Organization of the BcgI restriction–modification protein for the transfer of one methyl group to DNA

The Type IIB restriction–modification protein BcgI contains A and B subunits in a 2:1 ratio: A has the active sites for both endonuclease and methyltransferase functions while B recognizes the DNA. Like almost all Type IIB systems, BcgI needs two unmethylated sites for nuclease activity; it cuts both sites upstream and downstream of the recognition sequence, hydrolyzing eight phosphodiester bonds in a single synaptic complex. This complex may incorporate four A₂B protomers to give the eight catalytic centres (one per A subunit) needed to cut all eight bonds. The BcgI recognition sequence contains one adenine in each strand that can be N⁶-methylated. Although most DNA methyltransferases operate at both unmethylated and hemi-methylated sites, BcgI methyltransferase is only effective at hemi-methylated sites, where the nuclease component is inactive. Unlike the nuclease, the methyltransferase acts at solitary sites, functioning catalytically rather than stoichiometrically. Though it transfers one methyl group at a time, presumably through a single A subunit, BcgI methyltransferase can be activated by adding extra A subunits, either individually or as part of A₂B protomers, which indicates that it requires an assembly containing at least two A₂B units.     

Binding of MmeI Restriction–modification Enzyme to its Specific Recognition Sequence is Stimulated by <Emphasis Type="Italic">S</Emphasis>-adenosyl-<Emphasis Type="SmallCaps">l</Emphasis>-methionine

Restriction endonucleases serve as a very good model for studying specific protein–DNA interaction. MmeI is a very interesting restriction endonuclease, but although it is useful in Serial Analysis of Gene Expression, still very little is known about the mechanism of its interaction with DNA. MmeI is a unique enzyme as besides cleaving DNA it also methylates specific sequence. For endonucleolytic activity MmeI requires Mg(II) and S-adenosyl-l-methionine (AdoMet). AdoMet is a methyl donor in the methylation reaction, but its requirement for DNA cleavage remains unclear. In the present article we investigated MmeI interaction with DNA with the use of numerous methods. Our electrophoretic mobility shift assay revealed formation of two types of specific protein–DNA complexes. We speculate that faster migrating complex consists of one protein molecule and one DNA fragment whereas, slower migrating complex, which appears in the presence of AdoMet, may be a dimer or multimer form of MmeI interacting with specific DNA. Additionally, using spectrophotometric measurements we showed that in the presence of AdoMet, MmeI protein undergoes conformational changes. We think that such change in the enzyme structure, upon addition of AdoMet, may enhance its specific binding to DNA. In the absence of AdoMet MmeI binds DNA to the much lower extent.     

The methyl donor S-Adenosylmethionine inhibits active demethylation of DNA: a candidate novel mechanism for the pharmacological effects of S-Adenosylmethionine

S-Adenosylmethionine (AdoMet) is the methyl donor of numerous methylation reactions. The current model is that an increased concentration of AdoMet stimulates DNA methyltransferase reactions, triggering hypermethylation and protecting the genome against global hypomethylation, a hallmark of cancer. Using an assay of active demethylation in HEK 293 cells, we show that AdoMet inhibits active demethylation and expression of an ectopically methylated CMV-GFP (green fluorescent protein) plasmid in a dose-dependent manner. The inhibition of GFP expression is specific to methylated GFP; AdoMet does not inhibit an identical but unmethylated CMV-GFP plasmid. S-Adenosylhomocysteine (AdoHcy), the product of methyltransferase reactions utilizing AdoMet does not inhibit demethylation or expression of CMV-GFP. In vitro, AdoMet but not AdoHcy inhibits methylated DNA-binding protein 2/DNA demethylase as well as endogenous demethylase activity extracted from HEK 293, suggesting that AdoMet directly inhibits demethylase activity, and that the methyl residue on AdoMet is required for its interaction with demethylase. Taken together, our data support an alternative mechanism of action for AdoMet as an inhibitor of intracellular demethylase activity, which results in hypermethylation of DNA.     

Organization of the BcgI restriction-modification protein for the cleavage of eight phosphodiester bonds in DNA

Type IIB restriction-modification systems, such as BcgI, feature a single protein with both endonuclease and methyltransferase activities. Type IIB nucleases require two recognition sites and cut both strands on both sides of their unmodified sites. BcgI cuts all eight target phosphodiester bonds before dissociation. The BcgI protein contains A and B polypeptides in a 2:1 ratio: A has one catalytic centre for each activity; B recognizes the DNA. We show here that BcgI is organized as A₂B protomers, with B at its centre, but that these protomers self-associate to assemblies containing several A₂B units. Moreover, like the well known FokI nuclease, BcgI bound to its site has to recruit additional protomers before it can cut DNA. DNA-bound BcgI can alternatively be activated by excess A subunits, much like the activation of FokI by its catalytic domain. Eight A subunits, each with one centre for nuclease activity, are presumably needed to cut the eight bonds cleaved by BcgI. Its nuclease reaction may thus involve two A₂B units, each bound to a recognition site, with two more A₂B units bridging the complexes by protein–protein interactions between the nuclease domains.     

Activation of mammalian DNA methyltransferase by cleavage of a Zn binding regulatory domain.

Mammalian DNA (cytosine-5) methyltransferase contains a C-terminal domain that is closely related to bacterial cytosine-5 restriction methyltransferase. This methyltransferase domain is linked to a large N-terminal domain. It is shown here that the N-terminal domain contains a Zn binding site and that the N- and C-terminal domains can be separated by cleavage with trypsin or Staphylococcus aureus protease V8; the protease V8 cleavage site was determined by Edman degradation to lie 10 residues C-terminal of the run of alternating lysyl and glycyl residues which joins the two domains and six residues N-terminal of the first sequence motif conserved between the mammalian and bacterial cytosine methyltransferases. While the intact enzyme had little activity on unmethylated DNA substrates, cleavage between the domains caused a large stimulation of the initial velocity of methylation of unmethylated DNA without substantial change in the rate of methylation of hemimethylated DNA. These findings indicate that the N-terminal domain of DNA methyltransferase ensures the clonal propagation of methylation patterns through inhibition of the de novo activity of the C-terminal domain. Mammalian DNA methyltransferase is likely to have arisen via fusion of a prokaryotic-like restriction methyltransferase and an unrelated DNA binding protein. Stimulation of the de novo activity of DNA methyltransferase by proteolytic cleavage in vivo may contribute to the process of ectopic methylation observed in the DNA of aging animals, tumors and in lines of cultured cells.     

Characterization of the DNA methylase activity of the restriction enzyme from Escherichia coli K

The restriction endonuclease from Escherichia coli K is a multifunctional protein which efficiently methylates heteroduplex DNA (one strand modified and one strand unmodified) in the presence of S-adenosylmethionine (AdoMet), ATP, and Mg2+. The methylase activity is catalytic, and seems to modify different heteroduplex host specificity sites for E. coli K with equal efficiency. In the methylase reaction, both AdoMet and ATP (or its imido analog) act as allosteric effectors, but AdoMet also serves as a methyl donor. Preincubation of the enzyme with AdoMet eliminates the lag period observed in DNA methylation. The rate of enzyme activation was determined using the AdoMet analog Sinefungin. The result are consistent with the hypothesis that the early steps of AdoMet binding and enzyme activation are common to both restriction and modification reactions.     

Biotin-avidin microplate assay for the quantitative analysis of enzymatic methylation of DNA by DNA methyltransferases

An assay is described to measure methylation of biotinylated oligonucleotide substrates by DNA methyltransferases using [methyl-3H]-AdoMet. After the methylation reaction the oligonucleotides are immobilized on an avidin-coated microplate. The incorporation of [3H] into the DNA is quenched by addition of unlabeled AdoMet to the binding buffer. Unreacted AdoMet and enzyme are removed by washing. To release the radioactivity incorporated into the DNA, the wells are incubated with a non-specific endonuclease and the radioactivity determined by liquid scintillation counting. As an example, we have studied methylation of DNA by the EcoRV DNA methyltransferase. The reaction progress curves measured with this assay are linear with respect to time. Methylation rates linearly increase with enzyme concentration. The rates are comparable to results obtained with the same enzyme using a different assay. The biotin-avidin assay is inexpensive, convenient, quantitative, fast and well suited to process many samples in parallel. The accuracy of the assay is high, allowing to reproduce results within +/- 10%. The assay is very sensitive as demonstrated by the detection of incorporation of 0.8 fmol methyl groups into the DNA. Under the experimental conditions, this corresponds to methylation of only 0.03% of all target sites of the substrate. Using this assay, the DNA methylation activity of some M.EcoRV variants could be detected that was not visible by other in vitro methylation assays.     

Structure of pvu II DNA-(cytosine N4) methyltransferase, an example of domain permutation and protein fold assignment.

We have determined the structure of Pvu II methyltransferase (M. Pvu II) complexed with S -adenosyl-L-methionine (AdoMet) by multiwavelength anomalous diffraction, using a crystal of the selenomethionine-substituted protein. M. Pvu II catalyzes transfer of the methyl group from AdoMet to the exocyclic amino (N4) nitrogen of the central cytosine in its recognition sequence 5'-CAGCTG-3'. The protein is dominated by an open alpha/beta-sheet structure with a prominent V-shaped cleft: AdoMet and catalytic amino acids are located at the bottom of this cleft. The size and the basic nature of the cleft are consistent with duplex DNA binding. The target (methylatable) cytosine, if flipped out of the double helical DNA as seen for DNA methyltransferases that generate 5-methylcytosine, would fit into the concave active site next to the AdoMet. This M. Pvu IIalpha/beta-sheet structure is very similar to those of M. Hha I (a cytosine C5 methyltransferase) and M. Taq I (an adenine N6 methyltransferase), consistent with a model predicting that DNA methyltransferases share a common structural fold while having the major functional regions permuted into three distinct linear orders. The main feature of the common fold is a seven-stranded beta-sheet (6 7 5 4 1 2 3) formed by five parallel beta-strands and an antiparallel beta-hairpin. The beta-sheet is flanked by six parallel alpha-helices, three on each side. The AdoMet binding site is located at the C-terminal ends of strands beta1 and beta2 and the active site is at the C-terminal ends of strands beta4 and beta5 and the N-terminal end of strand beta7. The AdoMet-protein interactions are almost identical among M. Pvu II, M. Hha I and M. Taq I, as well as in an RNA methyltransferase and at least one small molecule methyltransferase. The structural similarity among the active sites of M. Pvu II, M. Taq I and M. Hha I reveals that catalytic amino acids essential for cytosine N4 and adenine N6 methylation coincide spatially with those for cytosine C5 methylation, suggesting a mechanism for amino methylation.     

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5’-ATCGAT-3’ sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection.     

Purification and characterization of HeLa endonuclease R. A G-specific mammalian endonuclease

We previously reported a double-stranded endonuclease from HeLa cells, endonuclease R (endo R), which specifically cleaves duplex DNA at sites rich in G.C base pairs. In this report we describe the purification of endo R to near homogeneity by conventional and affinity chromatography. The molecular mass of the active form of endo R is approximately 115-125 kDa. SDS-gel electrophoresis reveals a major protein species of 100 kDa. The enzyme requires Mg2+ as a cofactor and is equally active on closed circular and linear duplex DNA substrates that contain G-rich sequences. A 50% reduction in cleavage activity is observed with Ca2+ ions and no double-stranded cleavage occurs with Zn2+. Use of Mn2+ causes an altered specificity at low concentrations of enzyme or divalent metal ion and nonspecific degradation of the substrate at higher concentrations. Endo R is strongly inhibited by sodium or potassium chloride and exhibits a wide pH optimum of 6.0-9.0. The pI of the enzyme is between 6.5 and 7.0. A 2-fold stimulation is observed with the addition of dGTP or dATP but specific cleavage is inhibited by ATP at an equivalent concentration. Cleavage activity is competitively inhibited 10-fold more efficiently by single-stranded poly(dG)12 than by other DNA competitors. The ends of endo R cleavage products contain 5'-phosphate and 3'-hydroxyl groups, and a significant portion of these products were substrates for T4 DNA ligase. Endo R appears to be a previously uncharacterized mammalian endonuclease.     

Characterization of BseMII, a new type IV restriction-modification system, which recognizes the pentanucleotide sequence 5'-CTCAG(N)(10/8)/

We report the properties of the new BseMII restriction and modification enzymes from Bacillus stearothermophilus Isl 15-111, which recognize the 5'-CTCAG sequence, and the nucleotide sequence of the genes encoding them. The restriction endonuclease R.BseMII makes a staggered cut at the tenth base pair downstream of the recognition sequence on the upper strand, producing a two base 3'-protruding end. Magnesium ions and S:-adenosyl-L-methionine (AdoMet) are required for cleavage. S:-adenosylhomocysteine and sinefungin can replace AdoMet in the cleavage reaction. The BseMII methyltransferase modifies unique adenine residues in both strands of the target sequence 5'-CTCAG-3'/5'-CTGAG-3'. Monomeric R.BseMII in addition to endonucleolytic activity also possesses methyltransferase activity that modifies the A base only within the 5'-CTCAG strand of the target duplex. The deduced amino acid sequence of the restriction endonuclease contains conserved motifs of DNA N6-adenine methylases involved in S-adenosyl-L-methionine binding and catalysis. According to its structure and enzymatic properties, R.BseMII may be regarded as a representative of the type IV restriction endonucleases.     

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

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.     

6His-Eco29kI methyltransferase methylation site and kinetic mechanism characterization

A new type II 6His-Eco29kI DNA methyltransferase was tested for methylation site (CC(Me)GCGG) and catalytic reaction optimal conditions. With high substrate concentrations, an inhibitory effect of DNA, but not AdoMet, on its activity was observed. Isotope partitioning and substrate preincubation assays showed that the enzyme-AdoMet complex is catalytically active. Considering effect of different concentrations of DNA and AdoMet on initial velocity, ping-pong mechanisms were ruled out. According to data obtained, the enzyme appears to work by preferred ordered bi-bi mechanism with AdoMet as leading substrate.     

S-Adenosyl-L-methionine-dependent restriction enzymes

Restriction-modification (R-M) enzymes are classified into type I, II, III, and IV, based on their recognition sequence, subunit composition, cleavage position, and cofactor requirements. While the role of S-Adenosyl-L-methionine (AdoMet) as the methyl group donor in the methylation reaction is undisputed, its requirement in DNA cleavage reaction has been subject to intense study. AdoMet is a prerequisite for the DNA cleavage by most type I enzymes known so far, with the exception of R.EcoR124I. A number of new type II restriction enzymes belonging to the type IIB and IIG family were found to show AdoMet dependence for their cleavage reaction. The type III enzymes have been found to require AdoMet for their restriction function. AdoMet functions as an allosteric effector of the DNA cleavage reaction and has been shown to bring about conformational changes in the protein upon binding.     

Recombinant rat guanidinoacetate methyltransferase: structure and function of the NH2-terminal region as deduced by limited proteolysis

Recombinant rat liver guanidinoacetate methyltransferase, a monomeric protein with Mr 26,000, is inactivated upon incubation with low concentrations of trypsin. Examination of the reaction products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-performance liquid chromatography followed by amino acid analysis and sequencing of isolated peptides reveals that the inactivation is due to the cleavage of the NH2-terminal segment after Arg20. The cleaved peptide is not tightly associated with the rest of the protein. The rate of inactivation is not affected by the presence of either S-adenosylmethionine (AdoMet) or guanidinoacetate, but a substantial retardation of inactivation is observed when both substrates are present. The cleavage at Arg20 is also slowed by cross-linking Cys15 and Cys90 by a disulfide bond. An equilibrium binding study shows that guanidinoacetate methyltransferase in the free form binds AdoMet but not guanidinoacetate. The trypsin-modified enzyme, despite having no catalytic activity, can weakly bind AdoMet and guanidinoacetate in the presence of AdoMet. Chymotrypsin rapidly hydrolyzes the peptide bond after Trp19, and elastase cleaves the bond after Ala24, leading in both cases to loss of activity. The results obtained in this study suggest that the portion of the methyltransferase around residues 19-24 is highly exposed to the solvent and flexible. The results also indicate that the NH2-terminal region is not directly involved in substrate binding but plays a role in catalysis.     

The first structure of an RNA m5C methyltransferase, Fmu, provides insight into catalytic mechanism and specific binding of RNA substrate

The crystal structure of E. coli Fmu, determined at 1.65 A resolution for the apoenzyme and 2.1 A resolution in complex with AdoMet, is the first representative of the 5-methylcytosine RNA methyltransferase family that includes the human nucleolar proliferation-associated protein p120. Fmu contains three subdomains which share structural homology to DNA m(5)C methyltransferases and two RNA binding protein families. In the binary complex, the AdoMet cofactor is positioned within the active site near a novel arrangement of two conserved cysteines that function in cytosine methylation. The site is surrounded by a positively charged cleft large enough to bind its unique target stem loop within 16S rRNA. Docking of this stem loop RNA into the structure followed by molecular mechanics shows that the Fmu structure is consistent with binding to the folded RNA substrate.     

DNA containing 4'-thio-2'-deoxycytidine inhibits methylation by HhaI methyltransferase.

4'-Thio-2'-deoxycytidine was synthesized as a 5'- protected phosphoramidite compatible with solid phase DNA synthesis. When incorporated as the target cytosine (C*) in the GC*GC recognition sequence for the DNA methyltransferase M. HhaI, methyl transfer was strongly inhibited. In contrast, these same oligonucleotides were normal substrates for the cognate restriction endonuclease R. HhaI and its isoschizomer R. Hin P1I. M. HhaI was able to bind both 4'-thio-modified DNA and unmodified DNA to equivalent extents under equilibrium conditions. However, the presence of 4'-thio-2'-deoxycytidine decreased the half-life of the complex by >10-fold. The crystal structure of a ternary complex of M. HhaI, AdoMet and DNA containing 4'-thio-2'-deoxycytidine was solved at 2.05 A resolution with a crystallographic R-factor of 0.186 and R-free of 0.231. The structure is not grossly different from previously solved ternary complexes containing M. HhaI, DNA and AdoHcy. The difference electron density suggests partial methylation at C5 of the flipped target 4'-thio-2'-deoxycytidine. The inhibitory effect of the 4'sulfur atom on enzymatic activity may be traced to perturbation of a step in the methylation reaction after DNA binding but prior to methyl transfer. This inhibitory effect can be partially overcome after a considerably long time in the crystal environment where the packing prevents complex dissociation and the target is accurately positioned within the active site.     

Roles of divalent metal ions in flap endonuclease-substrate interactions

Flap endonucleases (FENs) have essential roles in DNA processing. They catalyze exonucleolytic and structure-specific endonucleolytic DNA cleavage reactions. Divalent metal ions are essential cofactors in both reactions. The crystal structure of FEN shows that the protein has two conserved metal-binding sites. Mutations in site I caused complete loss of catalytic activity. Mutation of crucial aspartates in site II abolished exonuclease action, but caused enzymes to retain structure-specific (flap endonuclease) activity. Isothermal titration calorimetry revealed that site I has a 30-fold higher affinity for cofactor than site II. Structure-specific endonuclease activity requires binding of a single metal ion in the high-affinity site, whereas exonuclease activity requires that both the high- and low-affinity sites be occupied by divalent cofactor. The data suggest that a novel two-metal mechanism operates in the FEN-catalyzed exonucleolytic reaction. These results raise the possibility that local concentrations of free cofactor could influence the endo- or exonucleolytic pathway in vivo.     

Biochemical characterization of a SET and transposase fusion protein, Metnase: its DNA binding and DNA cleavage activity

Metnase (SETMAR) is a SET and transposase fusion protein that promotes in vivo end joining activity and mediates genomic integration of foreign DNA. Recent studies showed that Metnase retained most of the transposase activities, including 5'-terminal inverted repeat (TIR)-specific binding and assembly of a paired end complex, and cleavage of the 5'-end of the TIR element. Here we show that R432 within the helix-turn-helix motif is critical for sequence-specific recognition, as the R432A mutation abolishes its TIR-specific DNA binding activity. Metnase possesses a unique DNA nicking and/or endonuclease activity that mediates cleavage of duplex DNA in the absence of the TIR sequence. While the HTH motif is essential for the Metnase-TIR interaction, it is not required for its DNA cleavage activity. The DDE-like motif is crucial for its DNA cleavage action as a point mutation at this motif (D483A) abolished its DNA cleavage activity. Together, our results suggest that Metnase's DNA cleavage activity, unlike those of other eukaryotic transposases, is not coupled to its sequence-specific DNA binding.