On the role of steric clashes in methylation control of restriction endonuclease activity

Restriction-modification systems digest non-methylated invading DNA, while protecting host DNA against the endonuclease activity by methylation. It is widely believed that the methylated DNA would not ‘fit’ into the binding site of the endonuclease in the productive orientation, and thus steric clashes should account for most of the protection. We test this concept statistically by grafting methyl groups in silico onto non-methylated DNA in co-crystal structures with restriction endonucleases. Clash scores are significantly higher for protective than non-protective methylation (P < 0.05% according to the Wilcoxon rank sum test). Structural data alone are sufficient to distinguish between protective and non-protective DNA methylation with 90% confidence and decision thresholds of 1.1 Å and 48 ų for the most severe distance-based and cumulative volume-based clash with the protein, respectively (0.1 Å was deducted from each interatomic distance to allow for coordinate errors). The most severe clashes are more pronounced for protective methyl groups attached to the nitrogen atoms (N6-methyladenines and N4-methylcytosines) than for C5-methyl groups on cytosines. Cumulative clashes are comparable for all three types of protective methylation.     

Substrate binding in vitro and kinetics of RsrI [N6-adenine] DNA methyltransferase

RsrI [N6-adenine] DNA methyltransferase (M·RsrI), which recognizes GAATTC and is a member of a restriction–modification system in Rhodobacter sphaeroides, was purified to >95% homogeneity using a simplified procedure involving two ion exchange chromatographic steps. Electrophoretic gel retardation assays with purified M·RsrI were performed on unmethylated, hemimethylated, dimethylated or non-specific target DNA duplexes (25 bp) in the presence of sinefungin, a potent inhibitory analog of AdoMet. M·RsrI binding was affected by the methylation status of the DNA substrate and was enhanced by the presence of the cofactor analog. M·RsrI bound DNA substrates in the presence of sinefungin with decreasing affinities: hemimethylated > unmethylated > dimethylated >> non-specific DNA. Gel retardation studies with DNA substrates containing an abasic site substituted for the target adenine DNA provided evidence consistent with M·RsrI extruding the target base from the duplex. Consistent with such base flipping, an ~1.7-fold fluorescence intensity increase was observed upon stoichiometric addition of M·RsrI to hemimethylated DNA containing the fluorescent analog 2-aminopurine in place of the target adenine. Pre-steady-state kinetic and isotope- partitioning experiments revealed that the enzyme displays burst kinetics, confirmed the catalytic competence of the M·RsrI–AdoMet complex and eliminated the possibility of an ordered mechanism where DNA is required to bind first. The equilibrium dissociation constants for AdoMet, AdoHcy and sinefungin were determined using an intrinsic tryptophan fluorescence-quenching assay.     

Structural and biochemical analyses of hemimethylated DNA binding by the SeqA protein

The Escherichia coli SeqA protein recognizes the 11 hemimethylated G-^mA-T-C sites in the oriC region of the chromosome, and prevents replication over-initiation within one cell cycle. The crystal structure of the SeqA C-terminal domain with hemimethylated DNA revealed the N⁶-methyladenine recognition mechanism; however, the mechanism of discrimination between the hemimethylated and fully methylated states has remained elusive. In the present study, we performed mutational analyses of hemimethylated G-^mA-T-C sequences with the minimal DNA-binding domain of SeqA (SeqA_71–181), and found that SeqA_71–181 specifically binds to hemimethylated DNA containing a sequence with a mismatched ^mA:G base pair [G-^mA(:G)-T-C] as efficiently as the normal hemimethylated G-^mA(:T)-T-C sequence. We determined the crystal structures of SeqA_71–181 complexed with the mismatched and normal hemimethylated DNAs at 2.5 and 3.0 Å resolutions, respectively, and found that the mismatched ^mA:G base pair and the normal ^mA:T base pair are recognized by SeqA in a similar manner. Furthermore, in both crystal structures, an electron density is present near the unmethylated adenine, which is only methylated in the fully methylated state. This electron density, which may be due to a water molecule or a metal ion, can exist in the hemimethylated state, but not in the fully methylated state, because of steric clash with the additional methyl group.     

Thermodynamic and hydration effects for the incorporation of a cationic 3-aminopropyl chain into DNA

The introduction of cationic 5-(ω-aminoalkyl)-2′-deoxypyrimidines into duplex DNA has been shown to induce DNA bending. In order to understand the energetic and hydration contributions for the incorporation of a cationic side chain in DNA a combination of spectroscopy, calorimetry and density techniques were used. Specifically, the temperature unfolding and isothermal formation was studied for a pair of duplexes with sequence d(CGTAGUCG TGC)/d(GCACGACTACG), where U represents 2′-deoxyuridine (‘control’) or 5-(3-aminopropyl)-2′-deoxyuridine (‘modified’). Continuous variation experiments confirmed 1:1 stoichiometries for each duplex and the circular dichroism spectra show that both duplexes adopted the B conformation. UV and differential scanning calorimetry melting experiments reveal that each duplex unfolds in two-state transitions. In low salt buffer, the ‘modified’ duplex is more stable and unfolds with a lower endothermic heat and lower release of counterion and water. This electrostatic stabilization is entropy driven and disappears at higher salt concentrations. Complete thermodynamic profiles at 15°C show that the favorable formation of each duplex results from the compensation of a favorable exothermic heat with an unfavorable entropy contribution. However, the isothermal profiles yielded a differential enthalpy of 8.8 kcal/mol, which is 4.3 kcal/mol higher than the differential enthalpy observed in the unfolding profiles. This indicates that the presence of the aminopropyl chain induces an increase in base stacking interactions in the modified single strand and a decrease in base stacking interactions in the modified duplex. Furthermore, the formation of the ‘control’ duplex releases water while the ‘modified’ duplex takes up water. Relative to the control duplex, formation of the modified duplex at 15°C yielded a marginal differential ΔG° term, positive ΔΔH_ITC–Δ(TΔS) compensation, negative ΔΔV and a net release of counterions. The opposite signs of the differential enthalpy–entropy compensation and differential volume change terms show a net uptake of structural water around polar and non-polar groups. This indicates that incorporation of the aminopropyl chain induces a higher exposure of aromatic bases to the solvent, which may be consistent with a small and local bend in the ‘modified’ duplex.     

Differential binding of Escherichia coli McrA protein to DNA sequences that contain the dinucleotide m5CpG

The Escherichia coli McrA protein, a putative C⁵-methylcytosine/C⁵-hydroxyl methylcytosine-specific nuclease, binds DNA with symmetrically methylated HpaII sequences (Cm5CGG), but its precise recognition sequence remains undefined. To determine McrA’s binding specificity, we cloned and expressed recombinant McrA with a C-terminal StrepII tag (rMcrA-S) to facilitate protein purification and affinity capture of human DNA fragments with m5C residues. Sequence analysis of a subset of these fragments and electrophoretic mobility shift assays with model methylated and unmethylated oligonucleotides suggest that N(Y > R) m5CGR is the canonical binding site for rMcrA-S. In addition to binding HpaII-methylated double-stranded DNA, rMcrA-S binds DNA containing a single, hemimethylated HpaII site; however, it does not bind if A, C, T or U is placed across from the m5C residue, but does if I is opposite the m5C. These results provide the first systematic analysis of McrA’s in vitro binding specificity.     

Biochemical characterization of I-CmoeI reveals that this H-N-H homing endonuclease shares functional similarities with H-N-H colicins

Endonuclease assays of the H-N-H proteins encoded by two group I introns in the Chlamydomonas moewusii chloroplast psbA gene revealed that the CmpsbA·1 intron specifies a site-specific DNA endonuclease, designated I-CmoeI. Like most previously reported intron-encoded endonucleases, I-CmoeI generates a double-strand break near the insertion site of its encoding intron, leaving 3′ extensions of 4 nt. This enzyme was purified from Escherichia coli as a fusion protein with a His tag at its N-terminus. The recombinant protein (rI-CmoeI) requires a divalent alkaline earth cation for DNA cleavage (Mg²⁺ > Ca²⁺ > Sr²⁺ > Ba²⁺). It also requires a metal cofactor for DNA binding, a property shared with H-N-H colicins but not with the homing endonucleases characterized to date. rI-CmoeI binds its recognition sequence as a monomer, as revealed by gel retardation assays. K_m and k_cat values of 100 ± 40 pM and 0.26 ± 0.04 min^–1, respectively, were determined. Replacement of the first histidine of the H-N-H motif by an alanine residue abolishes both rI-CmoeI activity and binding to its substrate. We propose that this conserved histidine residue plays a role in binding the metal cofactor and that such binding induces a structural modification of the enzyme which is required for DNA recognition.     

Solution structure of an arabinonucleic acid (ANA)/RNA duplex in a chimeric hairpin: comparison with 2′-fluoro-ANA/RNA and DNA/RNA hybrids

Hybrids of RNA and arabinonucleic acid (ANA) as well as the 2′-fluoro-ANA analog (2′F-ANA) were recently shown to be substrates of the enzyme RNase H. Although RNase H binds to double-stranded RNA, no cleavage occurs with such duplexes. Therefore, knowledge of the structure of ANA/RNA hybrids may prove helpful in the design of future antisense oligonucleotide analogs. In this study, we have determined the NMR solution structures of ANA/RNA and DNA/RNA hairpin duplexes and compared them to the recently published structure of a 2′F-ANA/RNA hairpin duplex. We demonstrate here that the sugars of RNA nucleotides of the ANA/RNA hairpin stem adopt the C3′-endo (north, A-form) conformation, whereas those of the ANA strand adopt a ‘rigid’ O4′-endo (east) sugar pucker. The DNA strand of the DNA/RNA hairpin stem is flexible, but the average DNA/RNA hairpin structural parameters are close to the ANA/RNA and 2′F-ANA/RNA hairpin parameters. The minor groove width of ANA/RNA, 2′F-ANA/RNA and DNA/RNA helices is 9.0 ± 0.5 Å, a value that is intermediate between that of A- and B-form duplexes. These results rationalize the ability of ANA/RNA and 2′F-ANA/RNA hybrids to elicit RNase H activity.     

Enzymatic properties of de novo-type mouse DNA (cytosine-5) methyltransferases

We have purified GST-fused recombinant mouse Dnmt3a and three isoforms of mouse Dnmt3b to near homogeneity. Dnmt3b3, an isoform of Dnmt3b, did not have DNA methylation activity. Dnmt3a, Dnmt3b1 or Dnmt3b2 showed similar activity toward poly(dG-dC)-poly(dG-dC) for measuring de novo methylation activity, and toward poly(dI-dC)-poly(dI-dC) for measuring total activity. This indicates that the enzymes are de novo-type DNA methyltransferases. The enzyme activity was inhibited by NaCl or KCl at concentrations >100 mM. The kinetic parameter, K_m^AdoMet, for Dnmt3a, Dnmt3b1 and Dnmt3b2 was 0.4, 1.2 and 0.9 µM when poly(dI-dC)-poly(dI-dC) was used, and 0.3, 1.2 and 0.8 µM when poly(dG-dC)-poly(dG-dC) was used, respectively. The K_m^DNA values for Dnmt3a, Dnmt3b1 and Dnmt3b2 were 2.7, 1.3 and 1.5 µM when poly(dI-dC)-poly(dI-dC) was used, and 3.5, 1.0 and 0.9 µM when poly(dG-dC)-poly(dG-dC) was used, respectively. For the methylation specificity, Dnmt3a significantly methylated CpG >> CpA. On the other hand, Dnmt3b1 methylated CpG > CpT ≥ CpA. Immuno-purified Dnmt3a, Myc-tagged and overexpressed in HEK 293T cells, methylated CpG >> CpA > CpT. Neither Dnmt3a nor Dnmt3b1 methylated the first cytosine of CpC.     

Degeneration of a homing endonuclease and its target sequence in a wild yeast strain

Mobile introns and inteins self-propagate by ‘homing’, a gene conversion process initiated by site-specific homing endonucleases. The VMA intein, which encodes the PI-SceI endonuclease in Saccharomyces cerevisiae, is present in several different yeast strains. Surprisingly, a wild wine yeast (DH1-1A) contains not only the intein⁺ allele, but also an inteinless allele that has not undergone gene conversion. To elucidate how these two alleles co-exist, we characterized the endonuclease encoded by the DH1-1A intein⁺ allele and the target site in the intein^– allele. Sequence analysis reveals seven mutations in the 31 bp recognition sequence, none of which occurs at positions that are individually critical for activity. However, binding and cleavage of the sequence by PI-SceI is reduced 10-fold compared to the S.cerevisiae target. The PI-SceI analog encoded by the DH1-1A intein⁺ allele contains 11 mutations at residues in the endonuclease and protein splicing domains. None affects protein splicing, but one, a R417Q substitution, accounts for most of the decrease in DNA cleavage and DNA binding activity of the DH1-1A protein. Loss of activity in the DH1-1A endonuclease and target site provides one explanation for co-existence of the intein⁺ and intein^– alleles.     

Different genome stability proteins underpin primed and na?ve adaptation in E. coli CRISPR-Cas immunity

CRISPR-Cas is a prokaryotic immune system built from capture and integration of invader DNA into CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) loci, termed ‘Adaptation’, which is dependent on Cas1 and Cas2 proteins. In Escherichia coli, Cascade-Cas3 degrades invader DNA to effect immunity, termed ‘Interference’. Adaptation can interact with interference (‘primed’), or is independent of it (‘naïve’). We demonstrate that primed adaptation requires the RecG helicase and PriA protein to be present. Genetic analysis of mutant phenotypes suggests that RecG is needed to dissipate R-loops at blocked replication forks. Additionally, we identify that DNA polymerase I is important for both primed and naive adaptation, and that RecB is needed for naïve adaptation. Purified Cas1-Cas2 protein shows specificity for binding to and nicking forked DNA within single strand gaps, and collapsing forks into DNA duplexes. The data suggest that different genome stability systems interact with primed or naïve adaptation when responding to blocked or collapsed invader DNA replication. In this model, RecG and Cas3 proteins respond to invader DNA replication forks that are blocked by Cascade interference, enabling DNA capture. RecBCD targets DNA ends at collapsed forks, enabling DNA capture without interference. DNA polymerase I is proposed to fill DNA gaps during spacer integration.     

Human DNA mismatch repair in vitro operates independently of methylation status at CpG sites

Whereas in Escherichia coli DNA mismatch repair is directed to the newly synthesized strand due to its transient lack of adenine methylation, the molecular determinants of strand discrimination in eukaryotes are presently unknown. In mammalian cells, cytosine methylation within CpG sites may represent an analogous and mechanistically plausible means of targeting mismatch correction. Using HeLa nuclear extracts, we conducted a systematic analysis in vitro to determine whether cytosine methylation participates in human DNA mismatch repair. We prepared a set of A·C heteroduplex molecules that were either unmethylated, hemimethylated or fully methylated at CpG sequences and found that the methylation status persisted under the assay conditions. However, no effect on either the time course or the magnitude of mismatch repair events was evident; only strand discontinuities contributed to strand bias. By western analysis we demonstrated that the HeLa extract contained MED1 protein, which interacts with MLH1 and binds to CpG-methylated DNA; supplementation with purified MED1 protein was without effect. In summary, human DNA mismatch repair operates independently of CpG methylation status, and we found no evidence supporting a role for CpG hemimethylation as a strand discrimination signal.     

The Helicobacter pylori HpyAXII restriction-modification system limits exogenous DNA uptake by targeting GTAC sites but shows asymmetric conservation of the DNA methyltransferase and restriction endonuclease components

The naturally competent organism Helicobacter pylori encodes a large number of restriction–modification (R–M) systems that consist of a restriction endonuclease and a DNA methyltransferase. R–M systems are not only believed to limit DNA exchange among bacteria but may also have other cellular functions. We report a previously uncharacterized H. pylori type II R–M system, M.HpyAXII/R.HpyAXII. We show that this system targets GTAC sites, which are rare in the H. pylori chromosome but numerous in ribosomal RNA genes. As predicted, this type II R–M system showed attributes of a selfish element. Deletion of the methyltransferase M.HpyAXII is lethal when associated with an active endonuclease R.HpyAXII unless compensated by adaptive mutation or gene amplification. R.HpyAXII effectively restricted both unmethylated plasmid and chromosomal DNA during natural transformation and was predicted to belong to the novel ‘half pipe’ structural family of endonucleases. Analysis of a panel of clinical isolates revealed that R.HpyAXII was functional in a small number of H. pylori strains (18.9%, n = 37), whereas the activity of M.HpyAXII was highly conserved (92%, n = 50), suggesting that GTAC methylation confers a selective advantage to H. pylori. However, M.HpyAXII activity did not enhance H. pylori fitness during stomach colonization of a mouse infection model.     

5-Methylcytosine DNA glycosylase activity is also present in the human MBD4 (G/T mismatch glycosylase) and in a related avian sequence

A 1468 bp cDNA coding for the chicken homolog of the human MBD4 G/T mismatch DNA glycosylase was isolated and sequenced. The derived amino acid sequence (416 amino acids) shows 46% identity with the human MBD4 and the conserved catalytic region at the C-terminal end (170 amino acids) has 90% identity. The non-conserved region of the avian protein has no consensus sequence for the methylated DNA binding domain. The recombinant proteins from human and chicken have G/T mismatch as well as 5-methylcytosine (5-MeC) DNA glycosylase activities. When tested by gel shift assays, human recombinant protein with or without the methylated DNA binding domain binds equally well to symmetrically, hemimethylated DNA and non-methylated DNA. However, the enzyme has only 5-MeC DNA glycosylase activity with the hemimethylated DNA. Footprinting of human MBD4 and of an N-terminal deletion mutant with partially depurinated and depyrimidinated substrate reveal a selective binding of the proteins to the modified substrate around the CpG. As for 5-MeC DNA glycosylase purified from chicken embryos, MBD4 does not use oligonucleotides containing mCpA, mCpT or mCpC as substrates. An mCpG within an A+T-rich oligonucleotide is a much better substrate than an A+T-poor sequence. The K_m of human MBD4 for hemimethylated DNA is ~10^–7 M with a V_max of ~10^–11 mol/h/µg protein. Deletion mutations show that G/T mismatch and 5-MeC DNA glycosylase are located in the C-terminal conserved region. In sharp contrast to the 5-MeC DNA glycosylase isolated from the chicken embryo DNA demethylation complex, the two enzymatic activities of MBD4 are strongly inhibited by RNA. In situ hybridization with antisense RNA indicate that MBD4 is only located in dividing cells of differentiating embryonic tissues.     

Massively parallel characterization of restriction endonucleases

Restriction endonucleases are highly specific in recognizing the particular DNA sequence they act on. However, their activity is affected by sequence context, enzyme concentration and buffer composition. Changes in these factors may lead to either ineffective cleavage at the cognate restriction site or relaxed specificity allowing cleavage of degenerate ‘star’ sites. Additionally, uncharacterized restriction endonucleases and engineered variants present novel activities. Traditionally, restriction endonuclease activity is assayed on simple substrates such as plasmids and synthesized oligonucleotides. We present and use high-throughput Illumina sequencing-based strategies to assay the sequence specificity and flanking sequence preference of restriction endonucleases. The techniques use fragmented DNA from sequenced genomes to quantify restriction endonuclease cleavage on a complex genomic DNA substrate in a single reaction. By mapping millions of restriction site–flanking reads back to the Escherichia coli and Drosophila melanogaster genomes we were able to quantitatively characterize the cognate and star site activity of EcoRI and MfeI and demonstrate genome-wide decreases in star activity with engineered high-fidelity variants EcoRI-HF and MfeI-HF, as well as quantify the influence on MfeI cleavage conferred by flanking nucleotides. The methods presented are readily applicable to all type II restriction endonucleases that cleave both strands of double-stranded DNA.     

Cation-dependent cleavage of the duplex form of the subtype-B HIV-1 RNA dimerization initiation site

The crystal structure of subtype-B HIV-1 genomic RNA Dimerization Initiation Site duplex revealed chain cleavage at a specific position resulting in 3′-phosphate and 5′-hydroxyl termini. A crystallographic analysis showed that Ba²⁺, Mn²⁺, Co²⁺ and Zn²⁺ bind specifically on a guanine base close to the cleaved position. The crystal structures also point to a necessary conformational change to induce an ‘in-line’ geometry at the cleavage site. In solution, divalent cations increased the rate of cleavage with pH/pKa compensation, indicating that a cation-bound hydroxide anion is responsible for the cleavage. We propose a ‘Trojan horse’ mechanism, possibly of general interest, wherein a doubly charged cation hosted near the cleavage site as a ‘harmless’ species is further transformed in situ into an ‘aggressive’ species carrying a hydroxide anion.     

Sugar composition of the pectic polysaccharides of charophytes,the closest algal relatives of land-plants: presence of 3-O-methyl-d-galactose residues

Background and Aims During evolution, plants have acquired and/or lost diverse sugar residues as cell-wall constituents. Of particular interest are primordial cell-wall features that existed, and in some cases abruptly changed, during the momentous step whereby land-plants arose from charophytic algal ancestors.Methods Polysaccharides were extracted from four charophyte orders [Chlorokybales (Chlorokybus atmophyticus), Klebsormidiales (Klebsormidium fluitans, K. subtile), Charales (Chara vulgaris, Nitella flexilis), Coleochaetales (Coleochaete scutata)] and an early-diverging land-plant (Anthoceros agrestis). ‘Pectins’ and ‘hemicelluloses’, operationally defined as extractable in oxalate (100 °C) and 6 m NaOH (37 °C), respectively, were acid- or Driselase-hydrolysed, and the monosaccharides analysed chromatographically. One unusual monosaccharide, ‘U’, was characterized by ¹H/¹³C-nuclear magnetic resonance spectroscopy and also enzymically.Key Results ‘U’ was identified as 3-O-methyl-d-galactose (3-MeGal). All pectins, except in Klebsormidium, contained acid- and Driselase-releasable galacturonate, suggesting homogalacturonan. All pectins, without exception, released rhamnose and galactose on acid hydrolysis; however, only in ‘higher’ charophytes (Charales, Coleochaetales) and Anthoceros were these sugars also efficiently released by Driselase, suggesting rhamnogalacturonan-I. Pectins of ‘higher’ charophytes, especially Chara, contained little arabinose, instead possessing 3-MeGal. Anthoceros hemicelluloses were rich in glucose, xylose, galactose and arabinose (suggesting xyloglucan and arabinoxylan), none of which was consistently present in charophyte hemicelluloses.Conclusions Homogalacturonan is an ancient streptophyte feature, albeit secondarily lost in Klebsormidium. When conquering the land, the first embryophytes already possessed rhamnogalacturonan-I. In contrast, charophyte and land-plant hemicelluloses differ substantially, indicating major changes during terrestrialization. The presence of 3-MeGal in charophytes and lycophytes but not in the ‘intervening’ bryophytes confirms that cell-wall chemistry changed drastically between major phylogenetic grades.     

Complementary action of restriction enzymes endo R-DpnI and Endo R-DpnII on bacteriophage f1 DNA

Bacteriophage f1 duplex DNA was isolated from Escherichia coli strains containing different DNA methylases and assayed for its sensitivity to endonucleolytic cleavage by the enzymes endo R · DpnI and endo R · DpnII. The former enzyme is specific for methylated DNA, the latter for unmethylated DNA (Lacks &; Greenberg, 1975). The E. coli dam methylase was found to be responsible for making f1 resistant to endo R · DpnII and sensitive to endo R · DpnI. Endo R · DpnI cleaved f 1 DNA from dam⁺ cells at four sites. Additional methylation by enzymes other than the dam methylase gave no further cleavage. Endo R · DpnII cleaved f1 DNA from dam^? cells also at four sites to give restriction fragments identical to those obtained with endo R · DpnI cleavage. Thus, the two enzymes are complementary in that they recognize and cleave within the same DNA sequence, one if the DNA is methylated, the other if it is unmethylated. DNA duplexes containing one methylated strand (dam ⁺) and one unmethylated strand (dam^?) were prepared in vitro. These methylated hybrids were refractory to endonucleolytic cleavage by both endo R · DpnI and endo R · DpnII. Neither enzyme, therefore, appears to make even a single strand break at a methylated/unmethylated hybrid site.