Cytosine modification in DNA by Bcn I methylase yields N 4 ‐methylcytosine

100

The DNA base composition, including the minor base content, of 26 strains of bacteria was determined. The studied bacteria are sources of widely used restriction endonucleases. Approximately 35% of the bacterial DNAs contained N4-methylcytosine, about 60% contained 5-methylcytosine, and about 90% had N6-methyladenine.Both N6-methyladenine (m6A) and 5-methylcytosine (m5C) have been long known to be minor bases in bacterial DNA (7,9,16). One or both of these methylated bases are present in most bacterial DNAs examined (4,11,33). Recently, a third minor base, N4-methylcytosine (m4C), has been found in DNA from eight types of thermophilic bacteria (11) and in that from one type of mesophilic bacteria (19,20). Previously, m4C residues had been detected only in the RNA of the small ribosomal subunit of bacteria and of mammalian and insect mitochondria (8,13,40). This modified base is not present in a variety of eucaryotic DNAs (13; Gehrke et al., unpublished results).The minor base composition of bacterial DNA is partially determined by restriction-modification systems (30). The level of minor bases should be consistent with the known specificities of the host restriction and modification enzymes. Also, it can reveal the presence of new DNA methyltransferases. For example, if the known restriction enzymes in a bacterium have recognition sites containing only G. C base pairs and if m6A as well as m5C is found in this cellular DNA, then one or more other unrelated modification pathways must be present. These could be involved in previously unidentified restriction-modification systems in the bacterium or in the control of various DNA functions (3,15,29,35).The present study demonstrates that genomic m4C is not mostly limited to the DNA of thermophilic bacteria. Rather, it is present as a minor base in the DNA of many bacterial mesophiles. Of the 26 bacterial species examined in this study, 9 contained m4C in their genomes. Most of the examined bacteria are mesophiles, and all of them are sources of commercially available restriction endonucleases. The prevalence of m4C as a minor base in these bacterial DNAs indicates that many restriction endonucleases may be inhibited by N4-methylation of cytosine residues in their recognition sites in vivo and that bacterial DNA cytosine methyltransferases must be carefully checked to determine whether they catalyze the formation of m4C. MATERIALS AND METHODSStrains. All bacteria came from the New England BioLabs strain collection except Xanthomonas campestris pv. oryzae, which was from the collection of M. Ehrlich. The * Corresponding author. strains are listed in Table 1 with the sources from which they were originally obtained and the temperature at which the cultures were grown.Purification of bacterial DNA. DNA was prepared from 10 g of wet packed cells suspended in 20 ml of 25% sucrose-50 mM Tris hydrochloride (pH 8.0), and then 10 ml of 0.25 M disodium EDTA (pH 8.0) and 6 ml of 10-mg/ml lysozyme in 0.25 M Tris (pH 8.0) were added. After 2 h at 0°C, 24 ml of 1% Triton X-100 in ...

Section: Resultssupporting

confidence: 75%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

N4-methylcytosine as a minor base in bacterial DNA

Ehrlich¹,

Wilson²,

Kuo³

et al. 1987

100

“…This fact raises the possibility that SuaI may generate 5mC at its recognition sites, which occur elsewhere in the S. acidocaldarius genome, and promote high rates of C-to-T transition at these sites. An alternative explanation is based on the observation that a number of R-M systems methylate the exocyclic N of cytosine to yield N 4 mC (14). This modified base occurs in the DNAs of both mesophiles and thermophiles (6) and thus does not seem to correlate strongly with high growth temperature.…”

mentioning

confidence: 99%

Cytosine Methylation by the Sua I Restriction-Modification System: Implications for Genetic Fidelity in a Hyperthermophilic Archaeon

Grogan

2003

5-Methylcytosine in chromosomal DNA represents a potential source of frequent spontaneous mutation for hyperthermophiles. To determine the relevance of this threat for the archaeon Sulfolobus acidocaldarius, the mode of GGCC methylation by its restriction-modification system, SuaI, was investigated. Distinct isoschizomers of the SuaI endonuclease were used to probe the methylation state of GGCC in native S. acidocaldarius DNA. In addition, the methylation sensitivity of the SuaI endonuclease was determined with synthetic oligonucleotide substrates and modified natural DNAs. The results show that the SuaI system uses N 4 methylation to block cleavage of its recognition site, thereby avoiding the creation of G ⅐ T mismatches by spontaneous deamination at extremely high temperature.

“…In general, two enzymes compose a restriction-modification system: an endonuclease or restriction enzyme that cleaves a specific DNA sequence and a methylase that modifies the same sequence by transferring methyl groups from Sadenosylmethionine to either adenine (N-6 position) or cytidine (C-5 or N-4 position [13]). Methylation blocks cleavage by the restriction enzyme and thereby prevents the destruction of cellular DNA.…”

mentioning

confidence: 99%

Site-specific methylases induce the SOS DNA repair response in Escherichia coli

Heitman¹,

Model²

1987

151

115

Expression of the site-specific adenine methylase Hhall (GmANTC, where me is methyl) or PstI (CTGCreAG) induced the SOS DNA repair response in Escherichia coli. In contrast, expression of methylases indigenous to E. coli either did not induce SOS (EcoRI [GA'mATTC] or induced SOS to a lesser extent (dam [GreATCJ). Recognition of adenine-methylated DNA required the product of a previously undescribed gene, which we named mrr (methylated adenine recognition and restriction). We suggest that mrr encodes an endonuclease that cleaves DNA containing N6-methyladenine and that DNA double-strand breaks induce the SOS response. Cytosine methylases foreign to E. coli (MspI [reCCGG] [9070][9071][9072][9073][9074] 1986).Many species of bacteria make restriction and modification enzymes to destroy foreign DNA that enters the cell (1). In general, two enzymes compose a restriction-modification system: an endonuclease or restriction enzyme that cleaves a specific DNA sequence and a methylase that modifies the same sequence by transferring methyl groups from Sadenosylmethionine to either adenine (N-6 position) or cytidine (C-5 or N-4 position [13]). Methylation blocks cleavage by the restriction enzyme and thereby prevents the destruction of cellular DNA. A restriction-modification system thus enables the cell to selectively destroy unmethylated foreign DNA.Restriction-modification systems from various bacterial species have been cloned in Escherichia coli (2,9,19,36). We have studied the HhaII system of Haemophilus haemolyticus, cloned by Mann et al. (19), and have found that HhaII methylase expression drastically inhibits growth of several standard E. coli K-12 lab strains. In this report, we show that this results from insults to the DNA which elicit the SOS DNA repair response.DNA-damaging agents (UV light and mitomycin C) or conditions which interfere with DNA replication (presence of nalidixic acid) induce a set of 20 or so genes responsible for a myriad of physiological effects termed the SOS response (for reviews, see references 39 and 40). Cell growth slows, the cellular replication machinery is altered, mutagenesis occurs, a number of DNA repair processes take effect, and X prophages are induced in lysogenic strains. Both A and the SOS genes are induced by a common cascade of steps initiated by the original DNA insult. RecA protein is activated to RecA* by damaged DNA, and RecA* then promotes the cleavage of the X cI repressor and the LexA protein, the transcriptional repressor of the SOS genes. In this manner, both SOS and X are induced by DNA damage.In this report, we show that, in general, methylases foreign to E. coli induced the SOS response. Recognition of adenine-methylated DNA required the product of a hitherto undescribed locus, which we named mrr (methylated adenine recognition and restriction). We suggest that mrr en-* Corresponding author.codes an endonuclease that cleaves adenine-methylated DNA and that DNA scission induces SOS. Similarly, the SOS response was induced by cytosine methylation, and this eff...