Effect of site-specific methylation on restriction endonucleases and DNA modification methyltransferases

Abstract

We present in Table I an updated list of the sensitivities of 298 restriction endonucleases and 20 DNA methyltransferases to sitespecific modification at 4-methylcytosine (m4C), 5-methylcytosine ('OC), 5-hydroxymethylcytosine (hm5C), and 6-methyladenine (m6A) (McC 14), four modifications that are common m the DNA of prokaryotes, eukaryotes, and their viruses (Mc2,Mc5,Mc8,Mcl 1,Ne3,Ne4). In addition, new information is included on restriction endonuclease cleavage at sites modified with 5-hydroxymethyluracil (h'5U). Knowledge of the sensitivity of restriction endonucleases to site-specific modification can be used to study cellular DNA methylation. Several restriction-modification enzymes share the same recognition sequence specificity, but have different sensitivities to site-specific methylation. Table II lists 32 known isoschizomer pairs and one isomethylator pair, along with the modified recognition sites at which they differ. The data presented here and an additional three other tables are available in printed form or as a text file on a 3.5" Macintosh diskette. The extra tables include Table III which is a list of over 205 characterized DNA methyltransferases. A detailed list of cloned restriction-modification genes has been made by Wilson (Wi4). Table IV lists the sensitivities of over 20 Type II DNA methyltransferases to m4C, m5C, hm5C, and m6A modification. Most DNA methyltransferases are sensitive to non-canonical modifications within their recognition sequences (Bu9,Mc10, Ne3,Po4), and this sensitivity often differs from that of their restriction endonuclease partners. Table V gives a list of restriction systems in this review alphabetized by recognition sequence. The data can be supplied as a Microsoft Word, Macwrite or MS-DOS file. Please contact Michael McClelland at CIBR, phone 619 535 5486, FAX 619 535 5472. Molecular basis for sensitivity restriction enzymes to methylation m4C, m5C, hm5C, and m6A are bulky alkyl substitutions in the major groove of DNA. Site-specific DNA methylation can interfere with many sequence-specific DNA binding proteins (e.g. St2,Wa8), including binding of restriction endonucleases and DNA methyltransferases. At the molecular level, the inability of restriction enzymes to cut modified DNA can be explained using EcoRI and EcoRV endonucleases as instructive models. DNA modification can interfere with substrate binding and/or conformational changes of the enzyme: substrate complex. Based on the EcoRI: DNA co-crystal structure (MclS,Ro8), methylation of either adenine (Gm6AATTC or GAm6ATTC)

Modification of Subtelomeric DNA

“…Neither MboI (GATC) nor NlaIII (CATG) are sensitive to cytidine methylation. NlaIII sites are sensitive to adenine methylation in bacterial cells (22,25), but such modifications have not been observed in mammalian DNA. The thymidine modification beta-D-glucosylhydroxymethyluracil (base J) is present in repetitive sequences, primarily telomeric DNA, of kinetoplastid protozoans such as trypanosomes (10,11,29).…”

Defining the Plasmid-Borne Restriction-Modification Systems of the Lyme Disease SpirocheteBorrelia burgdorferi

“…S3 and S4 in the supplemental material). RsaI cleavage is blocked by adenine methylation of a GTAC restriction site or by overlapping cytosine methylation of CGTAC or GTACG sequences (43). The latter sequences, however, are not among the protected RsaI sites present on B. burgdorferi shuttle vectors (Fig.…”

The Helicobacter pylori genome is modified at CATG by the product of hpyIM

“…M.HpyI also has strong homology to M.LlaI, M.LlaPI, M.FokI, and M.StsI, which have the same modification site, GGATG. FokI cleaves DNA specifically at GGATG sites, but when the adenine residues are methylated these sites are resistant to FokI digestion (36). Therefore, FokI was selected to test whether GGATG would be modified by M.HpyI and to test the specificity of modification at CATG sites by M.HpyI.…”

“…In N. lactamica, nlaIIIR is located immediately upstream of nlaIIIM (35) and encodes M.NlaIII, which is highly homologous to M.HpyI, which is encoded by hpyIM. That restriction and modification genes usually are closely linked in bacteria (36) suggests that iceA1 encodes a putative NlaIII-like restriction endonuclease. One explanation for the low frequency of obtaining hpyIM insertion mutants is that a second mutation presumed necessary inactivated iceA1 in the iceA1 strain to prevent digestion of unmodified DNA by an NlaIII-like endonuclease.…”