Site-Specific Methylation: Effect on DNA Modification Methyltransferases and Restriction Endonucleases

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

Site-specific methylation: effect on DNA modification methyltransferases 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 over sensitive to overlapping m5CG methylation at 240 restriction endonucleases to the site-specific DNA GGC"5CGN4GGCC sites in mammals and overlapping dcm modifications '14C, n-6C, h1"5C, and m6A, four modifications that methylation at GGC'5CWGGNNGGCC sequences in E. coli.. are common in DNA prokaryotes, eukaryotes, and their viruses (Mc2,Mc5,Mc8,Mcl 1 ,Ne3,Ne4). m4C and "n5C Cytosine modifications Table II is a list of over 130 characterized DNA In some cases, a restriction enzyme may differ with to sensitivity methyltransferases. A detailed list of cloned restriction- to m4C and m5C at a particular sequence. For example, BstNI modification genes has been made Wilson (Wi4). and MvaI cut I15C, but not m4C modified CCWGG sequences. Table m lists the sensitivities of over 20 Type II DNA KpnI cuts GGTACm5C but not GGTACm4C. BstYI cuts RG- methyltransferases to m4C, nSC, hm5C, and m6A modification. ATm5CY but not RGATm4CY. Restriction enzymes we have Most DNA methyltransferases are sensitive to non-canonical tested for sensitivity to m4C include: AatI, Afll, AlwI, AvaII, modifications within their recognition sequences BanI, BglI, BstI, BstNI, BstYI, DpnI, FokI, MboI, MvaI, NarI, (Bu5,MclO,Ne3,Po4), and this sensitivity may differ from that Ncil, PflMI, Sau3A, and ScrFI. of their restriction endonuclease partners. Finally, several restriction endonuclease isoschizomers are Rate of cleavage at methylated restriction sites known to differ in their ability to cleave DNA which has been m4C, m5C, hm5C, and m6A are bulky alkyl substitutions in the methylated. Table IV lists over 20 known isoschizomer pairs major groove of B-form DNA. It is therefore not surprising that and one isomethylator pair, along with the modified recognition site-specific DNA methylation can interfere with many sequence- sites at which they differ. specific DNA binding proteins (e.g. St2,Wa8) including binding of restriction endonucleases and DNA methyltransferases. DNA Effect of m5CG and m5CNG on restriction endonucleases methylation may cause long-range perturbations of DNA minor Enzymes that are not sensitive to site-specific methylation are and major grooves, and a range of rate effects are observed when particularly useful for achieving complete digestion of methylated modified substrates are used in restriction-modification reactions. DNA. For instance, endonucleases that are unaffected by mI5CG Results can be summarized as follows. and m5CNG are useful for digestion of plant DNA which is (1) Canonical site-specific methylation always inhibits DNA frequently methylated at these positions. Endonucleases that are cleavage by a restriction endonuclease. For example, M.BamHI unaffected by these two cytosine modifications include: Accll, methylase modifies GGATm4CC; and BamHI endonuclease AflH, AhaIH, AseI, Asp700I AsuH, BbuI, BclI, BspHI, BspNI, cannot cut this methylated sequence. BstElI, BstNI, CviQI, DpnI, DraI, EcoRV, HinCll, HpaI, KpnI, (2) In about one half of the cases tested, methylation at non- MboH, MseI, NdeI, NdeH, Pacd, RsaI, RspXI, SpeI, SphI, SspI, canonical sites inhibits the rate of duplex DNA cleavage at least Swal, TaqI, TthHBI and XmnI. ten-fold (Table I). However, in other cases non-canonical CpG sequences occur infrequently and are often methylated methylation has no effect on restriction cleavage. For example, in mammalian genomes (Mc9). Almost all the enzymes that could BamHI cuts DNA which has been modified at GGATCm4C or generate large fragments of mammalian DNA are blocked by GGATCm5C, but cannot cut DNA methylated at GGATm5CC. this m5CpG modification at overlapping sites, including AatII, (3) There are a few examples in which non-canonical ApeI, AvilI, BbeI, BmaDI, BssHll, BspMU, BstBI, ClaI, CspI, methylation slows the rate of cleavage or permits nicking of one Csp45I, EagI, EclXI, Eco47HI, FseI, FspI, Kpn2I MluI, strand of a hemi-methylated duplex. Examples of such rate effects Mlu9273I, Mlu9273H, MroI, NaeI, NarI, NotI, NruI, Pfid, PmlI, are presented in footnotes to Table I. PpuAI, PvuI, RsrH, Sall, SaIDI, Sbol3I, SfiI, SmaI, SnaBI, Spll, (4) Sometimes base modifications which lie outside a SpoI, XhoI and Xorfl (see Table I). recognition sequence can influence the rate of DNA cleavage by Only four enzymes suitable for pulsed field mapping of a restriction enzyme. For example, NarI does not cut at eukaryotic chromosomes are known to cut msCG-modified overlapping M.MvaI-NarI GGCGCCO4CCWGG sites (Nel); DNA: AcclI, AsuH, Cft9I and XinaI. It has been determined and HaeIII cannot cut certain GGCCmT sites, where mT are that SfiI is sensitive to n5C modification at the second cytosine modifed thymine residues (Wil). Such methylation-induced of its recognition sequence, GGCm5CN5GGCC. SfiI is therefore 'action at a distance' may be more common than has been

* To whom correspondence should be addressed 2046 Nucleic Acids Research, Vol. 19, Supplement previously appreciated. We have tested only a few enzymes for target sequences: mrr (m6A), mcrA (m5CG), mcr B (Rm5C) sensitivity to base modifications outside their canonical (Br5,Dil,He3,Ral,Ra2). In vivo or in vitro modified DNA is recognition sequences. inefficiently cloned into E. coli. For example, human DNA which is extensively methylated at "5CpG is restricted by mcrA (Wo2). Effect of site-specific methylation on DNA methyltransferases Appropriate non-restricting strains of E. coli (Go2,Krl, Ral,Ra2) Twenty-three Type II methyltransferases have been tested for should be chosen for efficient transformation and cloning of sensitivity to non-canonical DNA modifications, of which nine methylated DNA. Other species have such restriction systems were blocked (MclO and Table IE). As with restriction (e.g. Ma2). endonucleases, rate effects are sometimes seen with DNA methyltransferases at non-canonically modified sequences. For Engineered altered methylase specificities example, E. coli Dam methyltransferase is unaffected by G- Many methylase gene have now been sequenced. Extensive AT1mC, but methylates GATn'C relatively slowly. Such data is homologies between closely related enzymes (Wi3) or common summarized in Table II1 and footnotes to Table I. motifs (Po5,Sm3) allow new specificities to be developed (e.g. Ba4,Tr4). Methylase/endonuclease combinations can produce novel DNA cleavage specificities Data in electronic form Several strategies involving combinations of modification This paper is available as a text file on a 3.5' Macintosh diskette. methyltransferases and restriction endonucleases have been used The data can be supplied as a Microsoft Word, Macwrite or MS- to generate rare or novel DNA cleavage sites. DOS file. Please contact Michael McClelland at CIBR, phone For example, certain adenine methyltransferases may be used 619 535 5486, FAX 619 535 5472. There are tentative plans in conjunction with the methylation-dependent restriction to provide the supplement issue on a CD-ROM. endonuclease DpnI to create cleavages at eight- to twelve-base- pair sequences (Mc6,Mcl2). M ClaI and DpnI have been used to cut the 2.8 million base pair Staphylococcus aureus genome ACKNOWLEDGEMENTS into two pieces at the sequence ATCGATCGAT (Wel). Twelve- This work is supported by grants from the National Institutes base-pair TCTAGATCTAGA M XbaI/DpnI sites in a of Health and the U.S. Dept. of Energy. We gratefully transposon have been introduced into bacterial genomes and acknowledge the editorial assistance of Charlie Peterson, Mike permit cleavage one or more times depending on the number of Biros and Dotty Crosei. transposons integrated (HaS). 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Lunnen K. (unpublished). Ka7. Karreman C., Tandeau de Marsac N. and de Waard A.: Nucleic Acids Lu4. Lunnen K.D., Morgan R.D., Timan C.J., Krzycki J.A., Reeve J.N. Res. 14 (1986) 5199-5205. and Wilson G.G.: Gene 77 (1989) 11-20. Ka8. Karreman C. and de Waard A.: J. Bact. 172 (1989) 266-272 Lyl. Lynn S.P., Cohen L.K., Gardner J.F. and Kaplan S.: J. Bacteriol. 138 Ka9. Karyagina A.S., Lunin V.G. and Nikolskaya I.I.: Gene 87 (1990) (1979) 505-509. 113-118. Mal. Macdonald P.M. and Mosig G.: EMBO J. 3 (1984) 2863-2871. KalO. K4szubska W., Aiken C.R. and Gumport R.I.: Gene 74 (1988) 83-84. Ma2. MacNeil D.J.: J. Bact. 170 (1988) 5607-5612. Kel. Kelly S., Kaddurah D.R. and Smith H.O.: J. Biol. Chem. 260 (1985) Ma3. Mann M.B.: Gene Amplification and Analysis, Vol. 1 (1981)J. Chirikjian, 15339-15344. Ed., Elsevier, New York, pp229-237. Ke2. Keshet E. and Cedar H.: Nucleic Acids Res. 11 (1983) 3571-3580. Ma4. Mann M.B., Rao R.N. and Smith H.O.: Gene 3 (1978) 97-112. Ke3. Kessler C. and Holtke H-J.: Gene 33 (1985) 1-102. MaS. Mann M.B. and Smith H.O.: Nucleic Acids Res. 4 (1977) 4211-4221. Kil. Kim E.L. and Maliuta S.S.: FEBS Lett. 255 (1989) 361-364. Ma6. Mannarelli B.M., Balganesh T.S., Greenberg B., Springhorn S.S. and Ki2. Kiss A. and Baldauf F.: Gene 21 (1983) 111-119. Lacks S.A.: Proc. Natl. Acad. Sci. USA 82 (1985) 4468-4472. Ki3. Kiss A., Posfai G., Keller C.C., Venetianer P. and Roberts R.J.: Nucleic Ma7. Maher L.J., Wold B., and Dervan P.B.: Science 245 (1989) 725-730. Acids Res. 13 (1985) 6403-6421. Ma8. Matvienko N.I., Kramarov V.M. and Irismetov A.A.: Bioorg. Khim. Ki4. Kita K., Nobutsugu H., Oshima A., Kadonishi S. and Obayashi A: 11 (1985) 953-956. Nucleic Acids Res. 13 (1985) 8685 -8693. Ma9. Matvienko N.I., Kramarov V.M., and Pachkunov D.M.: Eur. J. Kll. Klimasauskas S., Butkus V. and Janulaitis A.: Mol. Biol. (Mosk). 21 Biochem. 165 (1987) 565-570. (1987) 87-92. MalO. May M.S. and Hattman S.: J. Bacteriol. 122 (1975) 129-138. K13. Klimasauskas S., Timinskas A., Menkevicius S., Butkiene D., Butkus Mcl. McClelland M.: (unpublished results). V. and Janulaitis A.: Eksperim. Biolog, Vilnius (in press). Mc3. McClelland M.: Nucleic Acids Res. 9 (1981) 6795-6804. Kol. Koncz C., Kiss A. and Venetianer P.: Eur. J. Biochem. 89 (1978) Mc2. McClelland M.: Nucleic Acids Res. 9 (1981) 5859-5866. 523 -529. Mc4. McClelland M.: J. Mol. Evol. 19 (1983) 346-354. Ko2. Koob M., Grimes E., and Szybalski W.: Science 241 (1988) 1084- 1086. McS. McClelland M.: Nucleic Acids Res. 10 (1983) rl69-r173. Ko3. Korch C. and Hagblom P.: Eur. J. Biochem. 161 (1986) 519-524. Mc6. McClelland M., Kessler L. and Bittner M.: Proc. Natl. Acad. Sci. USA Ko4. Korch C., Hagblom P. and Normark S.: J. Bacteriol. 161 (1985) 81 (1984) 983-987. 1236-1237. Mc7. McClelland M., and Nelson M.: (unpublished results). Ko5. Korch C., Hagblom P. and Normark S.: J. Bacteriol. 155 (1983) Mc8. McClelland M. and Nelson M.: Nucleic Acids Res. 13 (1985) 1324-1332. r201 -r207. Ko6. Kosykh V. G., Bur'yanov Ya.l. and Bayev A.A.: Mol. Gen. Genet. Mc9. McClelland M. and Nelson M.: Gene Amplification and Analysis,Vol. 178 (1980) 717-718. 5 (1987) J. Chirikjian, Ed., Elsevier, NY, pp257-282. Ko7. Kosykh V.G., Glinskaite I., Bur'yanov Ya.l. and Bayev A.A.: Doki. MclO. McClelland M. and Nelson M.: Gene 74 (1988) 169-176. Akad. Nauk SSSR 265 (1982) 727-730. Mc 1. McClelland M. and Nelson M.: Gene 74 (1988) 291-304. Ko8. Kosykh V.G., Solonin A.S., Bur'yanov Ya.l. and Bayev A.A.: Biochim. McI2. McClelland M., Nelson M. and Cantor C.R.: Nucleic Acids Res. 13 Biophys. Acta 655 (1981) 102-106. (1985) 7171-7182. Krl. Kretz P.L, Reid C.H., Greener A., J.M.Short (1989) Nucleic Acids Res. Mc13. McClelland M. and Patel Y.: (unpublished observations). 17: 5409. Mel. Meda M.M. and Perler F.B.: (unpublished). Kr2. Kramarov V.M. and Smolyaninov V.V.: Biokhimiya 46 (1981) Miu. Miner Z. and Hattman S.: J.Bacteriol. 170 (1988) 5177-5184 1526-1529. Mi2. Miner Z., Schlagman S.L. and Hatnman S.: Nucleic Acids Res. 17 (1989) Ku3. Kubareva E.A., Gromova E.S., Romanova E.A., Oretskaya T.S., 8149-8158. Shabarova Z.A.,: Bioorg. Khimiya (Russ.) 16 (1990) 501-506. Mol. Modrich P.: Quart. Rev. Biophys. 12 Kul. (1979) 315-369. Kubareva E.A., Pein C-D., Gromova E.S., Kuznezova S.A., Tashlitzki Mo2. Molloy P.L. and Watt F.: Nucleic Acids Res. 16 (1988) 2335. V.N., Cech D. and Shabarova Z.A.: Eur. J. Biochem. 175 (1988) Mo3. Morgan R.: (unpublished observations). 615-618. Mul. Mural R.J.: Nucleic Acids Res 15 (1987) 9085. Ku2. Kupper D., Jian-Guang Z., Kiss A. and Venetianer P.: Gene 74 (1988) Myl. Myers P.A. and Roberts R.J.: (unpublished results). 33. Nal. Nagaraja V., Shepherd J.C.W., Pripfl T. and Bickle T.A.: J. Mol. Biol. Lal. Labbe D., Holtke H.J. and Lau P.C.K.: Mol. Gen. Genet. 224 (1990) 182 (1985) 579-587. 101-110. Na2. Nagaraja V., Shepherd J.C.W. and Bickle T.A.: Nature 316(1985) La2. Labbe S., Xia Y. and Roy P.H.: Nucleic Acids Res.16 (1988) 7184. 371 -372. La3. Lacks S. and Greenberg B.: J. Mol. Biol. 114 (1977) 153-168. Na3. Nardone G.G. and Chirikjian J.G.: J. Biol. Chem. (1984)10357-10362. Nucleic Acids Research, Vol. 19, Supplement 2049

Na4. Narva K.E., Wendell D.L., Skrdla M.P. and Van Etten J.L.: Nucleic Ra2. Raleigh E.A. and Wilson G.: Proc. Natl. Acad. Sci. USA 83 (1986) Acids Res. 15 (1987) 9807-9823. 9070-9074. NaS. Nathans D. and Smith H.O.: Ann. Rev. Biochem. 44 (1974) 273-293. Ra3. Razin A., Urieli S., Pollack Y., Gruenbaum Y. and Glaser G.: Nucleic Nel. Nelson M: (unpublished results). Acids Res. 8 (1980) 1783-1792. Ne2. Nelson M., Christ C. and Schildkraut I.: Nucleic Acids Res. 12 (1984) Rel. Rees P.A. and Brenner J.S.: (unpublished). 5165-5173. Re2. Rees P., Nwankwo D.O., Wilson G.G. and J. Benner J.S.: Gene 74 Ne3. Nelson M. and McClelland M.: Nucleic Acids Res. 15 (1987) (1988) 37. r219-r230. Re3. Renbaum P., Abrahamove D., Fainsod A. and Wilson G.G.: Nucleic Ne4. Nelson M. and McClelland M.: Nucleic Acids Res. 17 (1987) r398-415. Acids Res. 18 (1990) 1145-1152. NeS. Nelson M. and McClelland M.: (unpublished observations). Re4. Revel H.R. and Georgeopoulos C.P.: Virology 39 (1969) 1-17. Ne6. Nelson M. and Schildkraut I.: Methods in Enzymology 155 (1987) Ril. Ritchot N. and Roy P.H.: Gene 86 (1990) 103-106. 31-48. Ri2. Richards D.F., Linnett P.E., Oultram J.D., and Young M.: J. Gen. Ne7. Nelson J.M., Miceli S.M., Lechevalier M.P. and Roberts R.J.: Nucleic Micro. 134 (1988) 3151-3157. Acids Res. 18 (1990) 2061-2064. Rol. Roberts R.J.: (unpublished results). Ne8. Nesterenko V.F., Bur'yanov Ya.I. and Bayev A. A.: Dokl. Akad. Nauk Ro2. Roberts R.J.: Nucleic Acids Res. 11 (1983) rl35-rl67. SSSR 250 (1980) 1265-1267. Ro3. Roberts R.J.: Nucleic Acids Res. 16 (1988) r271-r313. Ne9. Newman A.K., Rubin R.A., Kim S.H. and Modrich P.: J. Biol. Chem. Ro4. Rodicio M. and Chater K.F: Gene 74 (1988) 39-42. 256 (1981) 2131-2139. RoS. Rodicio M. and Chater K.F: Mol. Gen. Genet. 213 (1988) 346-353. Nil. Nikolskaya I.I., Karpetz L.Z., Kartashova I.M., Lopatina,N.G., Skripkin Ro6. Roy P.H. and Smith H.O.: J. Mol. Biol. 81 (1973) 427-444. E. A., Suchkov S.V., Uporova T.M., Gruber I.M. and Debov S.S.: Ro7. Roy P.H. and Smith H.O.: J. Mol. Biol. 81 (1973) 445-459. Mol. Genet. Mikrobiol. i Virusol. 12 (1983) 5-10. Rul. Rubin R.A. and Modrich P.J.: J. Biol. Chem. 252 (1977) 7265-7272. Ni2 Nikolskaya I.I., Lopatina N.G., Antikeicheva N.V. and Debov S.S.: Sal. Sain B. and Murray N.E.: Mol. Gen. Genet. 180 (1980) 35-46. Nucleic Acids Res. 7 (1979) 517-528. Sa2. Sano H. and Sager R.: Eur J. Biochem. 105 (1980) 471-480. Ni3. Nikolskaya I.I., Lopatina N.G., Suchkov S.V., Kartashova I.M. and Sa3. Sato S., Nakazawa K. and Shinomiya T.: J. Biochem. 88 (1980) Debov S.S.: Biochem. Int. 9 (1984) 771-781. 737-747. Ni4. Nikolskaya I.I., Lopatine N.G., Sharkova E.V., Suchkov S.V., Somodi Scl. Schertzer E., Auer B. and Schweiger M.: J. Biol. Chem. 262 (1987) P., Foldes I. and Debov S.S.: Biochem. Int. 10 (1985) 405-413. 15225-15231. Nol. Noyer-Weidner M., Jentsch S., Kupsch J., Bergbauer M. and Trautner Sc2. Schildkraut I., Morgan R. and Looney M.E.: (unpublished results). T.A.: Gene 35 (1985) 143-150. Sc3. Schlagman S.L., Miner Z., Feher Z. and Hattman S.: Gene 73 (1988) No2. Noyer-Weidner M., Jentsch S., Pawlek B., Gunthert U. and Trautner 517-530. T.A.: J. Virol. 46 (1983) 446-453. Sc4. Schlagman S.L. and Hattman S.: Gene 22 (1983) 139-156. No3. Noyer-Weidner M., Pawlek B., Jentsch S., Gunthert U. and Trautner ScS. Schlagman S.L. and Hattman S.: Nucleic Acids Res. 17 (1989) T. A.: J. Virol. 38 (1981) 1077-1080. 9101-9112. Nul. Nur I., Szyf M., Razin A., Glaser G., Rottem S. and Razin S.J.: Sc6. Schlagman S., Hattman S., May M.S. and Berger L.: J. Bacteriol. 126 Bacteriol. 164 (1985) 19-24. (1976) 990-996. Nwl. Nwankwo D.O. and Wilson G.G.: Mol. Gen. Genet. 209 (1987) Sc7. Schneider-Scherzer E., Auer B., De Groot E.J. and Schweiger M.: J. 570-574. Biol. Chem. 265 (1990) 6086-6091. Nw2. Nwankwo D.O. and Wilson G.G.: Gene 64 (1988) 1-8. Sc8. Schnetz K. and Rak B.: Nucleic Acids Res. 16 (1988) 1623. Onl. Ono A . and Ueda T.: Nucleic Acids Res. 15 (1987) 219-231. Sc9. Schoner B., Kelly S. and Smith H.O.: Gene 24 (1983) 227-236. Pal. Patel Y., Schildkraut I. and McClelland M.: (unpublished observations). Sc1O. Schoenfeld T., and Leland D.: (pers. comm.). Pa2. Patel Y., Nelson M. and McClelland M.: (unpublished observations). Scd 1. Schoenfeld T., Mead D.A., and Fiandt M.: Nucleic acids Res. 17 (1989) Pel. Pech M., Streeck R.E. and Zachau H.G.: Cell 18 (1979) 883-893. 4417. Pe2. Petrosyte M. and Janulaitis A.: Bioorg. Khim. 7 (1981) 1885- 1887. Sc12. Sciaky D. and Roberts R.J.: (unpublished results). Pil. Piekarowicz A. and Goguen J.D.: Eur. J. Biochem. 154 (1986) 295-298. Sel. Seeber S., Kessler C. and Gotz F.: Gene 94 (1990) 37-43. Pi2. Piekarowicz A. and Stein D.C.: (personal communication). Se2. Selker E.U., Cambareri E.B., Garrett P.W., Haack K.R., Jensen B.C. Pi3. Piekarowicz A., Yuan R. and Stein D.C.: Nucleic Acids Res. 16(1988) and Schabtach E.: Gene 74 (1988) 109-111. 5957-5972. Shl. Shibata T., Ikawa S. and Ando T.: Microbiology-1982, D. Schlessinger, Pi4. Piekarowicz A., Yuan R. and Stein D.C.: Nucleic Acids Res. 16 (1988) Ed., pp7l -74. 9868. Sh2. Shimizu-Kadota M. and Shibahara-Sone H.: Agric. Biol. Chem. 53 (1989) PiS. Piekarowicz A., Yuan R. and Stein D.C.: Nucleic Acids Res. 17 (1989) 2841-2842. 10132. Sh3. Shields S.L., Burbank D.E., Grabherrr R. and Van Etten J.L.: Virology Pi6. Pirrotta V.: Nucleic Acids Res. 3 (1976) 1747-1760. 176 (1990) 16-24 Pol. Posfai G., Baldauf F., Erdei S., Posfai J., Venetianer P. and Kiss A.: Sil. Silber K., Polisson C., Rees P., and Benner J.S.: Gene 74 (1988) 43-44. Nucleic Acids Res. 12 (1984) 9039-9049. Skl. Skrzypek E. and Piekarowicz A.: (unpublished). Po2. Posfai G., Kiss, A., Erdei, S., Posfai, J. and Venetianer, P.: J. Mol. Sll. Sladek T.L., Nowak J.A. and Maniloff J.: J. Bacteriol. 165 (1986) Biol. 170 (1983) 597-610. 219-225. Po3. Posfai, G. and Szybalski W.: Nucleic Acids. Res. 16 (1988) 6245. S12. Slatko B.E., Benner J.S., Jager-Quinton T., Moran L.S., Simcox T.G., Po4. Posfai G. and Szybalski W.: Gene 74 (1988) 179-181. Van Cott E.M. and Wilson G.G.: Nucleic Acids Res. 15 (1987) PoS. Posfai J., Bhagwat A.S., Posfai G. and Roberts R.J.: Nucleic Acids Res. 9781-9796. 17 (1989) 2421-2436. S13. Slatko B.E., Croft R., Moran L. and Wilson G.G.: Gene (1988) 45-50. Po6. Povilonis P., Vaisvila R., Lubys A. and Janulaitis A.: (personal Sml. Smith H.O.: Science 205 (1979) 455-462. communication). Sm2. Smith H.O. and Nathans D.: J. Mol. Biol. 81 (1973) 419-423. Prl. Prere M.F. and Fayet O.: Ann. Inst. Pasteur Microbiol. 136A (1985) Sm3. Smith H.O., Annau T.M. and Chandrasegaran S.: Proc. Natl. Acad. 323-338. Sci. USA 87 (1990) 826-830. Pr2. Price C. and Bickle T.A.: (unpublished). Sol. Som S., Bhagwat A.S. and Friedman S.: Nucleic Acids Res. 15 (1987) Pr3. Price C., Shepherd J.C.W. and Bickle T.A.: EMBO J. 6 (1987) 313-332. 1493-1497. So2. Sohail A., Bhagwat A.S. and Roberts R.J.: (unpublished). Qil. Qiang B-Q.: (personal communication). So3. Song Y-H., Rueter T. and Geiger R.: Nucleic Acids Res. 16 (1988) 2718. Qi2. Qiang B-Q. and Nelson M.: (personal communication). Sti. Stefan C., Xia Y., and Van Etten J.L.: Nucleic Acids Res. (1991) (in Qi3. Qiang B-Q., McClelland M., Poddar S., Spokauskas A. and Nelson M. press). (submitted). St2. Stemnberg N.: J. Bacteriol. 164 (1985) 490-493. Qul. Quint A. and Cedar H.: Nucleic Acids Res. 9 (1981) 633-646. St3. Streeck R.E.: Gene 12 (1980) 267-275. Ral. Raleigh E.A., Murray N.E., Revel H., Blumenthal R.M., Westaway St4. Striebel H.M., Schmnitz G.G., Kaluza K., Jarsch M. and Kessler C.: D., Reith A.D., Rigby P.W.J., Elhai J. and Hanahan D.: Nucleic Gene 91 (1990) 95-100. Acids Res. 16 (1988) 1563-1575. StS. Strobl J.S. and Gaido M.: (unpublished results). 2050 Nucleic Acids Research, Vol. 19, Supplement

St6. Strobl J.S. and Thompson E.B.: Nucleic Acids Res. 12 (1984) Wol. Wong K.K. and McClelland M.: Nucleic Acids Res. 19 (1991) (in press). 8073-8084. Wo2. Woodcock D.M., Crowther P.J., Diver W.P., Graham M., Batemen St7. Sturm R.A. and Yaciuk P.: Nucleic Acids Res. 17 (1989) 3615. C., Baker D.J. and Smith S.S.: Nucleic Acids Res. 16 (1988) Sul. Sugisaki H., Maekawa Y., Kanazawa and Takanami M.: Nucleic Acids 4465-4482. Res. 10 (1982) 5747-5752. Wul. Wu J.C. and Santi D.V.: Nucleic Acids Res. 16 (1988) 703-717. Su2. Sullivan K.M. and Saunders J.R. (1988). In 'Gonococci and Xil. Xia Y., Burbank D.E., Uher L., Rabussay D. and Van Etten J.L.: Mol. Meningococci' (1988) 329-334. Cell. Biol. 6 (1986) 1430-1439. Su3. Sullivan K.M. and Saunders J.R.: Nucleic Acids Res. 16 (1988) Xi2. Xia Y., Burbank D.E., Uher L., Rabussay D. and Van Etten J.L.: 4369-87. Nucleic Acids Res. 15 (1987) 6075-6090. Su4. Sullivan K.M. Saunders J.R.: Mol. Gen. Genet. 216 (1989) 380-387. Xi3. Xia Y., Burbank D.E. and Van Etten J.L.: Nucleic Acids Res. 14 (1986) Szl. Szilak L., Venetianer P. and Kiss A.: Nucleic Acids Res. (1990) 6017-6030. 4659-4664. Xi4. Xia Y., Morgan R., Schildkraut I. and Van Etten J.L.: Nucleic Acids Sz2. Sznyter L.A. and Brooks J.E.: Gene 74 (1988) 53. Res. 16 (1988) 9477-9487. Sz3. Sznyter L.A., Slatko B., Moran L., O'Donnell K.H. and Brooks J.E.: XiS. Xia Y., Narva K.E. and Van Etten J.L.: Nucleic Acids Res. 15 (1987) Nucleic Acids Res. 15 (1987) 8249-8266. 10063. Sz4. Szomolanyi E., Kiss A. and Venetianer P.: Gene 10 (1980) 219-225. Xi6. Xia Y. and Van Etten J.L.: Mol. Cell Biol. 6 (1986) 1440-1445. Tal. Tasseron-de Jong J.G., Aker J. and Giphart-Gassler M.: Gene 74 (1988) Xul. Xu G., Kapfer W., Walter J. and Trautner T.A.: (unpublished). 147-149. Yol. Yolov A.A., Vinogradova M.N., Gromova E.S., Rosenthal A., Cech Ta2. Tao T., Wiater J., Brennan K.J., Cotterman M.M. and Blumenthal R.M.: D., Veiko V.P., Metelev V.G., Buryanov Ya.I., Bayev A.A. and Nucleic Acids Res. 17 (1989) 4161-4176. Shabarova Z.A.: Nucleic Acids Res. 13 (1985) 8983-8998. Ta3. Tautz N., Kaluza G., Lane F., Frey B., Schmitz G., Jarsch M., Yo2. Yoo O.J. and Agarwal K.L.: J. Biol. Chem. 255 (1980) 6445-6449. Ankenbauer W., and Kessler C.: (unpublished). Yo3. Yoo O.J., Dwyer-Halquist P. and Agarwal K.L.: Nucleic Acids Res. Thl. Theriault G. and Roy P.H.: Gene 19 (1982) 355-359. 10 (1982) 6511-6520. Th2. Theriault G., Roy P.H., Howard K.A., Benner J.S., Brooks J.E., Waters Yo4. Yoshimori R., Roulland-Dussoix D. and Boyer H.W.: J. Bacteriol. 112 A.F. and Gingeras T.R.: Nucleic Acids Res. 13 (1985) 8441-8461. (1972) 1275-1279. Trl. Tran-Betcke A., Behrens B., Noyer-Weidner M. and Trautner T.A.: Yo5. Youssoufian H., Hammer S.M., Hirsch M.S. and Mulder C.: Proc. Natl. Gene 42 (1986) 89-96. Acad. Sci. USA 79 (1982) 2207-2210. Tr2. Trautner T.A.: Current Topics Microbiol. Immunol. 108 (1984) 11-22. Yo6. Youssoufian H. and Mulder C.: J. Mol. Biol. 150 (1981) 133-136. Tr3. Trautner T.A., Pawlek B., Gunthert U., Canosi U., Jentsch S. and Freund Zal. Zacharias W., Larson J.E., Kilpatrick M.W. and Wells R.D.: Nucleic M.: Mol. Gen. Genet. 180 (1980) 361-367. Acids Res. 12 (1984) 7677-7692. Tr4. Trautner T.A., Balganesh T.S., and Pawlek B.: Nucleic Acids Res. 16 Zil. Zieger M., Patillon M., Roizes G., Lerouge T., Dupret D. and Jeltsch (1988) 6649-6658. J.M.: Nucleic Acids Res. 15 (1987) 3919. Url. Urieli-Shoval S., Gruenbaum Y. and Razin A.: J. Bacteriol. 153 (1983) 274-280. Val. Van Cott E.M. and Wilson G.G.: Gene 74 (1988) 55-59. Va2. Van Cott E.M. and Wilson G.G.: (unpublished). Va3. Van der Ploeg L.H.T. and Flavell R.A.: Cell 19 (1980) 947-958. Va4. Van Etten J.L.: (personal communication). Va5. Vanyushin B.F. and Dobritsa A.P.: Biochim. Biophys. Acta 407 (1975) 61-72. Va6. VasquezC. and VicunaR.: Arch. Biol. Med. Exp. 15 (1982) 417-421. Vel. Venetianer P. and Kiss A.: Gene Amplification and Analysis, Vol. 1 (1981) J.Chirikjian, Ed., Elsevier, New York, 1981. pp209-215. Vii. Vinogradova M.N., Gomova E.S., Uporova T.M., Nikolskaya I.I., Shabarova Z.A., Debov S.S.: Doklady Akad. Nauk. SSSR 295 (1987) 732-736. Vol. Vovis G.F. and Lacks S.: J. Mol. Biol. 115 (1977) 525-538. Wal. Waalwijk C. and Flavell R.A.: Nucleic Acids Res. 5 (1978) 3231. Wa2. Walder R.Y.: Fed. Proc. 41 (1982) A5425. Wa3. Walder, R.Y., Hartley J.L., Donelson J.E. and Walder J.A.: Proc. Natl. Acad. Sci. USA 78 (1981) 1503-1507. Wa4. Walder R.Y., Hartley J.L., Donelson J.E. and Walder J.A.: Gene Ampfliicalion and Analysis, Vol. 1 (1981) J. Chirikjian,Ed., Elsevier, New York, pp217-227. WaS. Walder R.Y., Langtimm C.J., Chatterjee R. and Walder J.A.: J. Biol. Chem. 258 (1983) 1235-1241. Wa6. Walder R.Y., WalderJ.A. and Donelson J.E.: J. Biol. Chem. 259 (1984) 8015-8026. Wa7. Walter. J., Noyer-Weidner M. and Trautner T.A. EMBO J. (1990) 1007-1014. Wa8. Wang R.Y.H., Zhang X-Y., Khan R., Zhou Y., Huang L-H. and Ehrlich M.: Nucleic Acids Res. 14 (1987) 9843-9860. Wel. Weil M.D. and McClelland M.: Proc. Natl. Acad. Sci. USA 86 (1989) 51-55. We2. Weil M.D., Nelson M. and McClelland M.: (unpublished) Whl. Whitehead P.R. and Brown N.L.: FEBS Lett. 155 (1983) 97-101. Wh2. Whitehead P.R. and Brown N.L.: J. Gen. Microbiol. 131 (1985) 951-958. Wh3. Whitehead P., Jacobs D. and Brown N.L.: Nucleic Acids Res. 14 (1986) 7031-7045. Wil. Wiatr C.L. and Witmer H.J.: J. Virol. 52 (1984) 47-54. Wi2. Wigler M., Levy D. and Perucho M.: Cell 24 (1981) 33-40. Wi3. Wilke K., Rauhut E., Noyer-Weidner M., Lauster R., Pawlek B., Behrens B. and Trautner T.A.: EMBO J. 7 (1988) 2601-2609. Wi4. Wilson G.G.: Gene 74 (1988) 281-289. Nucleic Acids Research, Vol. 19, Supplement 2051 TABLE I: Methylation sensitivity of restriction endonucleases a Restriction Recogniton Sites Sites not References enzyme sequence cut cut%0"16 AacI CCWGG Cm5CWGG Br8 Aa-d AGGCCT AGGmSCCT Nel AGGCm5CT So3 AGGCm4CT Nel Aatill GACGTC GACGTTm5C Nel GAm5CGTC Fol A~I& GTMKAC GTMKm6AC# Lu2,Mc3 GTMKAm5C AccIl CGCG mSCGCG Ga2 TCCGGA Tm5CCGGA TCCGGm6A Ke3,La2,Sc2 TCmSCGGA AflI GGWCC GGWCm5C?? Mc 1,Wh2 GGWCm4C AflH ClTAAG I? m5CTrAAG Nel CIT-Am6AG ACRYGT Am5CRYGT Nel Ahall GRCGyC b GRm5CGYC Ka2,Hul GRCGYm5C AM AGCT m6AGCI Gr4,Mc 1 1 ,Ne2 AGm4CT AGm5CT# Hul,Wol AGhm5CT Bu5 AlwI GGATC GGm6ATC Ne4 GGATm4C AmaI TCGCGA TCGCGm6A Mc13 Aosll GRCGYC GRm5CGYC Eh2,Gr4,Va3 AaI GGGCCC GGGm5CCC# La9,Tr2 GGGCCm5C AxI ACGCGT Am5CGCGT Nel,Qi2 ,paLI GTGCAC GTGCm6AC GTGCAm5C Fol ,Hol,Ho2 CCWGG Cm5CWGGQ,b m5CCWGG K11,Mcl 1,Ra3 AguI CYCGRG m5CYCGRG# Ka7,Ka8 AseI ATTAAT ATpn6AAT Nel Asg7OOI GAAN4TTC GAm6AN4TTC Gm6AAN4TTC Nel GAAN4TPm5C As1718I GGTACC GGTm6Am5CC b GGTACm5C Mul,Ne4 GGTAm5Cm5C b AsuIl TTCGAA YfmSCGAA Nel Ata TGATCA TGm6ATCA Ro3,Scl2 AvaI CYCGRG Cm5CCGGG m5CYCGRG Eh2,Nel CYm5CGRG Ka4,Ka7,Mcl 1 CTCGm6AG b Ne2 2052 Nucleic Acids Research, Vol. 19, Supplement Restriction Recognition Sites Sites not References enzme sequence cut cut Aal GGWCC GGWCm4C b GGWm5CC Ba3,Ko3 GGWCm5C MclO,Mcl 1 GGWhmSChmSC Hul A3I AGCGCT m6AGCGC AGm5CGCT Nel BaII TGGCCA TGGm5CCA# Gil,Tr2 TGGCm5CA b famHl GGATCC GGATCm5C GGATmCc# Br8,Drl,Hal,Hul GGm6ATCC GGATm5CC La7 GGm6ATCm5C GGAltm5Chm5C GGATCm4C BpujF1 GGATCC GGm6ATCC GGATm4CC Anl,Shl IamK[ GGATCC GGm6ATCC GGATm4CC Anl,Shl BanI GGYRCC b GGm5CGCC Co3,Ka2 GGYRCm4C aII GRGCYC GRGCYm5C GRGm5CYC Fol,Ne2,Ne6 BIOI ATCGAT ATCGm6AT Sul Bj2eI GGCGCC GGCGm5CC GGm5CGCC Co3,Ne2,Sh2 GGCGCm5C BiHI GRCGYC GRm5CGYC Co3 BbrPI CACGTG m5CAm5CGTG Wol BbKI GAAGAC GAAGAm5C Fol BbvI GCWGC Gm5CWGC# Dol,Hal,Va5 BclI TGATCA b TGATm5CA TGm6ATCA Bi4,Br8,Eh3,Ro3 TGAThm5CA Hul BcnI CCSGG m5CCSGG Cm4CSGG# Ja3.,Ja6,KlI BZI CGCG m5CGCG Ku2 DftI CTTAAG m5C11AAG Wol B--II GCCN5GGC GCm5CN5GGC b Gm5CCN5GGC Kll,Ko3,Mcl1,Ne2 GCCN5GGm5C b GCm4CN5GGC b Bjl AGATCT b AGm6ATCT AGATm5CT Bi4,Br8,Drl,Dyl,Eh3 AGAPIm5cT Hul,Pi6 BinI GGATC ? GGm6ATC Bol BmaDI CGATCG CGm6ATCG CGATm6CG Qi2 Bfe2l6I GGWCC GGWCm5C Ma9 BnaI GGATCC GGm6ATCC GGATm4CC Nel GGATm5CC# I-W GGTCTC GGTCPwrm5C Fol BaAI YACGTR YAm5CGTR Fol BiOI GATN4ATC GATN4AT5C Fol BsmI GAATGC GAATGm5C Gm6AATGC Fol,Nel BsmAI GTCTC GTCTm5C Fol 106I ATCGAT ATCGm5AT# Ne5 Nucleic Acids Research, Vol. 19, Supplement 2053 Restriction Recognition Sites Sites not References enzyme sequence cut cut BS286I GDGCHC GDGCHm5C GDGmSCHC Fol,Ne2,Ne6 BZHI TCATGA TCm6ATGA Pa2 TCATGm6A Mci ACCTGC ACCTGm5C Fol SMlI TCCGGA TCCGGm6A T'5CCGGA La2,Sc2 TCm5CGGA SNI CCWGG m5CCWGG Ne4 CmSCWGG ATCGAT ATCGm6AT zil TGATCA T&m6ATCA zil RWHIItBHll GCGCGC b GmSCGCGC Ne4,Qi3 GGATCC GGm6ATCC GGATm4CC Ne4 GGATCmSC 'GGATm5CC LtBI GGATCm4C Nel TTCGAA TfCGm6AA Ne4 Trm5CGAA Wol BstEll GGTNACC GGTNAm5Cm5C b GGTNAhm5Chm5C Hu1,Mc11 GGTNACm4C Nel BstEfI GATC b Gm6ATC Myl,Ro3 BstGI TGATCA TGm6ATCA Ro3 CCWGG b m5CCWGG b hmsChmsCWGG Gr4,Hul,Mcl 1,Ro3 Cm5CWGG Cm4CWGG Nel m5CmSCWGG b .-lUI CGCG m5CGCG Ne5 BstXI CCAN6TGG m5CCAN6TGG Ne2 BstYI RGATCY RGm6ATCY RGAP114CY Ne4 RGAT 5CY Nel BsuBI CTGCAG CTGCm6AG# Gal,Jel,St5,Shl BsuEI CGCG m5CGCG# Gal ,Jel,St5,Shl BQlFI CCGG m5CCGG# Je1 CTCGAG CTm5CGAG# Jel RS-QI CCGG mCCGG Je2 DaRI GGCC GGm5CC# b Gu6,Ki2,Ki3 ciiI CTCGAG CTCGm6AG Nel CfoI GGGC Gm5CGC Ehl Ghm5CGhmsC Hul YGGCCR YGGm5CCR# Kll fr6I CAGCTG CAGm4CTG# Bu5 CAGmSCTG cft9I CCCGGG b CmSCCGGG m4CCCGGG Bu6 CCm5CGGG m5CCCGGG Cm4CCGGG# CCm4CGGG 2054 Nucleic Acids Research, Vol. 19, Supplement Restriction Recognition Sites Sites not References enzyme sequence cut cut £iOI RCCGGY Rm5CCGGY# Bi5,K1 f13I GGNCC GGNm5CC# Bi5,Kli Lia ATCGAT ')6ATCGAT Ca4,Mc1l,Mci2,Ne4 A1m5CGAT Woi ATCGm6AI* Mc3 TGATCA TGm6ATCA Fil,Ro3 CGGWCCG CGGWCm5CG CGGWmSCCG Mcii m5CGGWCCG Cz45I TTCGAA I? TTCGm6AA Ne4,Scl 1 GATC I? Gm6ATC# Ri2 GATC ? Gm6ATC Xii,Xi6 CviBI GANTC I? Gm6ANTC# Xi3 RGCY ? RGmSCY# Sh3,Xi2 CviPI m5cc# £NAPI cc cm5c Xi4 CviQI GTAC GTAm5C GTm6ACG Xi2,Xi5 DdI CTNAG I? m5CTNAG# Ho3,Ne2 b"n5CTNAG Hul DpnI Gm6ATC b Gm6ATC GATC La3,Mc 1i,Vol Gm6ATm5C b GATm4C Ne4 Gm6ATm4C GAT5C Ne5 GATC I? Gm6ATc# Del,La3,La4,La5,Ma6,Vo1 DraI TITAAA TITA6AA Nel RGGNCCY I? RGGNCm5CY Sc8 EareI YGGCCR I? YGGm5CCR# Ja2,Whl YGGCm5CR agI CGGCCG CGGm5CCG Mcii m5CGGCm5CG EarI GAAGAG Gm6AAGAG Ne4 GAAGm6AG m5C-wpn5C 1p5C Nel GGTANm6ACC GGTAm6ACC# Br2 CGGCCG m5CGGCm5CG Qi3 CGGm5CCG Eco47I GGWCC GGWCm5C Ja5 E&Q47M AGCGCT m6AGCGCT AGm5CGCT Nel,Ne4 GAGN7GTCA b Gm6AGN7GmTCA# b Bi2,Co6,Fu2 Eg&B TGAN8TGCT b TGm6AN8mTGCT # b Bi2,LalO,Lal 1 &QDXXU TCAN7AATC b TCAN7m6AAmTC # b Pil F,&2E GAGN7ATGC Gm6AGN7ATGC Co6,Fu2 F.gK AACN6GTGC b Am6ACN6GmTGC# b Bi2,Bi3,Kal E&20109I RGGNCCY RGGNCm5CY Sc8 F&2PI AGACC b AGAhm5CIh5C AGm6ACC# Bal,Ba2,Ha2,Re4 I&P15 CAGCAG b Cm6AGCAG# Hu2 Nucleic Acids Research, Vol. 19, Supplement 2055 Restriction Recognition Sites Sites not References enzyme sequence cut cut EQQRI GAAITC GAATrhm5C Gm6AATTC b Mci 1,Ne2,Rul GAm6ATrrd Brl,Br8,Dul GAATPm5C b Hul,Ka3,Tal EQRII CCWGG m5cCwGG b m4CCWGG Ku3,Yol Cm4CWGG Bu4,Na5,Ro3 Cm5CWGG# Bo5,Mcl 1 CCm6AGG Bu3 hm5Chm5CWGG Hul,Ka3 FQRV GATATC GATATm5C b Gm6ATATC# Mcl 1,Ne2,Wol GATm6ATC Fll FcoR124 GAAN6RTCG b GAm6AN6RTCG Pr2,Pr3 GAAN6RmTCG Bil EcoRl24/3 GAAN7RTCG b m6A Prl,Pr2 EheI GGCGCC GGCGCC Co2 FnIuI GCTNAGC GCTNAGm5C Gm5CTNAGC Ne4 Fnu4lH GCNGC Gm5CNGC Ko3,Tr2 GCNGm5C EnuDII CGCG m5CGCG Gal,Ga2,Ne2,Ne6,St6 CGm5CG EnEI GATC Gm6ATC b Lul,Ne2 FokI CATCC CATmSCC GGm6ATG Po3,Po4,Sc2 CATCm5C b Cm6ATCC CATCm4C Nel GGCCGGCC GGm5CCGGm5CC Ne7 GGCm5CGGCC GGm5CCGGCC FspI TGCGCA TGm5CGCA Ne4 ki=ll RGCGCY b RGm5CGCY Eh2,Gr4,Ka2,Ko3,Mc 1i,Pi5 RGhm5CGhm5CY Hul HaelII GGCC GGCm5C GGm5CC# b Ba3,Ka2,Ko3,Ma5 GGhm5Chm5C Hul CCGG Cm5CGG# Eh2,Wal HgI GACGC GAm5CGC Nel GACGm5C Mcii GRGCYC GRGCYm5C GRGm5CYC Fol,Ne2,Wh3 agicI GGYRCC GGYRCm5C Erl H.CII GGWCC GGWCm5C Erl HgiEI GGWCC GGWCm5C Erl HgJm GGYRCC GGYRCmSC Wh3 lIhaI GCGC Gm5CGC# Eh2,Ko3,Sml GCGm5C Mcil, Ghm5CGhm5C Hul 20S6 Nucleic Acids Research, Vol. 19, Supplement Restriction Recognition Sites Sites not References enzyme sequence cut cut jIll GANTC Gm6ANTC# Ma5 HizcIll GTYRAC GTYRAm5C GTYRm6AC Gr4,Ro7 GTYRAwm5C Hul Hindll GTYRAC GTYRm6AC* Ro7 GANTC GANTm5C,b Gm6ANTC Chl,Col,Ne2,Pel GANVmSC Hul Hindf AAGCIT m6AAGC1T# Br8,Gr4,Ro7 AAGm5CT Ne2 AAGhm5CT Hul,Ka3 HinPI GCGC Gm5CGC Mc 1,Ne6 GTTAAC GUTAAm5C G1TAm6ACt Br8,Gr4,Hul,Yo3 G1TAAhm5C Hul HD-an CCGG m4CCGG Be3,Bu6,Eh2,Ma5 m5CCGG b Ko3,Qul,Wa5 Cm4CGG b Cm5CGG# hm5Chm5CGG Hul HDhI TCACC TCACm5C PI5CACC# Fol,Mcl 1,Ne2 GGTGm6A GGTACC b GGTAm5CC GGTm6Am5CC Eh3,Mcl1,Ne2 GGTACm5C GGTACm4C Nel GGTAm5Cm5C b KDn2I TCCGGA TCCGGm6A 1m5CCGGA Mcl,Nel TCm5CGGA Nel LrI CCGCGG m5CCGCGG Nel Cm5CGCGG Qi2 MaeII ACGT Am5CGT b Mo2 MamI GATN4ATC Gm6ATN4m6ATC St4 MboI GATC b GATm4C Gm6ATC# Br5,Gel,Mc8 GATm5C b GAThmSC Hul,Ro3 MboII GAAGA Tm5CTPm15C b GAAGm6A# Ba3,Mcl 1,Mcl2,Ne2 Gm6AAGA mIm, RGATCY b RGm6ATCY Onl RGATn4CY RGATV5CY Lu~I ACGCGT m6ACGCGT Am5CGCGT Mcl 1,Shl,St5,Qi3 ?2UI9273I TCGCGA T'5CGCGA Nel Mlu927311 GCCGGC Gm5CCGGC Nel GCm5CGGC Mml GATC Gm6ATC Bo4 CCTC b m5CCTC Eh3,Mcll m5Cm5CTml5C Nucleic Acids Research, Vol. 19, Supplement 2057

Restriction Recognition Sites Sites not References enzyme sequence cut cut MphI CCWGG b Cm5CWGG Ro3 MI TCCGGA TCCGGm6A Pm5CCGGA Mcl,Nel TCm5CGGA Nel MseI TTAA '1116AA Nel MvaI CCGG b m4CCGG m5CCGG# Eh2,Je2,Va3,Wal,Wa5 Cm4CGG hm5Chm5CGG Bu6,Hul Cm5CGG Ms-tll CCTNAGG m5CCTNAGG Mcii CCWGG Cm5CWGG b Cm4CWGG# Bu4,Kul m5CCWGG CCm6AGG b Gr3,Ku3 m4CWGG b msCm5CWGG b Nel MvnI CGCG m5CGCG Nel NI GCCGGC b Gm5CCGGC Eh3,K1i,Mc 1i,Ne5 GCm5CGGC GCCGGm5C NanlI Gm6ATC b Gm6ATC GATC Pal,Ne5 Gm6ATm5C b GATm5C I GGCGCC GGCGCm5C GGm5CGCC Ko3,Mc1 l,Ne5 GGCGCm4C Nel NciI CCSGG m5CCSGG Cm4CSGG Br8,Ko3,Mc 1 1 Cm5CSGG b NcoI CCATGG CCm6ATGG m4CCATGG b Kll,Ne2,Ne4 m5CCATGG NcrI AGATCT AGm6ATCT b Qil NcuI GAAGA GAAGm6A Mci3 NdeI CATATG m5CATATG b m6A Be4,Mci 1 NdelI GATC GATm5C b Gm6ATC Mc9 Ng2BI TCACC Tm5CACC Pi3,Pi4 NgQPI RGCGCY RGm5CGCY Ko3,Ko5 NgoIPl GGCC GGm5CC# Ko3,Ko5 GGCm5Cb Su3,Su4 NheI GCTAGC GCTAGm5C Kli,Mc 1 ,Ne2 mam CATG Cm6ATG# Lal,Mo3 NmuDI Gm6ATC b Gm6ATC GATC Pal NmuEI Gm6ATC b Gm6ATC GATC Pal NotI GCGGCCGC GCGGCCGm5C GCGGm5CCGIC Mcii GCGGCm5CG;C St5,Qi2 NMI TCGCGA TCGm5CGA Tm5CGCGA Nel,Qi3 TCGCGm6A Ne2 NsiI ATGCAT ATGCm6AT Be5 ATGm5CAT Woi 2058 Nucleic Acids Research, Vol. 19, Supplement Restriction Recogniton Sites Sites not References enyme sequence cut cut NBII CMGCKG Cm5CGCKG Nel alM[ CCAN5TGG Cm4CAN5TGG Nel CmSCAN5TGG St7 GATC Gm6ATC Ro3 PfI CGTACG CGTAm5CG Nel nR7I CTCGAG CTCGm6AG# Gi3 CTm5CGAG Ghl CACGTG CAm5CGTG Fol P;LAI CGTACG CGTAm5CG Nel CTGCAG m5CTGCAG Dol ,Gr4,Mcl 1,Ne2 CTCGCm6AG# CGATCG b CGm6ATCG CGATm4CG Br8,Bu3,Eh3 CGATm5CG yIl CAGCTG CAGn4CTG# Br8,Bu5,Dol CAGm5CTG Eh3,Ja3,Rol &B4273I GTCGAC GTCGm6AC Ba6 RsaI GTAC b GTAmC b GTm6Am5C Eh3,Ne4,Ne5 RSIh CGATCG CGm6ATCG Lyl Rsj2KI TCATGA TCm6ATGA Pa2 TCATGm6A Ne4 RsrI GAATTC Gm6AATTC Mcli GAm6ATTC# b Ba5 CGGWCCG m5CGGWCCG Mc1 ,Qi3 CGGWm5CCG CGGWCm5CG SQI GAGCTC Gm6AGCTC GAGm5CTC Mcil CCGCGG I? m5CCGCGG Kll,Ne2 GTCGAC GTCGAm5C GTm5CGAC Br8,Eh2,Lu2,Qil GTCGm6AC# Mc3,Ro4,Ro5,Va4 SaDI TCGCGA TCGCGm6A T'5CGCGA Mcl3,Nel,Qi3 Sa3AI GATC b Gm6ATC GATm5C# b Drl,Eh2,Ja3,Mc3,Ro3,Se 1 GATm4C Ne5 GAThm5C Hul S96I GGNCC GGNm5CC# Ko3,Ne2,Pel GGNCm5C GGNhm5Chm5C Hul £213I TCGCGA TCGCGm6A T'5CGCGA Mc 1 1,Ne1 AGTACr AGTAm5CT Wol SaczFI CCNGG m5CCNGG Cm5CNGG Mc1 1,Ne2 Cm4CNGG Nel SfaNI GATGC GATGm5C Gm6ATGC Mcll1,Po4 ScI GGCCN5GGCC GGm5CCN5GGm5CC b GGCm5CN5GGCC Mcl l,Qi2 GGCCN5GGCm5C Sfll CrGCAGCTGCm6AG? Br8 Nucleic Acids Research, Vol. 19, Supplement 2059 Restiction Recognition Sites Sites not References enzyme sequence cut cut SaAI CRCCGGYG CRCm5CGGYG Ta3 Sid GGWCC GGWm5CC# Ka5,Ka6 SmaI CCCGGG Cm5CCGGG m4CCCGGG Br8,Bu6,Eh2,Ga4 m5yCCGGG b Ja3,Ka7,Mc3,Qul Cm4CCoGG b CCm4CGGG CCm5CGGG b SnaBI TACGTA TAm5CGTA Fol GTGCAC GTGm5CAm5C Ho2,Wol ACTAGT m6ACrAGT Hol S~I Am5CTAGT Wol II GCATGC GCATGm5C G(n6ATGC Mcl 1,Ne2,Mo3 Ghm5CATGhm5C CGTACG CGTm6ACG 9 Ne4,Qi3 TCGCGA TCGCGm6A T'5CGCGA Nel,Ne4 TCGm5CGA SsoIl CCNGG Cm5CNGG Vil m5CCNGG Gr3 GAATTC Gm6AA Ni4 sphI AATATT m6AATATT Nel GAGCTC GAGm5CTC Br8,Rol GAGhm5cThm5C Hul SQI AGGCCT AGGm5CCT Ca4,Mcll AGGCm5CT So3 AGGCm4CT Nel GAGN6RTAYG b Gm6AGN6RmTAYG# b Nal,Na2 RSPI AACN6GTRC b Am6ACN6GmTRC# b Nal,Na2 TQI TCGA 1m5CGA b TCGm6A# Gr4,Hul,Mc3,Va3 Tfun5CGA b Hul TaqII GACCGA Gm6ACCGA Ne4 CACCCA IaXI CCWGG m5C?CWG Grl Cm5CWGG TfiI GAWTC GAWTM5C Fol Tifi TCGA TCGm6A Sa3,Va6 CGCG m5CGCG m5CGCG Gal,Nel IfI Th,FJ hm5CGhm5CG Hul XbaI TCGA Tm5CGA TCGm6A# Sa3 flhHI TCTAGA TCTAGm6A# Mcl3,Wel Tm5CTAGA Gr4,Hul,Ne2 Thm5CTAGA 2060 Nucleic Acids Research, Vol. 19, Supplement Restriction Recognition Sites Sites not References enzy-me sequence cut cut XhQI CTCGAG b ? CTm5CGAG Br8,Eh2,Eh3,Ka7 CTCGm6AG Mc3,Va3 m5CTCGAG XhoII RGATCY RGm6ATCY RGATm5Cyb Br8 XmaI CCCGGG CCm'5CGGG b m4CCCGGG Bu6,Yo5,Yo6 m5CCCGGG Cm4CCGGG CCm4CGGG XmafiE CGGCCG ? CGGm5CCG Ne2,Tr2 XmnI GAAN4TTC GAm6AN4YrC Gm6AAN4TI C Mc1l,Ne2 GAAN4TP 5C b XorII CGATCG CGm6ATCG CGATm5CG Br8,Eh2 hm5CGAThm5CG Hul

FOOTNOTES a. # denotes canonical modification MTase 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,G or T, H = A,C or T. Sequences are in 5'-3' order. m4c= N4-methylcytosine; m5C = CS-methylcytosine; hm5C =hydroxymethylcytosine; mc= methylcytosine, N4 or C5-methylcytosine unspecified; m6A= N6-methyladenine. Nomenclature is according to (Sm2) and (Co4). b. AccI nicking occurs slowly in the unmethylated strand of the hemi-methylated sequence GTMKAm5C. Afll cuts slowly at GGWCn4C. Ahai (GRCGYC) will cut GRCGCC faster if these sites are methylated at GRCGm5CC (Ne5), but will not cut GRCGYm5C sites (Ne2,Ne5). Asp718I cuts M CviQI -modified (GTm6AC) Chlorella virus NY2A DNA. Asp7l.8I does not cut GGTACm5CWGG overlapping dcm sites (Mul) or m5C-substituted phage XP12 DNA, whereas 4Knl cuts XP12 readily (Ne4). AvaI nicking occurs slowly in the unmethylated strand of the hemi-methylated sequence CTCGm6AG/CTCGAG (NeS). AvaH cuts slowly at GGWCm4C. Bacillus species have been surveyed for Gm6ATC and Cm5CWGG specific methylases. Many species have Gm6ATC specific methylases but none had Cm5CWGG specific methylases (Di3). Ball sites overlapping dcm sites (TGGCm5CAGG) are 50-fold slower than unmethylated sites (Gil). BanI gives various rate effects when its recognition sequence is n4C- or m5C-methylated at different positions. BglI cleavage rate at certain GCm5CN5GGC, GCm4CN5GGC, and GCCN5GGm5C hemi-methylated sites is extremely slow. However, m5C bi-methylated MHaeIl-BglI sites are completely refractory to BglI (Ko3,Ne2). BssHIl does not cut M-HhaI-modified DNA, in which two different cytosine positions are hemi-methylated, Grm5CGCGC/GCGmICGC (Ne4). M BstI modifies the internal cytosine GGATmCC, but it is not known whether this modification is m C or m4C (Le2). BstEII cuts the fully m5C-substituted phage XP12 DNA (Ne5). BstNI cuts Cm5CWGG, n'5CCWGG and m5Cm5CWGG (Ne5). BstNI isoschizomers that are insensitive to Cm5CWGG include AorI, ApyI, BspNI, MvaI and TaqXI (Mc4). BsuRI nicking occurs in the unmethylated strand of the hemi-methylated sequence GG(m5CC/GGCC. Cfi9I, see reference Bu6 for rate effects. M CreI is from the unicellular eukaryote Chlamydomonas reinhardi (Sa2). DpnI requires adenine methylation on both DNA strands. Isoschizomers of DpnI include CfiI, NanII, NmuEI, NmuDI and NsuDI (Cal). DpnI cuts dam modified XP12 DNA (Ne6). M Eco dam modifies GATn5C at a reduced rate (NeS). Many other bacteria that modify their DNA at Gm6ATC are listed in references Bal and Lol. EcoA, EcoB, EcoD, EcoDXXI, EcoK are Type I restriction endonucleases. 'T represents a 6-methyladenine in the complementary strand. EcoPI is a Type HI restriction endonuclease (Ba2,Bal,Ha2). EcoP15 is a Type Im restriction endonuclease (Hu2). EcoRI cannot cut hemi-methylated Gm6AATTC/GAATTC sites. Bimethylated GAm6ATTC/GAm6ATTC sites are not cut by EcoRI or RsrI (NeS). EcoRI shows a reduced rate of cleavage at hemi-methylated GAATTm5C (Trl) and does not cut an oligonucleotide that contains GAATTm5C in both strands (Brl). EcoRII isoschizomers that are sensitive to Cm5CWGG include AtuBI, AtuIl, BstGH, BinSI, Cfr5I, Cfril I, EclII, EcalI, Eco27I, Eco38I and MphI (Ro3). EcoRJI shows reduced rate of cleavage at hemi-methylated m5CCWGG/CCWGG sites (Yol). EcoRV cuts the fully m5C-substituted phage XP12 DNA (NeS). EcoR124 and EcoR124/3 are Type I restriction endonucleases. mT represents a 6-methyladenine in the complementary strand. is a Type I restriction endonuclease. FokI cuts about two-fold to four-fold more slowly at CATCm5C than at unmodified sites (Ne5). M FokI in ref Po3 corresponds to M-FokIA in ref Po4. Haell show a reduction in rate of cleavage when its recognition sequence is modified at RGCGmSCY. HaeIi nicking occurs in the unmethylated strand of the hemi-methylated sequence GGm5CC/GGCC. Hinfl cuts GANTn5C, however, detectable rate differences are observed between unmethylated, hemi-methylated (GANTm5C/GANTC) and bi-methylated (GANTm5C/GANTm5C) target sequences. Hinfl does cut phage XP12 DNA, although at a reduced rate (Gr4,Ne5). Hinfl cuts unmethylated GANTC faster than hemi-methylated GANTm5C/GANTC, which is cut faster than GANTm C/GANTm C. However, the rate difference between unmethylated and fully methylated Hinfl sites is only about ten-fold (Hul,Ne5,Pel). Hpai nicking in the unmethylated strand of the hemi-methylated sequence m5CCGG/CCGG is in dispute (Be3,Bu6,Ko3). Hpall cuts hemimethylated mCCGG 50 times slower and fully methylated mCCGG 3000 times slower than unmethylated DNA (Ko3). See reference (Bu6) for HpaIl rate effects. Nucleic Acids Research, Vol. 19, Supplement 2061

KpnI sensitivity to hemi-methylated GGTAmSCC and GGTACm5C sites has been reported. KpnI efficiently cuts m5C-substituted phage XP12 DNA, but not Chlorella virus NY2A DNA, which carries both GTm6AC and m5CC modifications (Ne4). MaeII nicks slowly in the unmethylated strand of hemi-methylated Am5CGT/ACGT (Mo2). MboI isoschizomers that are sensitive to Gm6ATC include BssGH, BsaPI, Bsp74I, Bsp76I, BsplO5I, BstXll, BstEI1I,BssGII, CpaI, CtyI, CviAI, CviBII, CviHI, Dpnll, FnuAll, FnuCI, Had, Meul, MkrAI, Mmell, MnoIII, MosI, Msp67II, MthI, MthAI, NdeII, NflAII, NflBI, Nfl, NlaDI Nlal, NmeCI, NphI, NsiAI, NspAI, NsuI, PfaI, RlulI, SalAI, SalHI, Sau6782I, SinMI, Trull (Ro3). MboII cuts the fully m5C-substituted phage XP12 DNA (Ne5), although certain hemi-methylated m5C-containing substrates are reported not to be cut (Gr4). MflI cuts slowly at m6AGATCY sites (Onl). Mammalian methylase is the m5CG methyltransferase from Mus musculus. (mouse) (Be6). MspI cuts the hemi-methylated sequence CmSCGG/CCGG (WaS) and Cm4CGG/CCGG duplexes (Bu6). MspI cuts very slowly at GGCm5CGG (Bu2,Kel). An MMspI clone methylates m5(CG( (WaS,Wa2). However, there is a report that Moraxella sp. chromosomal DNA is methylated at m5Cm5CGG (Je2). MvaI nicking occurs in the unmethylated strand of the hemi-methylated sequence m4CCWGG/CCWGG and CCm6AGG/CCTGG (Ku3). MvaI cuts XP12 DNA very slowly at m5Cm5CWGG. NanH requires adenine methylation on both DNA strands (Cal). NanII cuts MEco dam modified XP12 DNA (Ne5). NciI may cut m5Cm5CGG methylated DNA (Br8,Je2). Possibly the second methylation negates the effect of Cm5CGG. NcoI is blocked by M SecI (CCNNGG) (Ne5). NcrI is a BgEl isoschizomer from Nocardia carnia Beijing (Qil). NdeI cuts the fully m5C-substituted phage XP12 DNA (Ne5). NdeHl 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. NgoPIl may be NgoIII. NgoPll does not cut overlapping dcm sites (Su4). NmuDI requires adenine methylation on both DNA strands (Cal). NmuEI requires adenine methylation on both DNA strands (Cal). PaeRI cuts hemimethylated CTm5CGAG/CTCGAG sites 100 fold slower and cuts fully methylated CTm5CGAG/CTm5CGAG 2900 fold slower than unmethylated sites (Ghl). Hemi- or full methylation at m6A completely protects against PaeR7 cleavage (Ghl). RsaI cuts the fully m5C-substituted phage XP12 DNA (Ne5), but does not cut Chlorella virus NY2A DNA, which is modified at GTm6AC (Ne4,Xil). DNA from Rhodopseudomonas sphaeroides species Kaplan is cut by Asp718I, but not by RsaI or KpnI (Ne4). It is likely that M RsaI specifies GTAm4C; and high levels of m4C are present in R. sphaeroides DNA (Eh3). RsrI cannot cut hemi-methylated Gm6AATTC/GAATTC sites. Sau3AI nicking occurs in the unmethylated strand of the hemi-methylated sequence GATm C/GATC (St3). Sau3AI cuts at a reduced rate at "6AGATC (OnI). Sau3AI isoschizomers that are insensitive to Gm6ATC include Bce243I, Bsp49I, Bsp5lI, Bsp52I,Bsp54I, Bsp57I, Bsp58I, Bsp59I,Bsp6OI, Bsp6lI, Bsp64I, Bsp65I, Bsp66I, Bsp67I, Bsp72I, BspAI, Bsp91I, BsrPH, Cpfl, Csp5I, CpeI, FnuEI, MspBI, SauCI, SauDI, SauEI, SauFI, SauGI and SauMI (Ro3). SfiI cannot cut M Bgll-modified DNA (Nel). SmiaI nicking occurs in the unmethylated strand of the hemi-methylated sequence CCmSCGGG/CCCGGG (Bu6,Wa5). SmaI may cut CmSCmSCGGG methylated DNA (Br8,Je2) Possibly the second methylation negates the effect of CCm5CGGG. There are conflicting results regarding SnaI: m5CCCGGG is not cut when modified by M AquI methyltransferase (Ka7) or at overlapping M HaeIII-SmiaI sites (GGm5CCCGGG, Ne5). Other investigators have reported that SmiaI cuts at a reduced rate at hemi-methylated m5CCCGGG sites (Bu6). SplI cuts GTm6AC-modified Chlorella virus NY2A DNA, but does not cut KpnI-digested XP12 DNA (Ne4). StySBI and StySPI are Type I restriction endonucleases. mT represents a 6-methyladenine in the complementary strand. TaqI cuts very slowly at Thm5CGA (Hul). TaqI cuts the fully m5C substituted phage XP12 DNA (Hul,Ne5). M-TaqI methylates Tm5CGA at least 20 fold slower that unmodified TCGA (Mc7). XbaI will cut Tm5CTAGA/TCTAGA hemi-methylated DNA at high enzyme levels (>100U Xba 1/ug), but will not cut this sequence in twenty to forty-fold overdigestions. XhoIl nicking occurs slowly in the unmethylated strand of the hemi-methylated sequence RGATmSCY/RGATCY. XmaI is claimed not cut CCm5CGGG in one report (Br8). See reference Bu6 for rate effects. XmnI cuts the fully m5C substituted phage XP12 DNA (Ne5). XmnI cuts slowly at some sites in DNA methylated on both strands at GAAN4TTm5C (Ne5). 2062 Nucleic Acids Research, Vol. 19, Supplement TABLE II: DNA methyltransferases and their modification specificities Cloned methylases in bold. Methylase a Specificity a References GACGTC Lu2 GTMKm6AC Lu2 M-A=I CITAAG (m6A) Lu2 M-AaK21 GATiP"C Sli M-&R AGmSCT Kr2,Lu2 M-Alw26I GT'i5CTC Bu4 and Gm6AGAC Bu4 M ApaI GGGm5CCC Mc8,Tr2 MAgul m5CYCGRG Ka7,Ka8 A1TAAT Mo3 M Asell CCSGG Mo3 M-AvaIM*RALu, CYCGRG Lu2 M.AI GGWCC Lu2 M*EAwI CYCGRG Lu3 MOAI TGGm5CCA Lu2,Mc8 Gm6ATC b Di3 M-BamHl GGATm4CC Hal,Lu2,Na3 GmCWGC? Hal M.A.=I GGYRCC Lu2 M-BalIs GRGCYC Lu2 M-BbvI Gm5CWGC Dol,Hal,Va5 M-BbvSI GmCWGC Hal,Va5 M-Bbv Gm6AT Hal M-'Bb Am6AG Hal Cm4CSGG Ja4,Ja6,Ja7,Pe2,Po6 M-BI m5CGCG Ku2 MIRa GCCN5GGC (m4C) Lu2 GGWCmC Ma9 M*D..a,I GGATmCC Kil M-BSRI GGm5CC Fe2,Ko1,Po2,Qi3,Sz4,Ve1 M.B106I ATCGm6AT Pa2 M.BA6I GCNCG Ja3 M-*I GGANPCC Le2 M*flVI CTCGm6AG Ba7 M-LYI RGATmCY Va2 M-BsuBI CTGCm6AG Xul M-*MEI m5CGCG Gal,,Gu7,Lkl,Jel M BzIFl m5CCGG Gu7,Ik1,Jel,Wa7 M*DhMI YTm5CGAR Gul,Gu2,Gu7,Jel,Shl M-'wp3T GGm5CC Bel,Gu5,Gu4,No2,No3 and Gm5CNGC Nol,Trl Nucleic Acids Research, Vol. 19, Supplement 2063 Methylase a Specificitv a Reference M Bslu.plI GGm5CC Gu4,Gu5,Gu7,Nol,No2 and Gm5CNGC M .lDjplls GGCC Bel and GDGCHC M-BMQI mCCGG Je2 M-BsuRI GGm5CC b Gu6,Ki2,Ki3 M-BjSPf3 GGm$CC Gu4,Gu5,Gu7,Je2,Ki2,N16 and Gm5CNGC No2,Trl,Tr3 M-ILMSPRI GGm5CC Bel,Gu5,Gu7,No2 and mSCmSCGG Pol and Cm5CWGG Be2,Bul,Gu3,Gu5,Ki2,Pol M *nSPRl91 m5Cm5CGG Je2,No2,Pol and CmCWGG M-BsuSPR83I GGm5CC Gu3 and Cm5CWGG M- A GCAN8GTGG Da2,Da3 M*fI YGGm5CCR Po6 M. 6I CAGm4CTG Bu5 M-Cfr9I Cm4CCGGG K13,Po6 M-L flOI Rm5CCGGY Po6 M-2 13I GGNm5CC Bi5 M-ClaI ATCGm6AT Mc3 M-CreI Tm5CR Sa2 (Chlamydomonas) M-Ctv Gm6ATC# Ri2 M CviBI Gm6ANTC X2 M *_viBIII TCGm6A Na4 M*fviJI RGm5Cy Sh3 MCviPI m5cc Xi4 M-CviQI GTm6AC Xi2,Xi5 M*£viRI TGCm6A Stl M-CviRII GTm6AC Stl M-DdIk m5CTNAG Ho3,Lu2,Sz3 M-D2gnII Gm6ATC Del,La3,La4,La5,Ma6 M-DInA ?Gm6ATC? Del M-EaeI YGGm5CCR Jal,Whl CGGCCG Sz2 MQIgM-EcaIdmIII GGTm6ACC Br2 Gm6ATC Br6,Bu9,Drl ,Gi2,Ha2,He2,Url M*LgM *EagImrdamI Cm5CWGG Bo5,MalO,So2,Url MEcaM-Ek dcm RmCCGG Bu8,Ne8 mCCWGG Ni2 M-Ef&A GGWCmC Mol,Ni2 Gm6AGN7GmTCA b Co7,Fu2 M-EQB TGm6AN8mTGCr b Go3 M-EcoD TTAN7GTCY b Go3 2064 Nucleic Acids Research, Vol. 19, Supplement Methylase a Specificitv a References M-*E&DXXI TCAN7ATTC b Skl GAGN7ATGC b Fu2 Am6ACN6GmTGC b Bo2,Go3,Kal,Lo2,Sal M*EM.cPI AGm6ACC b Ba2,Hu2 M-E.j Pl Gm6ATC b Co5 M.EoP1d5 Cm6AGCAG Hu2 M-E&QRIM-EnRlS GAm6ATTC Dul,Gr2,Ke2 Ne2,Ne9,Rul M-EcoRII Cm5CWGG Bh 1 ,Bu7,Bu8,Ko6,Ko7,Ko8 MalO,Sc6,Sol,Yo4 M-E.QRV Gm6ATATC Bo3 M As2R124 GAAN6RTCG (m6A) Pr2,Pr3 M-EcoR124/3 GAAN7RTCG (m6A) Pr2,Pr3 M.fEMT1 d. Gm6ATC Scl,Sc7 M'EFs&T2 ~z Gm6AT Br7,Ha2,Ha3,Mi 1,Sc4,Sc5 M-*EUT4iln Gm6ATC Ha4,Mal,Mi2,Sc3,Sc4,Sc5 M-Eco3 lI GGTm5CTC Bu4 and Gm6AGACC Bu4 M-E..v..47l GGNCC Po6 M-Eco5I CTGAAG (m6A) Po6 MM-Eg&57*coS7I CTGAAG (m6A) Po6 M-ESL64I GGYRCC Po6 M.ES&72I CACGTG (m5C) Po6 M -Er&98I AAGCT[ Po6 M*E. 105I TACGTA Po6 M-EM3I GGTm6CTC Ja3 GAGm6ACC M*EwiDI GGCC (m5C) Lu2,Val MEnuDIII m5CGCG Lu2,Nel M-EFokI GCGC Lu2 GGm6ATG La6,Lo3,Lu2,Ma8,Nwl M-E&kI and Cm6ATCC TGCGCA Mel ??Mr?? MTFV3 * - * * EsI (Frog virus) RGCGCY Lu2,S13 M-Ii.III GGm5CC b Lu2,Ma5,S13 M-HapII CmCGG Wal GACGC (mC) Lu2,Nwl M.kgCM-HdI GWGCWC Lu2 GGYRCm5C Er GGWCm5C Erl M-HgiEI GGWCm5C Er M-lhagI Gm5CGC Ba9,Ca3,Lu2,Sml,Wu 1 ,Zal M HhLIIn Gm6ANTC Chl,Kel,Ma3,Ma4,Sc9,Sml M*Hi,LCH GTYRm6AC Gr4,Mc8,,Ro7 Re2 Nucleic Acids Research, Vol. 19, Supplement 2065 Methylase a References M-lijndll GTYRm6AC Lu2,Re2,Ro6,Ro7 M-JjndIH m6AAGCIT Lu2,Ro6Ro7 MljfI Gm6ANTC Chl,Lu2 M-ElnPI GCGC Ba9,Lu2 GATATC (I6A) Dal M.H2 GTfAm6AC Br8,Yo3 cm5CGG Lu2,Ma5,Qul,Wi2,Yo2 M-HphI Tm5CACC Mc8,Ne2,Ne4 M-H2 GGCC La8 (Bacius phage) GCNGC GDGCHC M-Kpn2I TCCGGA Po6 Gm6ATC Mc8 M-MboII GAAGm6A Mcl2,Ne4,Ne2 M*MQll m5CG b Be6 (Mouse) m5CCGG b Eh2,Je2,Lu2,Nw2,Wal ,Wa5 M MstIT TGCGCA Mel M.MvaI Cm4CWGG K13,Po6 GCN7GC (m4C) Lu2,Lu4 GCCGGC Lu2,Val Neurospora ??m5.?? Se2 M .gI CCATGG (mC) Lu2,Val M-*I CATATG (m6A) Sil M-*NMVI GGNNCC b Pi5 M-.g2I RGCGCY b Ril M.NzgPI RGmCGCY b Su2 M-*2gII GGm5CC b Ko5,Ril M-NgoAI GGm5CC b Pi3 M.jgZPII GGm5CC b Su3,Su4 M*fQfi CCGCGG b Ko5,Ril M.NgQIV Gm5CCGGC b Ch2,Kol5,Ril MNjgqV GGNNm5CC b Ko5,Pi2 M.NgQVI Gm6ATC b Ko5 M-* VII GmCWGC b Ko5 M-NgQBI TP5CACC b Pi3 M-N2BII GTAN5m5CTC b Pi3 GGCC Mo3 M *laIII Cm6ATG Lal,Lu2,Mo3 M-NaIV Gm5CCGGC Lu2 GGNNm5 CC Mo3 M-EUR7 Ll1 cTCGm6AG Gi3,Thl,Th2 M*PaR7M-EmIx CTGCm6AG Lel,Wa3,Wa4,Wa6 M pvuII CAGm4CTG Bll,Ta2 M-E&4273I GTCGm6AC Ba6 2066 Nucleic Acids Research, Vol. 19, Supplement miScitv a GAm6ATTC Ba5,KalO M-iaII CCGCGG Lu2 M.hTI GTCGm6AC Lu2,Ro4,Ro5 M*53AM-Ifi GAPm5C Sel M*gg6I GGNm5CC Lu2,Nel,Szl M SfjI GGCCN5GGCC (m4C) Ba8 M*5jT GGWmSCC Ka5,Ka6 CCmCGGG Hel,Po6 M*XaI GCATGC Lu2 M*5a471 Gm6AATTC Ka9,Bu4 M'5o47II CmCNGG Ka9,Nil,Ni3 M*&MQI m5CG Nul,Pi5,Re3 CCWWGG Rel M*51ySBIM-T^li Gm6AGN6RmTYG b Ful,Fu3,Ga3,Nal,Na2 M*ityPI Am6ACN6GmTRC b Ful,Fu3,Nal,Na2 Am6ACN6RmTAyG b Ful,Fu3 Gm6AGN6GmTRC b Ga3 TCGm6A Lu2,Mc3,Sa3,S12 TCGm6A Mc3,Sa3 M*TflIM-MmaQ TCGm6A Sa3,Va6 Tetrahymena ??m6A??. Ca2,Gol TCrAGm6A Lu2,Mcl3,Val M*XmaI CCCGGG (m4C) Ba8 MXmaI CGGmCCG Mc8,Tr2 GAAN41TC Fel

NOTES a. See footnote "a" of Table I. b. See footnote "b" of Table I. Nucleic Acids Research, Vol. 19, Supplement 2067 TABLE III: Methylation sensitivity of Type 11 DNA methyltransferases. Methylase(specificity)a Not blocked by prior Blocked by prior References modification at b modification at b M*AluI (AGm5CT AGm4Cr Bu5 M-BamHI (GGATm4CC) GGm6ATCC GGATCmSC La7,MclO M-BI (GGATmCC)C GGm6ATCC Le2 M.Cft6I (CAGm4CTG) CAGm5CrG Bu5 M-CLaI (ATCGm6AT) m6ATCGAT M9,Mcl 1,Wel ATm5CGAT M-_viBIl (TCGm6A) Tm5CGA MclO,Va4 M*EoRI (GAm6ATTC) GAATIm5C Gm6AATTC Br2 M-*gRII (Cm5CWGG) Cm4CWGG Bu4 M-Eco dam (Gm6ATC) GATm5C c MclO GATtm5C GATm4C Ne4 M--EkIA (GGm6ATG)c CATCm5C CATm5CC Po3,Po4,Sc2 M-HhaI (Gm5CGC) GCGm5C Rol M.iUall (Gm6ANTC) GANTIm5C MclO M-paIl (Cm5CGG) m5CCGG Mc9,MclO M-HphI (Tm5CACC) GGTGm6A MclO M-MboI (Gm6ATC) GATm5C MclO M*MboII (GAAGm6A) T"5c1-1Tr5c MclO M-MsDI (m5CCGG) Cm5CGG MclO M-MvaI (Cm4CWGG) Cm5CWGG Bu4 m5cm5CwGG Nel M-PvwI (CAGm4CTG) CAGm5CTG Bu5 M-UT2 4am (Gm6ATY) GAThm5C Do2,Mil M-EcoT4 dam (Gm6ATC) GAThm5C Sc4 M-TaaI (TCGm6A) T5CGA c Mc7 a. See footnote "a" of Table L. b. An enzyme is classified as insensitive to methylation if it methylates the modified sequence at a rate that is at least one tenth the rate at which it methylates the unmodified sequence. An enzyme is classified as sensitive to methylation ifit is inhibited at least twenty-fold by methylation relative to the unmethylated sequence. c. See footnote "b" of Table I. 2068 Nucleic Acids Research, Vol. 19, Supplement TABLE IV: Isoschizomer/isomethylator pairs that differ in their sensitivity to sequence-specific methylationr. RLstQi iaWIKLQsAIn a,b Mcahykd sqam-ce I Cut by NQJcu by kfevnoe m4CCGG Nel MaulM-Ri Cm5CGG BanSnai Eh2,Mc1i Cm4CGG MaI Bu6 CCm5CGGG XmaTI (k9I) .Spl Bu6 Cm5CWGG LQRIISinaI Bu4 Gm6ATC SPM3Amm I:EmiEI) MboI (deIt) Gel,LulMc9,Ro3 GATm5C h3A Ne4 GAPm4C MboI Sa3A Ne4 GGCm5C Su4 GGTACm5C Mul GGTAmSCm5C HnoX Aau718I Ne4 GGWCm5C MI Avai (E&247 B3,Ja5,Wh2 RGm6ATCY X2LQo]1 W&tYI) Mc9,Ne4 Tm5CCGGA Acc]Ill mm La2,Sc2 TCm5CGGA Sc2 TCCGGm6A BsaMlI QMLQI) Ke3,Ne4 TCGCGm6A 13I (SaLI) LauMi Mcl 1,Ne4 lrsoCGAA As-ull Cau45I SclO CGGWCm5CG Rs-ril Qi3 Resiriction iso thylator pairs de Methylated sequence C methylated by NQt mehylated by References IP5CGA M.CviB[ (TCGm6A) M-IqI We2 a. In each row the first column lists a methylated sequence, the second column lists an isoschizomer that cuts this sequence, and the third column lists an isoschizomer that does not cut this sequence. b. An enzyme is classified as insensitive to methylation ifit cuts the methylated sequence at a rate that is at least one tenth the rate at which it cuts the unmethylated sequence. An enzyme is classified as sensitive to methylation if it is inhibited at least twenty-fold by methylation relative to the unmethylated sequence. c. See footnote "a" of Table I. d. In each row the first column lists a methylated sequence, the second column lists an isomethylator that modifies this sequence, and the third column lists an isomethylator that does not modify this sequence. e. An enzyme is classified as insensitive to methylation if it odifies the methylated sequence at a rae that is at least one tenth the rate at which it modifies the unmethylated sequence. An enzyme is classfied as sensitive to methylation if it is inhibited at least twenty-fold by methylation relative to the unmethylated sequence. Nucleic Acids Research, Vol. 19, Supplement 2069 TABLE V: List of restriction systems referred to in this paper.a Note: a. Restrction systems in Table V are arranged by recognition sequence length and alphabetically by recognition sequence to aid in identifying isoschizomers.

CC Cvi NY RGCY CATG Nl--- CCTC AGCr AluI CCGG BsuFI, sQI,HaPI, aII, MsI CGCG AccII, eI, BUI, _B_E, h-nDII ThaI Gm6ATC vnI,blanLL LaI,Nf uEI GATC Br243I, RuPI, R67L BAL RPII, RPli, DGI, DEII &LXII:,sCKQj_.I,, CviAI,, 12M.Hl, fMAII, f=CI, ErmaEI, MboIL M-m-eII, Avail,e2lM 6I, Eh,Q47I, hjjil, kIjgjE, SAInI I, fLIS S:inNff GCGC HhI PI GGCC GTAC CviQIP1 TCGA 1TAA mMsI CCNGG 5crFI CTNAG GANTC AviBI, InI, Hitl GGNCC H13I, Su96I CCWGG AacI, -AorI, AYI, A=BI, AWL RiSL, Bsj2NI, BsGIII, BhNI, 51I, ZIII,EcaII,EclII,EcoRII &g27I,, Ec3I M_I,MaI, T XI CCSGG Mki,BcnI, SI.Nxil GCAGC BgIJi GGWCC A-vaE[,TkI)igiliJB-me216I, EQ47I,, HdCIEI HgEl, SiLnl AGACC EcoPI ACCTGC CAGCAG HnPC15 CATCC FQkI GAAGA MbolII.NcuI GAAGAG EarI GAATGC BsmI GACCGA and CACCCA GACGC HgaI GATGC AMI GGATC GTCTC BamAI TCACC H12hl, NgoQBI GDGCHC S12861 CYCGRG &UL A-vaI GRCGYC GRCGYC GRGCYC AbA GTMKAC GTYRAC HLCH 2070 Nucleic Acids Research, Vol. 19, Supplement GWGCWC HgiM RCCGGY 210 RGCGCY HliMI, .jgPI RGATCY BWstYI, XMQIi, YGGCCR RGGNCCY Dril AAGQT WndIll ACGCGT ACTAGT S1I AGATCT AGCGCT Axll, L47fI AGGCCT SmI ATCGAT ATGCAT Lull, LaXI h CAGCTG CATATG NdI CCATGG £k911,"QI SinI, ma CCCGGG -CO Lml X-- CCGCGG CGATCG BmaDI, EnI, LhIi, XQEII CGGCCG CGTACG Eflt SDIi CTCGAG BsuMI, LuRII, PR7I, XhbI CTGCAG BUBI, FitI, SflI CrrAAG Afll GAATTC jQQRI, RLI, £bo47I GACGTC GAGCTC SAQ£I GATATC EQQRV GCATGC SahI GCCGGC Mb927311, NaeI GCGCGC BssHll GCTAGC NheI GGATCC BanHfl, LmFI, BamKI iamNi RLa Bil, 1503I GGCGCC fbQ&It Nir GGTACC As7181, KpnI GGGCCC A aI GTCGAC Rrh42731, SaI GTGCAC GTTAAC HpaI TCATGA BspHI, LaXI TCCGGA Accl, MI, Kpa2l, II TCGCGA AmzI, MhuI9273l TCGCGA NI, SJlDI, S13I, SpQj TCrAGA TGATCA AaCI, Li,BcIXllB BxGL -£pI TGCGCA TGGCCA aII rfCGAA Asall, fBLtBI, CW451 TTTAAA Diii CCAN6TGG WfaX,llI CCTNAGG MStlI GAAN4TTC XmnI GATN4ATC MimI GCCN5GGC Nucleic Acids Research, Vol. 19, Supplement 2071 GCTNAGC GGTNACC BEII, Ecal CGGWCCG X4VI, R Il GAAN6RTCG &QR124 GAAN7RTCG &gR124/3 AACN6GTGC EcgK AACN6GTRC StSPI GAGN7ATGC GAGN7GTGA EcoA GAGN6RTAYG StySBI TCAN7ATTC EcoDXXl TGAN8TGCT EcoB TTAN7GTCY EcoD CRGCGGYG SAI GGCCGGCC F&I GCGGCCGC NotI GGCCN5GGCC LiI