C.R.: Nucleic Acids Res. 13

(1985) 7171-7182.McClelland M. and Patel Y.: (unpublished observations).Meda M.M. and Perler F.B.: (unpublished).Miner Z. and Hattman S.: J.Bacteriol. 170 (1988) 5177-5184Miner Z., Schlagman S.L. and Hattman S.: Nudeic Acids Res. 17 (1989)

8149-8158.Modrich P.: Quart. Rev. Biophys. 12 (1979) 315-369.Molloy P.L. and Watt F.: Nucleic Acids Res. 16 (1988) 2335.Morgan R.: (unpublished observations).Mural R.J.: Nucleic Acids Res 15 (1987) 9085.Myers P.A. and Roberts RJ.: (unpublished results).Nagaraja V., Shepherd J.C.W., Pripfl T. and BickleT.A.: J. Mol. Biol.

182 (1985) 579-587.Nagaraja V., Shepherd J.C.W. and Bickle T.A.: Nature 316(1985)

371-372.NardoneG.G. and Chirikjian J.G.: J. Biol. Chem. (1984) 10357-10362.



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r219-r230. Re3.Ne4. Nelson M. and McOeUandM.: Nucleic Acids Res. 17 (1987) r398-415.Ne5. Nelson M. and McClelland M.: (unpublished observations). Re4.Ne6. Nelson M. and Schildkraut I.: Methods in Enzymology 155 (1987) Ril.

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E. A., Suchkov S.V., UporovaT.M., Gruber I.M. and Debov S.S.: Ro7.Mol. Genet. Mikrobiol. i Virusol. 12 (1983) 5-10. Rul.

Ni2 Nikolskaya I.I., Lopatina N.G., Antikeicheva N.V. and Debov S.S.: Sal.Nucleic Acids Res. 7 (1979) 517-528. Sa2.

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Nol. Noyer-WeidnerM., JentschS., KupschJ., BergbauerM. andTrautner Sc2.T.A.: Gene 35 (1985) 143-150. Sc3.

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Nwl. Nwankwo D.O. and Wilson G.G.: Mol. Gen. Genet. 209 (1987) Sc7.570-574.

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5957-5972. Shi.Pi4. Piekarowicz A., Yuan R. and Stein D.C: Nucleic Acids Res. 16(1988)

9868. Sh2.Pi5. Piekarowicz A., Yuan R. and Stein D.C: Nucleic Acids Res. 17 (1989)

10132. Sh3.Pi6. Pirrotta V.: Nucleic Acids Res. 3 (1976) 1747-1760.Pol. Pcsfai G., BaMauf F., Erdei S., Posfai J., Venetianer P. and Kiss A.: Sil.

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737-747.Schertzer E., Auer B. and Schweiger M.: J. Biol. Chem. 262 (1987)

15225-15231.Schildkraut I., Morgan R. and Looney M.E.: (unpublished results).Schlagman S.L., Miner Z., Feher Z. and Hattman S.: Gene 73 (1988)

517-530.Schlagman S.L. and Hattman S.: Gene 22 (1983) 139-156.Schlagman S.L. and Hattman S.: Nucleic Acids Res. 17 (1989)

9101-9112.Schlagman S., Hattman S., May M.S. and Berger L.: J. Bacteriol. 126

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4417.Sciaky D. and Roberts R.J.: (unpubUshed results).Seeber S., Kessler C. and Gotz F.: Gene 94 (1990) 37-43.Selker E.U., Cambareri E.B., Garrett P.W., Haack K.R., Jensen B.C.

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2841-2842.Shields S.L., BurbankD.E., GrabherrrR. and Van EttenJ.L.: Virology

176 (1990) 16-24Silber K., Polisson C , Rees P., and Benner J.S.: Gene 74 (1988) 43-44.Skrzypek E. and Piekarowicz A.: (unpublished).Sladek T.L., Nowak J.A. and Maniloff J.: J. Bacteriol. 165 (1986)

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10063.Xi6. Xia Y. and Van Etten J.L.: Mol. Cell Biol. 6 (1986) 1440-1445.Xul. Xu G., Kapfer W., Walter J. and Trautner T.A.: (unpublished).Yol. Yolov A.A., Vinogradova M.N., Gromova E.S., Rosenthal A., Cech

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(1972) 1275-1279.Yo5. Youssoufian H., Hammer S.M., Hirsch M.S. and Mulder C : Proc. Natl.

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J.M.: Nucleic Acids Res. 15 (1987) 3919.



TABLE I: Methylation sensitivity of restriction endonucleases a

Restriction RecognitionenzymeAacIA?tT

AgjH

AccI

AccnAccm

Afll

Ann

AfnnAhafl

Alul

Alwl

AmalAosIIApal

ApelApaLIApyJAquIAselAspJOOI

Asp718l

AsuIIAfliCIAval

sequenceCCWGGAGGCCT

GACGTC

GTMKAC

CGCGTCCGGA

GGWCC

CTTAAG

ACRYGTGRCGYC b

AGCT

GGATC

TCGCGAGRCGYCGGGCCC

ACGCGTGTGCACCCWGGCYCGRGATTAATGAAN4TTC

GGTACC

ITCGAATGATCACYCGRG

SitescutC^CWGG?

7

7

?T^CCGGATO^CGGAGGWC^C ??GGWCm4C?

7

?

7

7

TCGCGm6A7

7

?v T I v Tv A \ V

Cm5CWGGj)

?ATTÂATGAm6AN4TTC

GGTmeAmSccb

TP^CGAA?Cm5CCGGG

Sites notcut

7AGGntfCCTAGGO^CTAGGO^CTGACGT^CGA^CGTCGTMKÂC*GTMKA^CmSCGCGTCCGGm6A

?

n^CTTAAGCTTAm6AGAm5CRYGTGRm5CGYCGRCGYm5cm6AGCTAGm4CTA /~^ m T/~*( I 'JxAVv T (. 1

AGhm5CTGGm6ATCGGAT"1^?GRm5CGYCGGGm5CCC*GGGCCm5CA^CGCGTGTGCAm5Cm5CCWGGm5CYCGRG#

?

Gm6AAN4TTC

GGTAOn5C

GGTAm5Cm5C b

?TGÂTCAn^CYCGRGCYm5CGRGCTCGÂG b

References

Br8NelSo3NelNelFolLu2,Mc3

Ga2Ke3,La2,Sc2

Mcll,Wh2

Nel

NelKa2,Hul

Gr4,Mcll,Ne2

Hul.WolBu5Ne4

Mcl3Eh2,Gr4,Va3La9,Tr2

Nel,Qi2FolJIol,Ho2Kll,Mcll,Ra3Ka7,Ka8NelNel

Mul,Ne4

NelRo3,Scl2Eh2^NelKa4,Ka7,McllNe2



Restriction RecognitionenzvmeAvail

AvfflBall

BajnHI

BamFTBamTaBan!

BanllBanmBbel

SbJIBbrPIBbslBbvlBell

BcnlBepIBJjIBgll

figin

BinIBmaDIBjB£216l

Bnal

BsalB_saAIEsaBiBsmI

EsmAiBSD106I

sequenceGGWCC

AGCGCTTGGCCA

GGATCC

GGATCCGGATCCGGYRCCb

GRGCYCATCGATGGCGCC

GRCGYCCACGTGGAAGACGCWGCTGATCA b

CCSGGCGCGCTTAAGGCCN5GGC

AGATCTb

GGATCCGATCGGGWCCGGATCC

GGTCTCYACGTRGATN4ATCGAATGC

GTCTCATCGAT

Sitescut

GGWCm4C b

m6AGCGCT?

GGATO^CGGm6ATCCGGmÂTCmSCGGATC^CGGm6ATCCGGm6ATCCGGm5CGCCGGYRC^CGRGCY^C7GGCGm5CCGGCGCm5C77GAAGA^C7TGAT^SCA

m5CCSGG7?GCm5CN5GGC b

AGm6ATCT

?CGm6ATCG?GGm6ATCC

777GAATG^C

?7

Sites notcut

GGW^CCGGWO^CGGWhm5Chm5CAG^CGCTTGGm5CCA*TGGC^CA 5

GGATin4CC#

GGAT^SCCGGAThmSChn^C

GGAT^^CGGATm4CC?

GRGm5CYCATCGÂTGGm5CGCC

GRm5CGYCm5CAm5CGTG7Gm5CWGC#

TGÂTCATGAThm5CAC^CSGG*m5CGCGn^CTTAAGGm5CCN5GGCGCCN5GGm5C b

GCm4CN5GGC b

AGAT^CTAGATh^CTGGm6ATCCGATm6CGGGWC^CG G A T C C

GGTCT^CYA^CGTRGATRtAT^CGm6AATGC

GTCP^CATCGÂT*

References

Ba3JCo3^^f* 111 ^^f*l 1

HulNelGil,Tr2

Br8J)rlJIalJiulLa7

Anl.ShlAnl.ShlCo3,Ka2

Fol,Ne2,Ne6SulCo3,Ne2,Sh2

Co3WolFolDol,Hal,Va5Bi4,Br83h3,Ro3HulJa3,Ja6,KllKu2WolKll,Ko3,Mcll,Ne2

Bi4,Br8J)rl,Dyl^h3Hul,Pi6BolQi2Ma9Nel

FolFolFolFol.Nel

FolNe5



Restriction RecognitionenzvmeBspi286lBspHI

BspMIBspMII

BspNI

BspXIBspXnBssHIIBsjl

"D ctT3Tr> j \ ** i

BstEn

BstEfflB&GIMNI

B.5IUIBslXIBsjYI

BsuBIBsuEIBsuFIBsuMIBsuOIBsuRICcrlCfol

Cfel£fr6I

Cfr9I

sequenceGDGCHCTCATGA

ACCTGCTCCGGA

CCWGG

ATCGATTGATCAGCGCGCb

GGATCC

TTCGAA

GGTNACC

GATCb

TGATCACCWGG b

CGCGCCAN6TGGRGATCY

CTGCAGCGCGCCGGCTCGAGCCGGGGCCCTCGAGGCGC

YGGCCRCAGCTG

CCCGGGb

Sitescut

GDGCH^C?

ACCTO^CTCCGG111^

m5CCWGGC^CWGG???GGm6ATCCGGATO^C

?

G G T N A ^ C ^ C b

GGTNAO^C?7m5CCWGG b

Cm5CWGGm5Cm5CWGG b

77RGm6ATCYRGATm5CY7?7?7??7

?7

C^CCGGGCO"5CGGG

Sites notcut

GDGm5CHCTCÂTGATCATCHÂ?T^CCGGATO^CGGA?

A T C X J ^ A T

TGÂTCAG^CGCGCGGATm4CCGGAT^CCGGATO^CTTCGm6AATP^CGAAGGTNAhm5Chm5C

GÂTCTGÂTCAhm5Chm5CWGGCm4CWGG

"^CGCG"^CCANeTGGRGATm4CY

CTGCm6AG#

^CGCG*m5CCGG#

CF^CGAG*mCCGGGG m 5 CC # b

CTCGm6AGG^CGCGhm5CGhm5CYGGm5CCR#

CAG^CTG*CAG^CTGn^CCCGGG

C^CCGGG*CCra4CGGG

References

Folê2,Ne6Pa2MelFolLa2,Sc2

Ne4

ZilZilNe4,Qi3Ne4

NelNe4WolHul,McllNelMyl,Ro3Ro3Gr4JIul,Mcll,Ro3Nel

Ne5Ne2Ne4NelGal,Jel,St5,ShlGal,Jel,St5,ShlJelJelJe2Gu6,Ki2,Ki3NelEhlHulKllBu5

Bu6



Restriction Recognition Sitesenzyme sequence cut

Sites notcut

References

CjrlOIC&13IClal

CpelCspl

CtvlCviAI£viBICvUICviPICviQIDdel

DralDraH

Eael

Eagi

RCCGGYGGNCCATCGAT

TGATCACGGWCCG

TTCGAAGATCGATCGANTCRGCYCCGTACCTNAG

CGGWO^CG

77?

Dpnl Gm6ATCb Gm6ATC

EealEciXI

Eco47IEco47mEcoAEcoB

EcoE

EcoQlQ9lEcoPIEcoP15

GATCTTTAAARGGNCCYYGGCCR

CGGCCG

GAAGAG

GGTANÂCCCGGCCG

GGWCCAGCGCTGAGN7GTCA b

TGANgTGCT b

TCAN7AATC b

GAGN7ATGCAACN6GTGC b

RGGNCCYAGACCb

CAGCAGb

TTTA6AA?

m6AGCGCT

ntfATCGATAT^CGATATCGÂT*TGÂTCA

n^CGGWCCGTTCGm6AAGm6ATC#C^^TCGÂNTC*RG^CY*

111115 CTNAGGATCG A T m 4 C

GAT^CGIH6ATC*

RGGNO^CYYGGm5CCR#

CGGm5CCGm5CGGCm5CGGm6AAGAGGAAGm6AG

GGTAm6ACC#

CGGm5CCGGGWO^CAG^CGCT

TGm6AN8mTGCT # b

TCAN7m6AAmTC#b

Gm6AGN7ATGC

AGAhm5Chm5CRGGNC^CYAGÂCC*

BiSJQl

WolMc3Fil,Ro3Mcll

Ne4,ScllRi2

Xi3Sh3,Xi2Xi4

Ho3,Ne2HulLa3,Mcll,VolNe4Ne5

NelSc8Ja2,Whl

Mcll

Ne4

NelBr2Qi3

Ja5Nel,Ne4Bi2,Co6J7u2Bi2J Jal0âllPilCo6,Fu2Bi23i3,KalSc8Bal,Ba23a2,Re4Hu2



RestrictiorenzymeEcoRI

EcoRII

EcoRV

EcoRi24

EC0Rl24/3

EhelEssHFnu4HI

FnuDn

EnuEIFokl

Fsel

FsplXJo/*TTxiclw 11

Haem

HapnHgal

HgiAIHgiCIHelenHgiEIHgUnHhaj

i Recognitionsequence

GAATTC

CCWGG

GATATC

I » £\ LX ^U ^ | c 1 I 1 T

GAAN7RTCG b

GGCGCCGCTNAGCGCNGC

CGCG

GATCCATCC

GGCCGGCC

TGCGCARGCGCY b

GGCC

CCGGGACGC

GRGCYCGGYRCCGGWCCGGWCCGGYRCCGCGC

Sitescut

G A A T T hm5c

m5CCWGG b

GATAT^C b

7

7

7

GCTNAGm5C7

7

Gm6ATC b

CATmSCCCATCm5C b

CATCm4C7

7

7

GGCm5C

7

7

GRGCY^C7

7

7

7

7

Sites notcut

Gm6AATTC b

G A m 6 A T T C #

G A A Tpn5c b

n^CCWGGO^CWGGOn5CWGG#

CC^^AGGbmSQhmScwGGG m 6 A T A T c #

G ATm6ATCG A m6 A N 6 RTC G

GAAN6RmTCGm6 A

GGCGCCG^CTNAGCGm5CNGC

m5CGCG

7

GGm6ATG

GGm5CCGGm5CCGGCm5CGGCCGGm5CCGGCCTGm5CGCARGm5CGCYRGhm5CGhm5CYGGm5QC* b

GG h m 5 C h m 5 Cf IHJI T vl v~

GACG^CGRGm5CYCGGYRO71^GGWC^CGGWC^CGGYRCm5CG^CGC*

References

Mcll,Ne2,RulBrl,Br8JDulHulJCa3,TalKu3,YolBu4^Na5JRo3Bo5,McllBu3Hul,Ka3McllJSTe2,WolHIPr2,Pr3BilPrl,Pr2

Co2Ne4Ko3,Tr2

Gal,Ga2,Ne2,Ne6,St6

Lul,Ne2Po3,Po4,Sc2

NelNe7

Ne4Eh2,Gr4TKa2,Ko3,Mcll,Pi5HulBa3,Ka2,Ko3,Ma5HulEh2,WalNelMcllFol,Ne2,Wh3ErlErlErlWh3Eh2,Ko3,Sml

G h m 5 CG h m 5 CMcll,Hul



Restriction RecognitionenzymeHhallHincn

HiadnHinfl

Hindffl

HinPIHeal

HpaH

HphI

Kpnl

Kr>n2I

Kspl

MaellMamIMbol

MboII

MfU

MiniMlu9273lMJu.927311

MmellMnll

sequenceGANTCGTYRAC

GTYRACGANTC

AAGCTT

GCGCGTTAAC

CCGG

TCACC

GGTACC b

TCCGGA

CCGCGG

ACGTGATN4ATCG A T C b

GAAGA

RGATCY b

ACGCGTTCGCGAGCCGGC

GATCC C T C b

Sitescut

?GTYRA^C

?G A N T m 5 c b

7

7

GTTAA^C

?

TCAC™5C

GGTAm5CCGGTAC^C

TCCGGm6A

7

7

7

G A T m 4 C

GAT^C b

Tm5CTTm5Cb

GÂAGA?

m6ACGCGT7

??

Sites notcut

GÂNTC*GTYRÂCGTYRA^CGTYRm6AC#GÂNTCG A N T hm5cnÂAGCTT*AAG^CTTA A f^ nm ^t '^ 1 * 1 *

G^CGCG T T Am6Ac#G T T A A hm5cn^CCGG

C^4CG^ b

Sc^CGGT^CACC*GGTGm6AGGTm6Am5cCGGTACm4C

T^CCGGATCm5CGGA"^CCGCGGO^CGCGGAm5cGT b

Gm6ATN4m6ATC

(3m6Axc#GAThm5cGAACHÂ*

RGm6ATCYRGATm4CYRGAT^CYA^CGCGTT^CGCGAGm5CCGGCGCm5CGGCGm6ATCn^CCTC

References

Ma5Gr4ô7HulRo7Chl,Col,Ne2JPelHulBr8,Gr4,Ro7Ne2

Mcll,Ne6Br8,Gr4JIul,Yo3HulBe33u63h2,Ma5Ko3,Qul,Wa5

HulFol,Mcll,Ne2

Eh3,Mcll,Ne2Nel

MclJSTelNelNelQi2Mo2St4Br5,Gel,Mc8Hul,Ro3Ba3,Mcll>lcl2ê2

Onl

Mcll,Shl,St5,Qi3NelNel

Bo4Eh3>Icll



Restriction RecognitionenzvmeMphJEMrol

MsdMspl

MstnMval

MvnlNael

Nanll

Narl

Neil

Ncol

NcrlNculNdelNdenNgoBINgoPINgoPn

NhclMainNmuDINmuElNotI

Mrui

Nsil

seauenceCCWGGb

TCCGGA

TTAACCGGb

CCTNAGGCCWGG

CGCGGCCGGCb

Gm6ATC b

GGCGCC

CCSGG

CCATGG

AGATCTGAAGACATATGGATCTCACCRGCGCYGGCC

GCTAGCCATGGm6ATC b

Gm6ATC b

GCGGCCGC

TCGCGA

ATGCAT

Sitescut

?TCCGGm6A

TFÂAm4CCGGCm4CGGCm5CGGn^CCTNAGGO ^ C W G G 5

m5CCWGG

77

Gm6ATCGmeATn^C b

GGCGCm5C

m5CCSGG

Ccm6ATGG

AGm6ATCT b

GAAGm6A"^CATATG b

GAT^c b

7??

7?Gm6ATCGm6ATCGCGGCCGm5C

TCG^CGA

?

Sites notcut

Cm5CWGGT^CCGGATO^CGGA

m5GCGG#

hm5chnx5CGG

?O^CWGG*C O n ^ G G b

m 4CCWGG b

m 5Cm 5CWGGb

n»5CGCGGm5CCGGCGCm5CGGCGCCGGm5CGATCG A T ^ CGGm5CGCCGGCGCm4CCm4CSGGCm5CSGG b

n^CCATGG b

m5CCATGG??m6AGm6ATCT^CACCRGm5CGCY

GGCm 5Cb

GCTAGm5CCm6ATG#

GATCGATCGCGG^CCGCGCGGOnSCGCT^CGCGATCGCGm6AATGOÂTATG^CAT

References

Ro3Mcl^felNelNelEh2Je2,Va3,Wal,Wa5Bu6,Hul

McllBu4,KulGr3,Ku3

NelNelEh3,Kll,Mcll,Ne5

Pal,Ne5

Ko3,Mcll,Ne5NelBr8,Ko3,Mcll

Kllê2,Ne4

OilMcl3Be4>lcllMc9Pi3JPi4Ko3,Ko5Ko3,Ko5Su3,Su4Kll,Mcll,Ne2LalMo3PalPalMcllSt5,Qi2Nel,Qi3Ne2Be5Wol



Restriction RecognitionenzvmeNspBIIEflMI

PfalPfulPaeR7I

EmUPpuAIp s t i

Pvul

Pvun

Rlh4273IRsalRshIRspXI

RsrI

Rsrll

SacISacHSail

SalDiSau3Ai

Sau96l

S12Q13ISealSciFI

SfaNiSfil

sequenceCMGCKGCCAN5TGG

GATCCGTACGCTCGAG

CACGTGCGTACGCTGCAG

CGATCG b

CAGCTG

GTCGACGTACb

CGATCGTCATGA

GAATTC

CGGWCCG

GAGCTCCCGCGGGTCGAC

TCGCGAGATC b

GGNCC

TCGCGAAGTACTCCNGG

GATGCGGCCNsGGCC

Sitescut

Cm5CGCKG?

GÂTC??

??7

CGm6ATCG

7

?G T Am5c b

CGm6ATCG7

?

7

Gm6AGCTC7GTCGA^C

TCGCGm6AGm6ATC

?

TCGCGm6AAGTAm5cTm5CCNGG

GATG^CGG^CCNsGG1115!

Sites notcut

7

O^CANsTGGO^CANsTGG7CGTAm5CGCTCGÂG*CTm5CGAGCA^CGTGCGTAm5CGn^CTGCAGCTGCm6AG#

CGATm4CGCXjATm5CGCAG^CTG*CAG^CTGGTCGm6ACGTm6Am5c?TOÂTGATCATG"1^Gm6AATTCGAm6ATTC# b

n^CGGWCCGCGGWm5CCGCGGWCm5CGGAG^CTCmSCCGCGG

GTm5cGACGTCGÂC^T^CGCGAG A Tm5c#bG A 'pn4G

GArj 'hm5£

GGNm5CC#

GGNCm5C

T^CGCGA?Cm5CNGGC^CNGGGm6ATGC

CC b GGC^CNsGGCC

References

NelNelSt7Ro3NelGi3GhlFolNelDol,Gr4,Mcll,Ne2

Br8,Bu3,Eh3

Br8,Bu5JDolEh3,Ja3,RolBa6Eh3,Ne4,Ne5LylPa2Ne4McllBa5Mcll,Qi3

McllKll,Ne2Br8,Eh2,Lu2,QilMc3,Ro4,Ro5,Va4Mcl3^fel,Qi3Drl,Eh2fJa3,Mc3,Ro3,SelNe5HulKo3,Ne2J>el

HulMcllJSfelWolMcll,Ne2NelMel 1^04

! Mcll,Qi2

Sfll CTGCAGGGCCNsGGC^C? CTGCm6AG Br8



Restriction RecognitionenzymeSffiAISjnJSmal

SnaBISnolSpel

Spkl

SpilSfioJ

Sson

Sso47ISsplSstI

Smi

SttSBI (S&SPITaqI

Tagil

laaxi

IfHIfUThai

UhJiBIXbal

sequenceCRCCGGYGGGWCCCCCGGG

TACGTAGTGCACACTAGT

GCATGC

CGTACGTCGCGA

CCNGG

GAATTCAATATTGAGCTC

AGGCCT

JAGNARTAYG b

AACN6GTRC b

TCGA

GACCGACACCCACCWGG

GAWTCTCGACGCG

TCGATCTAGA

Sitescut

?7

Cm5CCGGG

?77

GCATG^CahmSc A TY"ihnU ULA1 \j

CCPÂCGTCGCGm6A

7

7m6AATATT7

7

7

7

Tn^CGA b

Thm5CGA b

7

m5CCWGGC^CWGGGAWTm5C?m5CGCG

T^CGA7

Sites notcut

CRO^CGGYGGGWm5CC#

n^CCCGGG"^CCCGGG b

Cm4CCGGG b

CCm4CGGGCCm5CGGG b

TA^CGTAGTGm5CAm5C"ÂCTAGTAm5CTAGT

7

T^CGCGATCG^CGACm5CNGGm5CCNGGGm6AATTC#

7

GAG^CTCGAGhm5CThm5CAGGm5CCTAGGC^CTAGGO^CT

Gm6AGN6RmTAYG# b

Am6ACN6GmTRC# b

TCGm6A#

Gm6ACCGA

7

7

TCGm6Am5CGCGhm5cGhm5CGTCGm6A#

TCTAGÂ*T^CTAGA

References

Ta3Ka5,Ka6Br8,Bu6,Eh2,Ga4Ja3,Ka7,Mc3,Qul

FolHo2,WolHoiWolMcll,Ne2,Mo3

Ne4,Qi3Nel,Ne4

VilGr3Ni4NelBr8,RolHulCa4,McllSo3NelNal,Na2Nal,Na2Gr4,Hul,Mc3,Va3HulNe4

Grl

FolSa3,Va6Gal,NelHulSa3Mcl3,WelGr4,Hul,Ne2



Restriction Recognition Sites Sites not Referencesenzyme sequence cut cuj

CTCGAGb ?

RGATCY ROÂTCYCCCGGG nrm5r>nnr, b

Xmani CGGCCG ?XmnT GAAN4TTC GAm6AN4lTC

CGATCG CGm6ATCG

CT^CGAGCTCGm6AG"^CTCGAGRGAT^CY b

n^CCCGGGntfCCCGGGCm4CCGGGCCm4CGGGCGGm5CCGGm6AAN41TCGAAN4TTm5C b

CGATm5CGhm5r;GAT'un5CG

Br8JEh2^h3,Ka7Mc3,Va3

Br8Bu6,Yo5,Yo6

Ne2,Tr2McllJSTe2

Br8JEh2Hul

FOOTNOTESa. # denotes canonical modification MTasc specificity. M= A or C, K= G or T, N= A,C,G, or T, R= A or G, Y= C or T, W= A or T, S= G or C, D =A.GorT, H = A.CorT. Sequences are in 5'-3' order. m4C= N4-methylcytosine; m 3C= C5-methylcytosine; hm5C=hydroxymethylcytosine; mC= methylcytosine,N4 or C5-methylcytosine unspecified; m 6A= N6-methyladerune. Nomenclature is according to (Sm2) and (Co4).b. Accl nicking occurs slowly in the unmethylated strand of the hemi-methylated sequence GTMKA^C.Afll cuts slowly at GGWC^C.AhaU (GRCGYC) will cut GRCGCC Joner if these sites are methylated at GRCG^CC (Ne5), but wUl not cut GRCGY^C sites (Ne2,Ne5).Aip718I cuts M CviQI -modified ( G T ^ A Q ChlortUa virus NY2A DNA. Asp718I does not cut GGTAC^CWGG overlapping dan sites (Mul) or "^C-substituted

phage XP12 DNA, whereas Kpnl cuts XP12 readily (Ne4).Aval nicking occurs slowly in the unmethylated strand of the hemi-methylated sequence CTCCÂG/CTCGAG (Ne5).^vtiH cuts slowly at GGWC^C.Bacillus species have been surveyed for GÂTC and C^CWGG specific methylases. Many species have GÂTC specific methylases but none had C^CWGG

specific methylases (Di3).Ball sites overlapping dan sites (TGGC^CAGG) are 50-fold slower than unmethylated sites (Gil).Banl gives various rate effects when its recognition sequence is "^C- or '"'C-methylated at different positions.Bgll cleavage rate at certain GC^CNjGGC, GCm4CNJGGC, and GCCNjGG^C hemi-methylated sites is extremely slow. However, "^C bi-methylated

M HaeHl-Bgtl sites are completely refractory to Bgll (Ko3,Ne2).BssHU does not cut M /flial-modified DNA, in which two different cytosine positions are hemi-methvlated, G^CGCGC/GCG^CGC (Ne4).Mflwl modifies the internal cytosine GGAT^CC, but it is not known whether this modification is ""C or "^C (Le2).BstEU cuts the fully m5C-substituted phage XP12 DNA (Ne5).BstNl cuts C^CWGG, "^CCWGG and '"'C'^CWGG (Ne5). finNl isoschizomers that are insensitive to C^CWGG include Aori, Apyl, flspNI, Mval and TaqXl

(Mc4).BsuRl nicking occurs in the unmethylated strand of the hemi-methylated sequence GG^CC/GGCC.Cfr9l, see reference Bu6 for rate effects.MCrel is from the unicellular eukaryote Chiamydomonas reinhardi (Sa2).Dpnl requires adenine methylation on both DNA strands. Isoschizomers of Dpnl include CJul, Nanll, NmuEl, NmuDl and NsuDl (Cal). Dpnl cuts dam modified

XP12 DNA (Ne6)-MEco dam modifies GAT^C at a reduced rate (Ne5). Many other bacteria that modify their DNA at GÂTC are listed in references Bal and Lol.£coA, £coB, EcoD, EcoDXXl, EcoK are Type 1 restriction endonucleases. "T represents a 6-methyladenine in the complementary strand.EcoPl is a Type ni restriction endonuclease (Ba2,Bal,Ha2).£coP15 is a Type ID restriction endonuclease (Hu2).EcoRl cannot cut hemi-methylated GÂATTC/GAATTC sites. Bimethylated GAtD6ATTC/GAm6ATTC sites are not cut by EcoRl or Rsri (Ne5). EcoRl shows

a reduced rate of cleavage at hemi-methylated GAATT^C (Trl) arid does not cut an oligonucleotide that contains GAATT^C in both strands (Brl).£coRL isoschizomers that are sensitive to C^CWGG include Aruftl, AtuU, BstGU, BinSl, Cfiil, CfiV I, £cfll, EcaU, Ecolll, EcolSl and Mphl (Ro3). EcoWA

shows reduced rate of cleavage at hemi-methylated "^CCWGG/CCWGG sites (Yol).EcoRV cuts the fully "^C-substituted phage XP12 DNA (Ne5).£coR124 and £coR124/3 are Type I restriction endonucleases. T represents a 6-methyladenine in the complementary strand,is a Type I restriction endonuclease.Fokl cuts about two-fold to four-fold more slowly at CATC^C than at unmodified sites (Ne5).MFOJU in ref Po3 corresponds to MFoklA in ref Po4.HaeU show a reduction in rate of cleavage when its recognition sequence is modified at RGCff^CY.HaeUl nicking occurs in the unmethylated strand of the hemi-methylated sequence GG^CC/GGCC.Hinfl cuts GANT^C, however, detectable rate differences are observed between unmethylated, hemi-methylated (GANT^C/GANTC) and bi-methylated

(GANTm5C/GANTm3C) target sequences. Hinfl does cut phage XP12 DNA, although at a reduced rate (Gr4,Ne5). tfinfl cuts unmethylated GANTC faster thanhemi-methylated GANT^C/GANTC, which is cut faster than GANTm3C/GANTmiC. However, the rate difference between unmethylated and fully methylatedHinfl sites is only about ten-fold (Hul,Ne5,Pel).

HpaU nicking in the unmethylated strand of the hemi-methylated sequence "^CCGG/CCGG is in dispute (Be3,Bu6,Ko3). HpaD cuts hemimethylated mCCGG 50times slower and fully methylated mCCGG 3000 times slower than unmethylated DNA (Ko3). See reference (Bu6) for HpaU rate effects.



Kpnl sensitivity to hemi-methylated GGTA^CC and GGTAC^C sites has been reported. Kpnl efficiently cuts m5C-substituted phage XP12 DNA, but not ChloreUavims NY2A DNA, which carries both GTÂC and "^CC modifications (Ne4).

MaeU nicks slowly in the unmethylated strand of hemi-methylated A^CGT/ACGT (Mo2).Mbol isoschizomers that are sensitive to GÂTC include BssGU, BsaPl, Esp74I, flsp76I, fisplO5I, BslXH, &rEm,fiisGII, Cpal, Ctyl, CviAl, CviBU, CvOU, DpnU,

FnuAll, FnuCl, HacI, Meul, MkrAI, MmeH, MnoHl, Mos\, Msp67n, MthI, MthAI, NdeU, NflAQ, NflBl, Nfll, NlaDl Mall, NmeCl, Nph\, NsiM, NspM,Nsul, Pfal, Rlull, SalM, So/Hi, Sau61X2l, SinMl, TruU (Ro3).

MboU cuts the fully "^C-substituted phage XP12 DNA (Ne5), although certain hemi-methylated ra5C<ontaining substrates are reported not to be cut (Gr4).Mfll cuts slowly at m6AGATCY sites (Onl).Mammalian methylase is the '"'CG methyltransferase from Mus musculus. (mouse) (Be6).Mspl cuts the hemi-methylflted sequence C^CGG/CCGG (Wa5) and C^CGG/CCGG duplexes (Bu6). Mspl cuts very slowly at GGC^CGG (Bu2,Kel). An M Mspl

clone methylatcs "^CCGG (Wa5,Wa2). However, there is a report that Moraxella sp. chromosomal DNA is methylated at "^C^CGG (Je2).Mval nicking occurs in the unmethylated strand of the hemi-methylated sequence m4CCWGG/CCWGG and CCÂGG/CCTGG (Ku3). Mval cuts XP12 DNA very

slowly at "^C^CWGG.MuiII requires adenine methylation on both DNA strands (Cal). NanU cuts M Eco dam modified XP12 DNA fNe5).Neil may cut m5Cm5CGG methylated DNA (Br8 Je2). Possibly the second methylation negates the effect of C^CGG.Ncol is blocked by MSecl (CCNNGG) (Ne5).Ncrt is a BglH isoschizomcr from Nocardia carnia Beijing (Qil).Nde\ cuts the fully "'C-substituted phage XP12 DNA (Ne5).Nden cuts the fully m5C-substituted phage XP12 DNA (Ne5).Ngo. There is some confusion about naming restriction enzymes from these strains. NgoPII, NgoII and NgoSI may be the same. NgoPIII may be NgoIH.NgoPH does not cut overlapping dem sites (Su4).MnuDI requires adenine methylation on both DNA strands (Cal).MnuEI requires adenine methylation on both DNA strands (Cal).PaeRl cuts hemimethylated CT^CGAG/CTCGAG sites 100 fold slower and cuts fully methylated CTmiCGAG/CTmiCGAG 2900 fold slower than unmethylated

sites (Ghl). Hemi- or full methylation at "Â completely protects against PaeR7 cleavage (Ghl).Rsal cuts the fully ""^-substituted phage XP12 DNA (Ne5), but does not cut Chlorella virus NY2A DNA, which is modified at GTÂC (Ne4,Xil). DNA from

Rhodopscudomonas sphaeroides species Kaplan is cut by Asp718I, but not by Rsal or Kpnl (Ne4). It is likely that M Rsal specifies GTA^C; and high levelsof '"'C are present in R. sphaeroides DNA (Eh3).

Rsrl cannot cut hemi-methylated GÂATTC/GAATTC sites.Sau3Al nicking occurs in the unmethylated strand of the hemi-methylated sequence GAT^C/GATC (St3). Sau3M cuts at a reduced rate at '"'AGATC (Onl). Sau3M

lsoschizomers that are insensitive to Gm6ATC include BcelMl, flsp49I, flsp51I, flr/j52I,fisp54I, &p57I, fisp58I, fisp59I,fi!p60I, flsp61I, fisp64I, fiip65I, fl^66I,Bsp61l, Bsp72l, fispAI, flsp91I, fiyrPU, Cpfl, Csp5l, Cpel, FnuEl, MspBl, SauCl, SauDl, SauEl, SauPl, SauGl and SauMl (Ro3).

Sfil cannot cut M B«/I-modified DNA (Nel).Smal nicking occurs in the unmethylated strand of the hemi-methylated sequence CC^CGGG/CCCGGG (Bu6,Wa5). Sma\ may cut C^C^CGGG methylated DNA

(Br8 Je2) Possibly the second methylation negates the effect of CC^CGGG. There are conflicting results regarding Smal: "^CCCGGG is not cut when modifiedby MAqul methyltransferase (Ka7) or at overlapping M HaeW-Smal sites (GG^CCCGGG, Ne5). Other investigators have reported that Smal cuts at a reducedrate at hemi-methylated "^CCCGGG sites (Bu6).

Spa cuts GTÂC-modified ChloreUa virus NY2A DNA, but does not cut tf/wl-digested XP12 DNA (Ne4)SfySBI and SfvSPI are Type I restriction endonucleases. "T represents a 6-methyladenine in the complementary strand.Taql cuts very slowly at T ^ C G A (Hul). Taal cuts the fully "^C substituted phage XP12 DNA (Hul,Ne5).M Taql methylates T^'CGA at least 20 fold slower that unmodified TCGA (Mc7).Xbal will cut T^CTAGA/TCTAGA hemi-methylated DNA at high enzyme levels (> 100U Xba I/ug), but will not cut this sequence in twenty to forty-fold

overdigestions.XhoU nicking occurs slowly in the unmethylated strand of the hemi-methylated sequence RGAT^CY/RGATCY.Xmal is claimed not cut CC^CGGG in one report (Br8). See reference Bu6 for rate effects.Xmnl cuts the fully "^C substituted phage XP12 DNA (Ne5). Xmnl cuts slowly at some sites in DNA methylated on both strands at GAAN/TT^C (Ne5).



TABLE II: DNA methyltransferases and their modification specificitiesQoned methylases in bold.

Methvlase a

MAatnM'AccIM-AflllM-AjaK21MAluIMAlw26I

MApalM-AaulM-AselMAsellM-AvalM-AialiM-AliIMRallBacillusMBamHIMBamHIIM-BanIMBanllMBbvIMBbvSIMBbvMBbvMficjilMBepIMBgllM.BJB£216I

MBnalMfififtRIM.BsplO6lM.Bsp6l

MBsiVIM-fisJLYIMBsuBIMBsuEIM-fisjiFIMBsuMIM-Bsuq>3T

Specificity a

GACGTCGTMKm6ACCTTAAG (m6A)GAT^CAG^CTGT^CTCandGm6AGACGGGm5CCC"rfCYCGRGATTAATCCSGGCYCGRGGGWCCCYCGRGTGGm5CCAGm6ATC b

GGATm4CCGmCWGC?GGYRCCGRGCYCGm5CWGCGmCWGCGm6ATAm6AGCm4CSGGm5CGCGGCCN5GGC (m4C)GGWC^CGGATmCCGGm5CCATCGm^TGCNCGGGATmCCCTCGm6AGRGAI^CYCTGCm^AGm5CGCGm5CCGGYT^CGARGG^CCand Gm5CNGC

ReferencesLu2Lu2Lu2SllKr2,Lu2Bu4Bu4Mc8,Tr2Ka7,Ka8Mo3Mo3Lu2Lu2Lu3Lu2>Ic8Di3Hal,Lu2,Na3HalLu2Lu2DolJial,Va5Hal,Va5HalHalJa4,Ja6Ja7,Pe2JJo6Ku2Lu2Ma9KilFe2^Co 1 ,Po2,Qi3,S z4, Ve 1Pa2Ja3U2Ba7Va2XulGal,Gu7,Ikl,JelGu7JkUel,Wa7Gul,Gu2,Gu7,Jel,ShlBel,Gu5,Gu4,No2^o3Nol.Trl



Methvlase a

M-BsupllI

M-Bsuplls

MBsuQIMBsyRIM-BsuSPP

M-BsuSPRI

M-fifiiiSPR191

MBsuSPR83I

MCfrAMCfrlM-£fr,6IMCfr9IMCfrlOIMCfrl 31MCJalMCrelMCtvIMCviBIM£viBIIIM-CviJIM£viPIMCviQIMCviRIMCviRHMDdelMDpnIIM-DpnAM-EagiM-EagIMEcalM-Eco damM*Eco dcmlMEco HcmnM*Eco dcminM-EcodcmlVM-EcoAM-EcoBM-EcoD

Specificity a

GGm5CCand Gm5CNGCGGCCandGDGCHCmCCGGGGm5CC b

GG^CCand Gm5CNGCGG^CCand m5Cm5CGGand Cm5CWGGm5cm5CGGand OnCWGGGG^CCand (Jn5CWGGGCAN8GTGGYGG^CCRCAGm4CTGCm4CCGGGRm5CCGGYGGNm5CCATCGm6ATXm5CRGm6ATC#Gm6ANTCTCGm6ARGm5CYm 5CCGTm6ACTGCm6AG Tm6Acm5CTNAGGm6ATC?Gm6ATC?YGG^CCRCGGCCGG G T m 6 A C C

Gm6ATCCm5CWGGRmCCGGmCCWGGGGWC^CGm6AGN7GmTCA b

TGm6AN8mTGCT b

TTANTGTCY b

ReferencesGu4,Gu5,Gu7,Nol ,No2

Bel

Je2Gu6,Ki2^Ki3Gu4,Gu5,Gu7rTe2^Ki2,Nl 6No2,Trl,Tr3Bel,Gu5,Gu7^fo2PolBe23ul , Gu3,Gu5,Ki2,PolJe2,No2,Pol

Gu3

Da2JDa3P06Bu5103^06P06Bi5Mc3Sa2 (Chlamydomonas)

Ri2X2Na4Sh3Xi4Xi2^G5StlStlHo3J.u2.Sz3Del Jî3 Jâ4 Jâ5>Ia6DelJal.WhlSz2Br2Br63u9 JDrl ,Gi2,Ha2 JIe2,UrlBo5,MalO,So2,UrlBu8,Ne8Ni2

0)7^112Go3Go3



Methvlase a

M-EfiftDXXIM-EcoEMEcoKM-EcoPIM-Eco PI damMEcoPISMEcoRI

MEcoRII

M'EcoRVM'EcoR124M-ECQR124/3

M-EcoTl damM-EcoT2 damM-EcoT4 damMEco31I

M-Eco47IIM-Eco51IM*Eco57IMEco64IM.Eco72IM'Eco98IM -£€01051M»Esp3I

M-FniiPIM-FnuDIIMFnuDIIIMFokl

M-FsplMFV3MHaenM-HaeIIIMHapHMHyalMHgiAIMHgiCIMHgiCIIMHgiEIMHhalM*lilia.IIM-HincII

Specificity a

TCAN7ATTC bGAGN7ATGC bAm6ACN6GmTGC b

AGm6ACC b

Gm6ATC bQn^GCAGGA^ATTC

Cm5CWGG

Gm6ATATCGAAN6RTCG (^6A)GAAN7RTCG (ntA)Gm6ATCGm6ATGm6ATCGGTm5CTCandGm6AGACCGGNCCCTGAAG (m6A)CTGAAG (™6A)GGYRCCCACGTG (m 5QAAGCTTTACGTAGGTm6CTCGAGm6ACCGGCC (m5C)m5CGCGGCGCGGm6ATGand Cm6ATCCTGCGCA??mC??RGCGCYGGm5CC b

CmCGGGACGC (mC)GWGCWCGGYRCm5CGGWO^^CGGWC^^CGm5CGCGm6ANTCGTYRm6AC

ReferencesSkiFu2Bo2,Go3,Kal ,Lo2,SalBa2JIu2Co5Hu2Dul,Gr2JCe2Ne2,Ne9,RulBhl,Bu7,Bu8,Ko6^Ko7^KoMalb,Sc6,Sol,Yc4Bo3Pr2JPr3Pr2,Pr3Scl,Sc7Br7,Ha2 JIa3,Mi 1 ,Sc4,Sc5Ha4,Mal,Mi2,Sc3,Sc4,Sc5Bu4Bu4P06P06P06P06P06P06P06Ja3

Lu2,ValLu2,NelLu2La6 J^o3,Lu2,Ma8,Nw 1

MelEsl (Frog virus)Lu2,S13Lu2,Ma5,S13WaiLu2,NwlLu2ErlErlErlBa9,Ca3,Lu2,Sml,Wul,ZalChlJCel,Ma3,Ma4,Sc9,SmlGr4J4c8.RO7 Re2



Methvlase a

MHindllMHindinMHinflM-HinPIMHjalMHpalMHpallMHphIM-H2

MKpn2IMMbolM-MboIIMflrnrna'<J

M-MSDI

MMstIM'MvalM-MwolM-NaelNeurosporaMNcolMNdelM-NgoMVIMNgolM.NgoPIMNgoIIM-NpoAIM.NyoPHM'NgoinM'NgoIVMNgoVM-NgoVIM-Npo VIIM-NgoBIM-NgoBIIMNlalM-NlainM-NlaIVM-NlaVMNlaXM-PaeR7IMPstIMPvuIIM-Rjrti4273I

Specificity a

GTYRm6ACm6AAGCTTGm6ANTCGCGCGATATC (^A)GTTAm6ACOnSCGGT^CACCGGCCGCNGCGDGCHCTCCGGAGm6ATCGAAGm6Am 5 CG b

m 5CCGGb

TGCGCAC^CWGGGCN7GC (n^C)GCCGGC??m5C??CCATGG (mC)CATATG (m6A)GGNNCCb

RGCGCY b

RGmCGCY b

GGm5CC b

GGm5CC b

GGm5CC b

CCGCGG b

Gm5CCGGC b

GGNNm5CC b

Gm6ATC b

GmCWGC b

T^CACCbGTANs^CTC b

GGCCO^TGGm5CCGGCGGNfN^CC??mC??CTCGm6AGCTGC^^GCAG^CTGG T C G r a 6 A C

ReferencesLu2Jle2,Ro6Jlo7Lu2Jlo6Jlo7Chl,Lu2Ba9JLu2DalBr8,Yo3Lu2>Ia5,Qul,Wi2,Yo2Mc8,Ne2,Ne4La8 (Bacillus phage)

P06Mc8

Be6 (Mouse)Eh2 Je2 J>u2,Nw2,Wal ,Wa5MelK13J»o6Lu2J.u4Lu2,ValSe2Lu2,ValSilPi5RilSu2Ko5,RilPi3Su3,Su4Ko5,RilCh2,Kol5,RilKo5,Pi2Ko5Ko5Pi3Pi3Mo3LalJ.u2>Io3Lu2Mo3LaiGi3,Thl,Th2Lel,Wa3,Wa4,Wa6Bll,Ta2Ba6



Methvlase a

MRsrlMSacIIMSallM-Sau3AMSau96IM-SfilMSjalM-SmalMSphIMSso47IMSso47IIMSspMOIM-Sj^IMStvSBIMStvSPIM-StvSOMStvS.IMTaa lMTthHBIM-lflJTetrahymenaM X h a lM-2CmaiM-XmamM-Xmtf

Specificity a

GA^ATTCCCGCGGGTCGm6ACGAT^CGGNm5CCGGCCN5GGCC (m4C)GGW^CCCOCGGGGCATGCGm6AATTCf 1111 f^l ft T

m5cGCCWWGGGm6AGN6RmTYG b

Am6ACN6GmTRC b

Am6ACN6RmTAYG b

Gm6AGN6GmTRC b

TCGm6ATCGm6ATCGm6A??m6A??TCTAGm6ACCCGGG (m4C)

GAAN4TTC

ReferencesBa5JCalOLu2Lu2,Ro4^Ro5SelLu2^Srel,SzlBa8Ka5,Ka6He 1^06Lu2Ka9,Bu4Ka9JNil,Ni3Nul,Pi5,Re3RelFul,Fu3,Ga3,Nal,Na2Ful,Fu3,Nal,Na2Ful,Fu3Ga3Lu2>tc3,Sa3,S12Mc3,Sa3Sa3,Va6Ca2,GolLu2,Mcl3,ValBa8Mc8,Tr2Fel

NOTES a. See footnote "a" of Table I. b. See footnote "b" of Table I.



TABLE HI: Metbylation sensitivity of Type II DNA methyltransferases.

Methylase(specificity)a Not blocked by prior Blocked by priormodification at b mortification at b

References

MAluI (AGm5CT)MBamHI (GGAT™4CQ GGm6ATCC

GGm6ATCCM£fr6I (CAGm4CTG)MClal (ATCGm6AT)

AT^CGAT(TCGm6A)

M-EgoRI (GAm6ATTC)M-EcfiRH (Cm5CWGG)MEco dam (Gm6ATQ

GAATT^C

(GGm6ATG)c

M-Hhal (Gm5CGC)M-ISian (Gm6ANTC)MHpan (Cm5CGG)

G A T m 4 C

CATO^C

GGTGm6AM-MbQl (Gm6ATC)M-MboII (GAAGm6A)MMspI (mSCCGG)MMval (C^CWGG) Cm5CWGG

m5Cm5CWGGM-Pvun (CAGm4CTG)MEcoT2 iJam (Gm6ATY) GATh^CMEcoT4 dam (Gm6ATC)MTaal (TCGm6A)

AGm4CTGGATO^C

CAG^CTG

Gm6AATTC

m5CCGG

CAG^CTG

Bu5La7,MclOLe2Bu5M9,Mcll,Wel

MclO,Va4Br2Bu4MclO

Ne4Po3,Po4,Sc2RolMclOMc9,MclOMclOMclOMclOMclOBu4NelBu5Do2.MilSc4Mc7

a. See footnote "a" of Table Lb. An enzyme is classified as insensitive to methylation if it methylates the modified sequence at arate that is at least one tenth the rate at which it methylates the unmodified sequence. An enzyme isclassified as sensitive to methylation if it is inhibited at least twenty-fold by methylation relative tothe unmethylated sequence.c. See footnote "b" of Table L



TABLE IV: Isoschizomer/isomethylator pairs that differ in their sensitivityto sequence-specific methylation.

Methylated sequence c

m4CCGGO^CGGC^GGCC™5CGGGO^CWGGGm6ATCG A T ^ CG A T m 4 C

GGCra5CGGTAO^CGGTAm^Cm^CGGW^O^CRG^ATCY" P ^ C C X J G A

TO^CGGATCCGGm6ATCGCGm6ATpnSCGAACGGWOnScG

Methylated sequence c

T^CGA

Restriction isoschizomer pairs ^Cut bv Not cut bvMspl HpaHMspl Hpall (HapIDMspl HpaflXmaI(Cfi9D SmalBstNI (MvaD EfijRHSau3A (FnuED Mbol (NdelDMbol Sau3AMbol Sflll3AHagin NgoPIIKpnl Asp7i8IKpnl Asp7i8IAfll Avan (Eco47DXhon (SsiYI) MfllAccIII BJCMII (MroDAccm BspMII (MroDBspMJ (MroD AccmSbol3I (SalDD NmlAsuH Csp45ICspl Rsrn

Restriction isomethvlator pairs *e

methylated bv Not methvlated bv

M.CviBm (TCGm6A) Mlaal

ReferencesNelEh2,McllBu6Bu6Bu4Gel,Lul,Mc9,Ro3Ne4Ne4Su4MulNe4B3,Ja5,Wh2Mc9JSfe4La2,Sc2Sc2Ke3,Ne4Mcll,Ne4SclOQi3

References

We2

a. In each row the first column lists a methylated sequence, the second column lists anisoschizomer that cuts this sequence, and the third column lists an isoschizomer that does not cutthis sequence.b. An enzyme is classified as insensitive to methylation if it cuts the methylated sequence at a ratethat is at least one tenth the rate at which it cuts the unmethylated sequence. An enzyme is classifiedas sensitive to methylation if it is inhibited at least twenty-fold by methylation relative to theunmethylated sequence.c. See footnote "a" of Table I.d. In each row the first column lists a methylated sequence, the second column lists anisomethylator that modifies this sequence, and the third column lists an isomethylator that does notmodify this sequence.e. An enzyme is classified as insensitive to methylation if it modifies the methylated sequence at arate that is at least one tenth the rate at which it modifies the unmethylated sequence. An enzyme isclassified as sensitive to methylation if it is inhibited at least twenty-fold by methylation relative tothe unmethylated sequence.



TABLE V: List of restriction systems referred to in this paper.a

Note:a. Restriction systems in Table V are arranged by recognition sequence length and alphabetically byrecognition sequence

CCRGCYCATGCCTCAGCTCCGGCGCGGm6ATCGATC

GCGCGGCCGTACTCGATTAACCNGGCTNAGGANTCGGNCCCCWGG

CCSGGGCAGCGGWCCAGACCACCTGCCAGCAGCATCCGAAGAGAAGAGGAATGCGACCGAandCACCCAGACGCGATGCGGATCGTCTCTCACCGDGCHCCYCGRGGRCGYCGRCGYCGRGCYCGTMKACGTYRAC

to aid in identifying lsoschizomers.

CviNYCviJINlamMnllAlulBsuFL BsuOI. HapII. Hpall. MsplAccE, figgl, BsiUI, BsuEH. EnuDII, ThaiDpnI. NanIL NmuDI. NmuEI£££2431, BsaPL Bsp67I. BspAI. BspPH. BsrPEL £ss.GII, BstEIHBstXII, Cpal. Ctyl. CviAI, DpnIL FnuAH FnuCI. FnuEI. Mbol. Mmell.Mnom. Mosl. Mthl. Ndell. Nfll. Nlall. NsiAI. Nsul. Pfal. £au3A,SinMIHhaI,HinPIBsuRI. HaelH NgoPIIC_viQI, RsalTaql. IfU, TihlMselScrFIDdel£viBI, Hhall. HinflCfrl3I. Sau96IAacl. AorL Apvl. AtuBI. AtuII. BinSL BspNL fisjGn, £siNI, Ci[5I,CjfcH I, Ecan. EfilJI, E£O_RII, Ecc27I, E£Q38I, Mphl. Mval. laflXIBcnI. NeilBbvlAvail. Bme216I. Eco47I. HgiCII, HgiEI. SinIEcoPIBspMIECJ2P15FoklMboII. NculEarlBsmITaqfl

HgaiSfaNIAlwI. BinIBsmAIHphl. NgoBIBsp 12861Aqul. AvalAhallAosII. Bbill. BanllHgiJIIAccIHinCn



GWGCWCRCCGGYRGCGCYRGATCYYGGCCRRGGNCCYAAGCTTACGCGTACTAGTAGATCTAGCX3CTAGGCCTATCGATATGCATCAGCTGCATATGCCATGGCCCGGGCCGCGGCGATCGCGGCCGCGTACGCTCGAGCTGCAGCTTAAGGAATTCGACGTCGAGCTCGATATCGCATGCGCCGGCGCGCGCGCTAGCGGATCCGGCGCCGGTACCGGGCCCGTCGACGTGCACGTTAACTCATGATCCGGATCGCGATCGCGATCTAGATGATCATGCX}CATGGCCATTCGAATTTAAACCAN6TGGCCTNAGGGAAN4TTCGATN4ATCGCCN5GGC

HgiAIC&10Hacn. NgoPIBstYLMfllXhonCfrL EaelPralliiindmMMSpelBgUI. NcrlAviH Eco47m

Banffl. BspXL QalNsilCfr6I. PvuH

NcolCfr9L SmaL XmalSacnBmaDI. Pvul. Rshl. XorUEagL X m #Pful. SplIBsuMI, EsuRn, EasR7i, XhoiBsuBI. P51I, SfllMnEcoRI. RsrL Sso47IAatnSad. SstIEcoRVSphIMlu9273n. NaelBssHHNhelBamHI, BamFI. BamKI, BamNI. BstL Bstl. B511503IBbel. NarlAsp718I. KpnlApalRrii4273I, M IApaLJHpalBspHI. RspXIAccm. B§nMII, Kpn2I. MrolAmal. MluI9273INrul. SalDL Sbol3I. SpQlXbalAtuCI. Bell. BspXn. BstGL QsslFsplIanAsuH. BstBI. Csp45IPralPflMI.fistXIMstPXmnIMamIBgUDownloaded from https://academic.oup.com/nar/article-abstract/19/suppl/2045/1058107

by gueston 29 January 2018


GCTNAGCGGTNACCCGGWCCGGAAN6RTCGGAAN7RTCGAACN6GTGCAACN6GTRCGAGN7ATGCGAGN7GTCAGAGN6RTAYGTCANvATTCTGANgTGCTTTAN7GTCYCRGCGGYGGGCCGGCCGCGGCCGCGGCCN5GGCC

EsplBstEII. EcalCspl. RsrHEC2R124g£oR124/3EcoKSttSPIEcoEEcoAStySBIESQDXXIEcoBEcoDSsrAIFscINod


site-specific methylation: effect on dna modification

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