A novel activity in Escherichia coli K-12 that directs restriction of DNA modified at CG dinucleotides

Abstract

The restriction systems McrA and McrB of Escherichia coli K-12 are known to attack DNA containing modified cytosine. In strains lacking both activities, however, we observed that DNA methylated at CG dinucleotides (as is mammalian DNA) was still significantly restricted. We show that this substantial barrier to the acceptance of 5-methylcytosine-containing DNA is attributable to a hitherto unknown activity of the Mrr restriction system. Strikingly, the multiple systems used by this gut inhabitant to determine the fate of invading DNA will all limit genetic exchange with its mammalian host(s), reinforcing the idea that one role of DNA methylation is to serve as a "molecular passport" (E.

In Vitro CpG Methylation Increases the Transformation Efficiency ofBorrelia burgdorferiStrains Harboring the Endogenous Linear Plasmid lp56

“…The commercially available CpG methylase M.SssI, which methylates both unmethylated and hemimethylated DNA and requires only a 2-nucleotide sequence (CG) for recognition (24), is capable of blocking a wide range of restriction enzymes. CpG methylation confers sensitivity to some restriction enzymes, such as the E. coli McrA, McrBC, and Mrr restriction systems (12,38), but identifiable homologs of these enzymes are not present in the B. burgdorferi genome (8; http://cmr .tigr.org). The widely used Kan r B. burgdorferi shuttle plasmid pBSV2 (36), a pBSV2-derived Str r plasmid, pKFSS1 (7), and its derivative, pKFSS1k32 (see Table 3 below), were modified using M.SssI.…”

Characterization and expression of the Escherichia coli Mrr restriction system

“…We had initially screened many cytosine methylase-containing constructs but found none that were strongly restricted. Kelleher and Raleigh's (23) was noted in the efficiency of transformation of an NlaIII methylase clone, p(pUC)NlaIIIM-H64. In RR1 containing p(pACYC184)mrr6.3-4, the methylase-carrying plasmid was restricted 20-fold compared with restriction in RR1.…”

Transposon-Mediated Linker Insertion Scanning Mutagenesis of the Escherichia coli McrA Endonuclease

“…A further inference can be drawn; there may be a privileged location in that contains CCGG and is particularly sensitive to interference by DNA binding proteins. This inference rests on the observation that not all McrBC-sensitive methyltransferases give substrates that behave like .MspI (7,14). Thus, binding of McrB at different locations, as dictated by a different methylation patterns, yields different behaviors, but binding of a wholly different protein to a similar set (or subset) of sites yields similar behavior.…”

“…Furthermore, evidence that McrA actually cleaves DNA is indirect, consisting of the bacteriophage restriction phenomenon and the ability of a partially disabled mcrA gene to mediate induction of a DNA damage reporter in the presence of a sensitive methyltransferase gene (22). Substrates sensitive to McrA restriction in vivo include those modified by M.HpaII (C 5m CGG), M.Eco1831I (C 5m CSGG), and M.SssI ( 5m CG) (14,16,27); however, a comprehensive survey of 5-methylcytosine methyltransferases with regard to McrA restriction has not yet been undertaken, so the precise recognition sequence remains unclear.…”