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

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

Richard D Morgan et al. Nucleic Acids Res. 2008 Nov.

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.

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Figures

Figure 1.
Figure 1.

DNA sequence containing MmeI and predicted flanking genes from M. methylotrophus genomic locus. (A–D) are uncharacterized putative ORFs. RE sites are numbered from the 5′-EcoRI site (in bp). The four MmeI sites are listed with the arrow indicating the orientation of the site: 5′-TCCRACN20/N18-3′.

Figure 2.
Figure 2.

Detection of MmeI modification using antibodies specific for N6-methyladenine (m6A) and N4-methylcytosine (m4C). (Top row) unmethylated DNA; (second row) MmeI in vitro modified DNA; (third row) M.EcoRI in vitro modified DNA; (fourth row) M.BamHI in vitro modified DNA. Positive controls: m6A antibody panel; (fifth row) dam (m6A) in vivo methylated pUC19 DNA (400–25 ng dilution), m4C antibody panel; (fifth row) M.EsaBC3I (m4C) in vivo methylated plasmid DNA (400–25 ng dilution).

Figure 3.
Figure 3.

Endonuclease digestion of MmeI in vitro modified or unmodified DNA (150-bp PCR product across pMMHI polylinker region) containing a HinfI site overlapping the top strand of the MmeI site: 5′-TCC

GACTC

-3′ and an MboI site overlapping the bottom strand of the MmeI site: 5′-GTCG

GATC

-3′. The restriction endonucleases were mixed with buffer, aliquoted into three reactions to which were added MmeI modified DNA, unmodified DNA or both DNAs. Size standard: pBR322-MspI.

Figure 4.
Figure 4.

(A) In vivo MmeI Modification. MmeI endonuclease digestion of purified plasmid DNA from the construct expressing active or inactive MmeI. ‘Active’ is the MmeI expression plasmid, pTBMmeI.1. ‘Inactive’ is a plasmid DNA derived from pTBMmeI.1 that carries a single point mutation (N773D) that renders MmeI inactive. Addition of PhiX174 DNA verifies that the added MmeI endonuclease is active in the reaction wherein pTBMmeI.1 DNA is not cut. (B) In vitro MmeI modification. pMMH1 DNA from a dam-deficient host, either modified in vitro by MmeI or not modified, digested with MmeI, HinfI or MboI. (C) pMMH1 DNA from a dam-proficient host digested with MmeI, MboI and DpnI. M: Size standards: Lambda-HindIII, PhiX174-HaeIII.

Figure 5.
Figure 5.

Digestion of synthetic DNAs containing modified or unmodified adenines in the MmeI recognition sequence. (A) UU, fully unmethylated DNA. (B) UM, containing N6-methyl adenine in the bottom strand, 5′-GTYGG(m6A)-3′, of the MmeI recognition sequence. (C) MU containing N6-methyl adenine in the top strand, 5′-TCCR(m6A)C-3′, of the MmeI recognition sequence. (D) MM containing N6-methyl adenine in the both strands of the MmeI recognition sequence. Lanes: 1 = no enzyme; 2 = MmeI (TCCRAC); 3 = Hpy188I (TCNGA); 4 = HinfI (GANTC); 5 = MboI (GATC); 6 = BfuCI (GATC). M: pBR322-MspI size standard. DNA recognition sites: 6 = m6-adenine, MmeI site 5′-TCCGAC-3′ in ‘bold’, HinfI site 5′-GANTC-3′ top strand underlined, MboI and BfuCI site 5′-GATC-3′ bottom strand underlined.

Figure 6.
Figure 6.

MmeI modification following endonuclease cleavage. ‘Cut’ indicates lambda DNA digested with MmeI (2 U/μg DNA) for 1, 5, 15 and 60 min; ‘Lig’ indicates the cut lambda DNA purified from MmeI and ligated into concatamers; ‘Recut’ indicates subsequent MmeI digestion of the ligated DNA concatamers.

Figure 7.
Figure 7.

Relative rates of MmeI endonuclease cleavage and DNA methyltransferase activities. MmeI (2 Us/μg) digestion of Phix174 DNA with 1 μM [methyl-3H]-AdoMet over a time course of 0, 0.25, 1, 5, 15, 60 and 120 min. 3H counts are reported for an equal amount of DNA to that on the gel. The extent of endonuclease cleavage was estimated from the MmeI fragment pattern. The extent of methylation was calculated as a percent of the maximal counts reached at 60 min, subtracting the background counts from each count.

Figure 8.
Figure 8.

MmeI cleavage of a single site substrate is incomplete but can be stimulated by in trans DNA containing an MmeI site. (A–E) MmeI digestion in a 2-fold serial dilution from 16 U/μg DNA to 0.125 U/μg DNA on p996SS1 DNA previously linearized by digestion with PstI. (A) no trans DNA; (B) 0.2 μM M12 trans DNA; (C) 0.02 μM M12 trans DNA; (D) 0.2 μM NmeA30 trans DNA that does not contain an MmeI site; (E) 0.2 μM N6-adenine methylated (5′-TCCG(m6A)C-3′) M12 trans DNA; (F) 2-fold serial dilution of the M12 trans DNA from 0.16 μM to 0.001 μM in a digestion reaction containing 4 U (0.026 μM) MmeI/μg substrate DNA. Arrows indicate ‘linear’ DNA (uncut by MmeI) and ‘cut’ DNA that is the MmeI cleavage products.

Figure 9.
Figure 9.

MmeI cleaves the two DNA strands at one site simultaneously. (A) Time course of MmeI digestion of supercoiled pUC19 DNA (2 U/μg) for 10 s, 20 s, 30 s, 1, 3 10, 20, 30 and 60 min. Supercoiled plasmid (sc) is converted directly to linear (lin) DNA, with no accumulation of open circular DNA (oc). ‘A+B cut’ indicates the 184-bp product of MmeI cleavage at both pUC19 sites. (B) MmeI digestion of linear pUC19 DNA (previously cut with PstI), in a 2-fold serial dilution from 8 to 0.03 U/μg. MmeI cuts at a one site, forming products from either site A or site B, before forming product from cleavage at both sites (A+B). ‘lin’ indicates linear pUC19, ‘A-R’ and ‘A-L’ indicate the cleavage products from MmeI cutting at the 996 site, ‘B-R’ and ‘B-L’ indicate the cleavage products from MmeI cutting at the 1180 site, while ‘A+B’ indicates the cleavage product from MmeI cutting at both sites.

Figure 10.
Figure 10.

MmeI cleaves DNA containing a newly synthesized (unmodified) top strand and a genomic M. methylotrophus bottom strand. MmeI digestion of DNAs from one round of primer extension. Lanes 2–5 are the newly synthesized top strand, while lanes 6–10 are the newly synthesized bottom strand. Lanes 2 = uncut; 3 = 20 U MmeI; 4 = 5 U MmeI; 5 = 10 U BspHI; 6 = uncut; 7 = 20 U MmeI; 8 = 5 U MmeI; 9 = 20 U MmeI digestion of both newly synthesized bottom strand and top strand DNA (as a positive control for MmeI activity); 10 = 10 U BspHI. Lanes 1 and 11: PhiX174-HaeIII size standard. MmeI cuts the DNA containing a genomic M. methylotrophus bottom strand and an unmodified top strand (lanes 3, 4 and 9), but not the DNA containing a genomic M. methylotrophus top strand and an unmodified bottom strand (lanes 7, 8 and 9). The native host DNA from M. methylotrophus is thus modified to prevent MmeI cleavage only in the top DNA strand of the MmeI recognition sequence.

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