MmeI: a minimal Type II restriction-modification system that only modifies one DNA strand for host protection

Abstract

MmeI is an unusual Type II restriction enzyme that is useful for generating long sequence tags. We have cloned the MmeI restriction-modification (R-M) system and found it to consist of a single protein having both endonuclease and DNA methyltransferase activities. The protein comprises an amino-terminal endonuclease domain, a central DNA methyltransferase domain and C-terminal DNA recognition domain. The endonuclease cuts the two DNA strands at one site simultaneously, with enzyme bound at two sites interacting to accomplish scission. Cleavage occurs more rapidly than methyl transfer on unmodified DNA. MmeI modifies only the adenine in the top strand, 5′-TCCRAC-3′. MmeI endonuclease activity is blocked by this top strand adenine methylation and is unaffected by methylation of the adenine in the complementary strand, 5′-GTYGGA-3′. There is no additional DNA modification associated with the MmeI R-M system, as is required for previously characterized Type IIG R-M systems. The MmeI R-M system thus uses modification on only one of the two DNA strands for host protection. The MmeI architecture represents a minimal approach to assembling a restriction-modification system wherein a single DNA recognition domain targets both the endonuclease and DNA methyltransferase activities.

The MmeI family: type II restriction–modification enzymes that employ single-strand modification for host protection

“…For RpaB5I the concentration of sites supplied in trans needed to stimulate cutting of the single site DNA was approximately equimolar (0.01 µM) with the concentration of recognition sites (0.007 µM) in the single site DNA (Figure 5C), in a reaction that contained two units of RpaB5I. These results are quite similar to those obtained for MmeI (12).

Figure 5.Cleavage of a single site substrate is incomplete but can be stimulated by in trans DNA containing a specific recognition site.

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Next-generation DNA sequencing of paired-end tags (PET) for transcriptome and genome analyses

“…MmeI cuts DNA 18/20 bp downstream of its recognition site (Morgan et al 2008). Use of MmeI enabled the development of LongSAGE to produce 20-bp tags (Saha et al 2002).…”

Bifunctional Prokaryotic DNA-Methyltransferases

“…As can be seen from Table 1, the enzymes were grouped according to their domain organisation as it was presented in Conserved Domain Database [9]. As could be judged from their domain organisation, 20 new bifunctional REases are thought to represent the fusion of a REase with MTase and target recognition subunits of the type I restriction-modification systems (R-M-S structure), having similar organisation with the known type IIC bifunctional enzymes such as AloI [10], CjeI [11], MmeI [12], PpiI [13], TstI [13] and TspGWI [14]. Type I RMS enzymes are multisubunit proteins that function as a single protein complex, consisting of R, M and S subunits [3].…”

“…This is perhaps why it is the only found example of natural bifunctional RMS originating from type II RMS. The newly found potential type IIC proteins are good candidates to expand on the current list of 12 bifunctional enzymes: AloI [10], BcgI [17], BseMII [18], BseRI [19], BspLU11III [20], CjeI [11], Eco57I [15], HaeIV [21], MmeI [12], PpiI [13], TstI [13] and TspGWI [14]. Taking into consideration intensiveness with what new microbial genomes have been sequencing during the last decade, new bifunctional RMS could be discovered very soon.…”