Structural adaptations in the interaction of EcoRI endonuclease with methylated GAATTC sites.

We have studied the interaction of EcoRI endonuclease with oligonucleotides containing GAATTC sites bearing one or two adenine-N6-methyl groups, which would be in steric conflict with key protein side chains involved in recognition and/or catalysis in the canonical complex. Single-strand methylation of either adenine produces small penalties in binding free energy (deltadeltaG0(S) approximately +1.4 kcal/mol), but elicits asymmetric structural adaptations in the complex, such that cleavage rate constants are strongly inhibited and unequal in the two DNA strands. The dependences of cleavage rate constants on the concentration of the Mg2+ cofactor are unaltered. When either adenine is methylated on both DNA strands, deltadeltaG0(S) (approximately +4 kcal/mol) is larger than the expected sum of the deltadeltaG0(S) values for the single-strand methylations, because the asymmetric adaptations cannot occur. Cleavage rate constants are reduced by 600 000-fold for the biologically relevant GAmATTC/CTTmAAG site, but the GmAATTC/CTTAmAG site forms only a non-specific complex that cannot be cleaved. These observations provide a detailed thermodynamic and kinetic explanation of how single-strand and double-strand methylation protect against endonuclease cleavage in vivo. We propose that non-additive effects on binding and structural 'adaptations' are important in understanding how DNA methylation modulates the biological activities of non-catalytic DNA binding proteins.     

Modulation of Escherichia coli DNA methyltransferase activity by biologically derived GATC-flanking sequences

Escherichia coli DNA adenine methyltransferase (EcoDam) methylates the N-6 position of the adenine in the sequence 5'-GATC-3' and plays vital roles in gene regulation, mismatch repair, and DNA replication. It remains unclear how the small number of critical GATC sites involved in the regulation of replication and gene expression are differentially methylated, whereas the approximately 20,000 GATCs important for mismatch repair and dispersed throughout the genome are extensively methylated. Our prior work, limited to the pap regulon, showed that methylation efficiency is controlled by sequences immediately flanking the GATC sites. We extend these studies to include GATC sites involved in diverse gene regulatory and DNA replication pathways as well as sites previously shown to undergo differential in vivo methylation but whose function remains to be assigned. EcoDam shows no change in affinity with variations in flanking sequences derived from these sources, but methylation kinetics varied 12-fold. A-tracts immediately adjacent to the GATC site contribute significantly to these differences in methylation kinetics. Interestingly, only when the poly(A) is located 5' of the GATC are the changes in methylation kinetics revealed. Preferential methylation is obscured when two GATC sites are positioned on the same DNA molecule, unless both sites are surrounded by large amounts of nonspecific DNA. Thus, facilitated diffusion and sequences immediately flanking target sites contribute to higher order specificity for EcoDam; we suggest that the diverse biological roles of the enzyme are in part regulated by these two factors, which may be important for other enzymes that sequence-specifically modify DNA.     

Accuracy of the EcoRI restriction endonuclease: binding and cleavage studies with oligodeoxynucleotide substrates containing degenerate recognition sequences

We have synthesized a series of 18 nonpalindromic oligodeoxynucleotides that carry all possible base changes within the recognition sequence of EcoRI. These single strands can be combined with their complementary single strands to obtain all possible EcoRI sequences (left), or they can be combined with a single strand containing the canonical sequence to obtain double strands with all possible mismatches within the recognition sequence (right): (sequence; see text) The rate of phosphodiester bond cleavage of these oligodeoxynucleotides by EcoRI was determined in single-turnover experiments under normal buffer conditions in order to find out to what extent the canonical recognition site can be distorted and yet serve as a substrate for EcoRI. Our results show that oligodeoxynucleotides containing mismatch base pairs are in general more readily attacked by EcoRI than oligodeoxynucleotides containing EcoRI sites and that the rates of cleavage of the two complementary strands of degenerate oligodeoxynucleotides are quite different. We have also determined the affinities of these oligodeoxynucleotides to EcoRI. They are higher for oligodeoxynucleotides carrying a mismatch within the EcoRI recognition site than for oligodeoxynucleotides containing an EcoRI site but otherwise do not correlate with the rate with which these oligodeoxynucleotides are cleaved by EcoRI. Our results allow details to be given for the probability of EcoRI making mistakes in cleaving DNA not only in its recognition sequence but also in sequences closely related to it. Due to the fact that the rates of cleavage in the two strands of a degenerate sequence generally are widely different, these mistakes are most likely not occurring in vivo, since nicked intermediates can be repaired by DNA ligase.     

Kinetic mechanism of the EcoRI DNA methyltransferase

We present a kinetic analysis of the EcoRI DNA N6-adenosine methyltransferase (Mtase). The enzyme catalyzes the S-adenosylmethionine (AdoMet)-dependent methylation of a short, synthetic 14 base pair DNA substrate and plasmid pBR322 DNA substrate with kcat/Km values of 0.51 X 10(8) and 4.1 X 10(8) s-1 M-1, respectively. The Mtase is thus one of the most efficient biocatalysts known. Our data are consistent with an ordered bi-bi steady-state mechanism in which AdoMet binds first, followed by DNA addition. One of the reaction products, S-adenosylhomocysteine (AdoHcy), is an uncompetitive inhibitor with respect to DNA and a competitive inhibitor with respect to AdoMet. Thus, initial DNA binding followed by AdoHcy binding leads to formation of a ternary dead-end complex (Mtase-DNA-AdoHcy). We suggest that the product inhibition patterns and apparent order of substrate binding can be reconciled by a mechanism in which the Mtase binds AdoMet and noncanonical DNA randomly but that recognition of the canonical site requires AdoMet to be bound. Pre-steady-state and isotope partition analyses starting with the binary Mtase-AdoMet complex confirm its catalytic competence. Moreover, the methyl transfer step is at least 10 times faster than catalytic turnover.     

In vivo existence of left-handed DNA

A genetic-biochemical assay has been developed to investigate the in vivo existence and consequences of unusual DNA structures. Left-handed DNA was shown to exist in living Escherichia coli. The EcoRI methyltransferase gene (temperature-sensitive) was cloned to serve as a probe for perturbed GAATTC sites in vivo. This plasmid was cotransformed with different plasmids containing inserts that had varying capacities to form left-handed helices or cruciforms with a target EcoRI site in the center or at the ends of the inserts. Inhibition of methylation in vivo was found for the stable inserts with the longest left-handed helices. In vitro methylation with the purified M.EcoRI enzyme agreed with the in vivo results.     

A DNA adenine methyltransferase of Escherichia coli that is cell cycle regulated and essential for viability

DNA sequence analysis revealed that the putative yhdJ DNA methyltransferase gene of Escherichia coli is 55% identical to the Nostoc sp. strain PCC7120 gene encoding DNA methyltransferase AvaIII, which methylates adenine in the recognition sequence, ATGCAT. The yhdJ gene was cloned, and the enzyme was overexpressed and purified. Methylation and restriction analysis showed that the DNA methyltransferase methylates the first adenine in the sequence ATGCAT. This DNA methylation was found to be regulated during the cell cycle, and the DNA adenine methyltransferase was designated M.EcoKCcrM (for "cell cycle-regulated methyltransferase"). The CcrM DNA adenine methyltransferase is required for viability in E. coli, as a strain lacking a functional genomic copy of ccrM can be isolated only in the presence of an additional copy of ccrM supplied in trans. The cells of such a knockout strain stopped growing when expression of the inducible plasmid ccrM gene was shut off. Overexpression of M.EcoKCcrM slowed bacterial growth, and the ATGCAT sites became fully methylated throughout the cell cycle; a high proportion of cells with an anomalous size distribution and DNA content was found in this population. Thus, the temporal control of this methyltransferase may contribute to accurate cell cycle control of cell division and cellular morphology. Homologs of M.EcoKCcrM are present in other bacteria belonging to the gamma subdivision of the class Proteobacteria, suggesting that methylation at ATGCAT sites may have similar functions in other members of this group.     

Fidelity of DNA recognition by the EcoRV restriction/modification system in vivo

The EcoRV restriction/modification system consists of two enzymes that recognize the DNA sequence GATATC. The EcoRV restriction endonuclease cleaves DNA at this site, but the DNA of Escherichia coli carrying the EcoRV system is protected from this reaction by the EcoRV methyltransferase. However, in vitro, the EcoRV nuclease also cleaves DNA at most sites that differ from the recognition sequence by one base pair. Though the reaction of the nuclease at these sites is much slower than that at the cognate site, it still appears to be fast enough to cleave the chromosome of the cell into many fragments. The possibility that the EcoRV methyltransferase also protects the noncognate sites on the chromosome was examined. The modification enzyme methylated alternate sites in vivo, but these were not the same as the alternate sites for the nuclease. The excess methylation was found at GATC sequences, which are also the targets for the dam methyltransferase of E. coli, a protein that is homologous to the EcoRV methyltransferase. Methylation at these sites gave virtually no protection against the EcoRV nuclease: even when the EcoRV methyltransferase had been overproduced, the cellular DNA remained sensitive to the EcoRV nuclease at its noncognate sites. The viability of E. coli carrying the EcoRV restriction/modification system was found instead to depend on the activity of DNA ligase. Ligase appears to proofread the EcoRV R/M system in vivo: DNA, cut initially in one strand at a noncognate site for the nuclease, is presumably repaired by ligase before the scission of the second strand.     

Promiscuous methylation of non-canonical DNA sites by HaeIII methyltransferase

The cytosine C5 methyltransferase M.HaeIII recognises and methylates the central cytosine of its canonical site GGCC. Here we report that M.HaeIII can also, with lower efficiency, methylate cytosines located in a wide range of non-canonical sequences. Using bisulphite sequencing we mapped the methyl- cytosine residues in DNA methylated in vitro and in vivo by M.HaeIII. Methyl-cytosine residues were observed in multiple sequence contexts, most commonly, but not exclusively, at star sites (sites differing by a single base from the canonical sequence). The most frequently used star sites had changes at positions 1 and 4, but there is little or no methylation at star sites changed at position 2. The rate of methylation of non-canonical sites can be quite significant: a DNA substrate lacking a canonical site was methylated by M.HaeIII in vitro at a rate only an order of magnitude slower than an otherwise identical substrate containing the canonical site. In vivo methylation of non-canonical sites may therefore be significant and may have provided the starting point for the evolution of restriction–modification systems with novel sequence specificities.     

Evidence that Escherichia coli virus T1 induces a DNA methyltransferase

DNA of Escherichia coli virus T1 is resistant to MboI cleavage and appears to be heavily methylated. Analysis of methylation by the isoschizomeric restriction enzymes Sau3AI and DpnI revealed that recognition sites for E. coli DNA adenine methylase (dam methylase) are methylated. The same methylation pattern was found for virus T1 DNA grown on an E. coli dam host, indicating a T1-specific DNA methyltransferase.     

Overmethylation of DNAs by the EcoRI methylase.

EcoRI methylase is able to catalyze methy incorporation into DNA at sequences other than the canonical EcoRI site. At high enzyme concentrations and over a wide range of pH and ionic strengths, EcoRI methylase modifies polyoma DNA (which contains one EcoRI site) at a number of sites. This modification prevents EcoRI endonuclease activity, and thus is presumably at or near the EcoRI sequences (5') NAATTN.     

The combination of DNA methylation and H1 histone binding inhibits the action of a restriction nuclease on plasmid DNA.

To investigate the potentials of DNA methylation and H1 histone in regulating the action of DNA binding proteins, well ordered complexes were formed by slow salt gradient dialysis of mixtures of H1 histone with either methylated or nonmethylated DNA. The sites methylated in the plasmids were CCGG. Methylation of cytosine in this site protects the DNA against HpaII endonuclease but not against MspI. However, when the methylated DNA was complexed to H1, it was protected against MspI. The protection was only effective for a subset of the MspI restriction sites. The protection of DNA afforded by the combination of H1 binding and DNA methylation did not apply to EcoRI, PstI, or BamHI sites and so did not seem to be due to aggregation of the DNA by H1 histone. Gel retardation assays indicated that the affinity of H1 for methylated DNA was not detectably different from its affinity for nonmethylated DNA. Probably methylated DNA when bound to H1 is in a conformation that is resistant to MspI endonuclease. Such conformational changes induced by DNA methylation and H1 binding might affect the action of other DNA binding proteins, perhaps in chromatin as well as in H1.DNA complexes.     

GATC flanking sequences regulate Dam activity: evidence for how Dam specificity may influence pap expression

Escherichia coli DNA adenine methyltransferase (Dam) plays essential roles in DNA replication, mismatch repair and gene regulation. The differential methylation by Dam of the two GATC sequences in the pap promoter regulates the expression of pili genes necessary for uropathogenic E.coli cellular adhesion. Dam processively methylates GATC sites in various DNA substrates, yet the two pap GATC sites are not processively methylated. We previously proposed that the flanking sequences surrounding the two pap GATC sites contribute to the enzyme's distributive methylation. We show here that replacement of the poorly methylated pap GATC sites with sites predicted to be processively methylated indeed results in an increase in Dam processivity. The increased processivity is due to a change in the methyltransfer kinetics and not the binding efficiency of Dam. A competition experiment in which the flanking sequences of only one pap GATC site were altered demonstrates that the GATC flanking sequences directly regulate the enzyme's catalytic efficiency. The GATC flanking sequences in Dam-regulated promoters in E.coli and other bacteria are similar to those in the pap promoter. Gene regulation from some of these promoters involves mechanisms and proteins that are quite different from those in the pap operon. Further, GATC sequences previously identified to remain unmethylated within the E.coli genome, but whose function remains largely unassigned, are flanked by sequences predicted to be poorly methylated. We conclude that the GATC flanking sequences may be critical for expression of pap and other Dam-regulated genes by affecting the activity of Dam at such sites and, thus, its processivity. A model is proposed, illustrating how the sequences flanking the GATC sites in Dam-regulated promoters may contribute to this epigenetic mechanism of gene expression, and how flanking sequences contribute to the diverse biological roles of Dam.     

Maize chromomethylase Zea methyltransferase2 is required for CpNpG methylation

A cytosine DNA methyltransferase containing a chromodomain, Zea methyltransferase2 (Zmet2), was cloned from maize. The sequence of ZMET2 is similar to that of the Arabidopsis chromomethylases CMT1 and CMT3, with C-terminal motifs characteristic of eukaryotic and prokaryotic DNA methyltransferases. We used a reverse genetics approach to determine the function of the Zmet2 gene. Plants homozygous for a Mutator transposable element insertion into motif IX had a 13% reduction in methylated cytosines. DNA gel blot analysis of these plants with methylation-sensitive restriction enzymes and bisulfite sequencing of a 180-bp knob sequence showed reduced methylation only at CpNpG sites. No reductions in methylation were observed at CpG or asymmetric sites in heterozygous or homozygous mutant plants. Our research shows that chromomethylase Zmet2 is required for in vivo methylation of CpNpG sequences.     

Core sequence in the RNA motif recognized by the ErmE methyltransferase revealed by relaxing the fidelity of the enzyme for its target.

Under physiological conditions, the ErmE methyltransferase specifically modifies a single adenosine within ribosomal RNA (rRNA), and thereby confers resistance to multiple antibiotics. The adenosine (A2058 in Escherichia coli 23S rRNA) lies within a highly conserved structure, and is methylated efficiently, and with equally high fidelity, in rRNAs from phylogenetically diverse bacteria. However, the fidelity of ErmE is reduced when magnesium is removed, and over twenty new sites of ErmE methylation appear in E. coli 16S and 23S rRNAs. These sites show widely different degrees of reactivity to ErmE. The canonical A2058 site is largely unaffected by magnesium depletion and remains the most reactive site in the rRNA. This suggests that methylation at the new sites results from changes in the RNA substrate rather than the methyltransferase. Chemical probing confirms that the rRNA structure opens upon magnesium depletion, exposing potential new interaction sites to the enzyme. The new ErmE sites show homology with the canonical A2058 site, and have the consensus sequence aNNNcgGAHAg (ErmE methylation occurs exclusively at adenosines (underlined); these are preceded by a guanosine, equivalent to G2057; there is a high preference for the adenosine equivalent to A2060; H is any nucleotide except G; N is any nucleotide; and there are slight preferences for the nucleotides shown in lower case). This consensus is believed to represent the core of the motif that Erm methyltransferases recognize at their canonical A2058 site. The data also reveal constraints on the higher order structure of the motif that affect methyltransferase recognition.     

Studies on the biological role of dna methylation; IV. Mode of methylation of DNA in E. coli cells

Two pairs of restriction enzyme isoschizomers were used to study in vivo methylation of E. coli and extrachromosomal DNA. By use of the restriction enzymes MboI (which cleaves only the unmethylated GATC sequence) and its isoschizomer Sau3A (indifferent to methylated adenine at this sequence), we found that all the GATC sites in E. coli and in extrachromosomal DNAs are symmetrically methylated on both strands. The calculated number of GATC sites in E. coli DNA can account for all its m6Ade residues. Foreign DNA, like mouse mtDNA, which is not methylated at GATC sites became fully methylated at these sequences when introduced by transfection into E. coli cells. This experiment provides the first evidence for the operation of a de novo methylation mechanism for E. coli methylases not involved in restriction modification. When the two restriction enzyme isoschizomers, EcoRII and ApyI, were used to analyze the methylation pattern of CCTAGG sequences in E. coli C and phi X174 DNA, it was found that all these sites are methylated. The number of CCTAGG sites in E. coli C DNA does not account for all m5Cyt residues.     

Thermodynamic and kinetic basis for the relaxed DNA sequence specificity of "promiscuous" mutant EcoRI endonucleases

Promiscuous mutant EcoRI endonucleases produce lethal to sublethal effects because they cleave Escherichia coli DNA despite the presence of the EcoRI methylase. Three promiscuous mutant forms, Ala138Thr, Glu192Lys and His114Tyr, have been characterized with respect to their binding affinities and first-order cleavage rate constants towards the three classes of DNA sites: specific, miscognate (EcoRI*) and non-specific. We have made the unanticipated and counterintuitive observations that the mutant restriction endonucleases that exhibit relaxed specificity in vivo nevertheless bind more tightly than the wild-type enzyme to the specific recognition sequence in vitro, and show even greater preference for binding to the cognate GAATTC site over miscognate sites. Binding preference for EcoRI* over non-specific DNA is also improved. The first-order cleavage rate constants of the mutant enzymes are normal for the cognate site GAATTC, but are greater than those of the wild-type enzyme at EcoRI* sites. Thus, the mutant enzymes use two mechanisms to partially bypass the multiple fail-safe mechanisms that protect against cleavage of genomic DNA in cells carrying the wild-type EcoRI restriction-modification system: (a) binding to EcoRI* sites is more probable than for wild-type enzyme because non-specific DNA is less effective as a competitive inhibitor; (b) the combination of increased affinity and elevated cleavage rate constants at EcoRI* sites makes double-strand cleavage of these sites a more probable outcome than it is for the wild-type enzyme. Semi-quantitative estimates of rates of EcoRI* site cleavage in vivo, predicted using the binding and cleavage constants measured in vitro, are in accord with the observed lethal phenotypes associated with the three mutations.