Restriction endonuclease isoschizomers ItaI, BsoFI and Fsp4HI are characterised by differences in their sensitivities to CpG methylation

BsoFI , ItaI and Fsp4HI are isoshizomers of Fnu4HI (5'-GC NGC-3'). Both Fnu4HI and BsoFI have previously been shown to be inhibited by cytosine-specific methylation within the recognition sequence. Fnu4HI is inhibited if either the internal cytosine at position 2 or the external cytosine at position 5 of the restriction sequence is methylated, but the precise nature of the methylation sensitivity of BsoFI is unclear from the literature. The methylation sensitivities of ItaI and Fsp4HI have not previously been reported. By methylating the plasmid pUC18 with M.SssI (a DNA cytosine-5'-methyltransferase with a specificity for CpG), we have determined that ItaI is sensitive only to methylation of internal CpG sites within the restriction sequence. The methylation sensitivity of Fsp4HI is identical to that of Fnu4HI, being inhibited by methylation of either internal CpG sites or overlapping CpG sites. BsoFI , like the other isoschizomers tested, is sensitive to a combination of internal and overlapping CpG methylation. BsoFI is also sensitive to overlapping CpG methylation (in the absence of internal CpG methylation) if CpG overlap with both sides of the recognition sequence. Sites containing one overlapping CpG (in the absence of internal CpG) are cut when methylated but show marked individual variation in their rates of cleavage. Considerable variation in the rate of cleavage by BsoFI is also observed at sites containing only internal methylated CpG. Some sites are cut slowly, whilst others fail to cut even after prolonged incubation with excess of enzyme.     

Effect of reproduction of the LPP-3 cyanophage on glutamate dehydrogenase and glutamine synthetase activity in the cyanobacterium Plectonema boryanum

The effect of cyanophage LPP-3 reproduction on glutamate dehydrogenase and glutamine synthetase (GS) in P boryanum cells have been studied. It was determined that the both reactions are intensified by 135% and 220%, accordingly. Isoenzymes of GS were purified from native and infected cell of cyanobacteria. Their physical-and-chemical properties are different. The cyanophage development probably causes specific modification of the cell enzymes.     

Rapid analysis of DNA methylation using new restriction enzyme sites created by bisulfite modification

Bisulfite converts non-methylated cytosine in DNA to uracil leaving 5-methylcytosine unaltered. Here, predicted changes in restriction enzyme sites following reaction of genomic DNA with bisulfite and amplification of the product by the polymerase chain reaction (PCR) were used to assess the methylation of CpG sites. This procedure differs from conventional DNA methylation analysis by methylation-sensitive restriction enzymes because it does not rely on an absence of cleavage to detect methylated sites, the two strands of DNA produce different restriction enzyme sites and may be differentially analyzed, and closely related sequences may be separately analyzed by using specific PCR primers.     

Position effect takes precedence over target sequence in determination of adenine methylation patterns in the nuclear genome of a eukaryote, Tetrahymena thermophila

Approximately 0.8% of the adenine residues in the macronuclear DNA of the ciliated protozoan Tetrahymena thermophila are modified to N 6-methyladenine. DNA methylation is site specific and the pattern of methylation is constant between clonal cell lines. In vivo, modification of adenine residues appears to occur exclusively in the sequence 5'-NAT-3', but no consensus sequence for modified sites has been found. In this study, DNA fragments containing a site that is uniformly methylated on the 50 copies of the macronuclear chromosome were cloned into the extrachromosomal rDNA. In the novel location on the rDNA minichromosome, the site was unmethylated. The result was the same whether the sequences were introduced in a methylated or unmethylated state and regardless of the orientation of the sequence with respect to the origin of DNA replication. The data show that sequence is insufficient to account for site-specific methylation in Tetrahymena and argue that other factors determine the pattern of DNA methylation.     

The cyanobacterium Synechocystis sp. strain PCC 6803 expresses a DNA methyltransferase specific for the recognition sequence of the restriction endonuclease PvuI

By use of restriction endonucleases, the DNA of the cyanobacterium Synechocystis sp. strain PCC 6803 was analyzed for DNA-specific methylation. Three different recognition sites of methyltransferases, a dam-like site including N6-methyladenosine and two other sites with methylcytosine, were identified, whereas no activities of restriction endonucleases could be detected in this strain. slr0214, a Synechocystis gene encoding a putative methyltransferase that shows significant similarities to C5-methylcytosine-synthesizing enzymes, was amplified by PCR and cloned for further characterization. Mutations in slr0214 were generated by the insertion of an aphII gene cassette. Analyses of chromosomal DNAs of such mutants demonstrated that the methylation pattern was changed. The recognition sequence of the methyltransferase was identified as 5'-CGATCG-3', corresponding to the recognition sequence of PvuI. The specific methyltransferase activity was significantly reduced in protein extracts obtained from mutant cells. Mutation of slr0214 also led to changed growth characteristics of the cells compared to wild-type cells. These alterations led to the conclusion that the methyltransferase Slr0214 might play a regulatory role in Synechocystis. The Slr0214 protein was also overexpressed in Escherichia coli, and the purified protein demonstrated methyltransferase activity and specificity for PvuI recognition sequences in vitro. We propose the designation M.Ssp6803I [corrected] (Synechocystis methyltransferase I) for the slr0214-encoded enzyme.     

DNA methylation patterns in the calcitonin gene region at first diagnosis and at relapse of acute lymphoblastic leukemia (ALL)

Aberrant DNA methylation can occur early in neoplastic transformation and may lead to the development of cancer. We describe the alterations of methylation patterns at the DNA sequence level which occurred in the 5' region of the calcitonin gene in lymphoblasts from 14 pediatric patients with acute lymphoblastic leukemia (ALL). The DNA methylation status of 25 CpG sites was determined by sequence analysis after bisulfite treatment of the DNA. This method showed that 13 out of 14 patients had increased numbers of methylated CpG sites in the calcitonin gene region at initial diagnosis when compared to control DNA from healthy individuals. The 5' region of the calcitonin gene appears to be methylated to a significantly higher degree in T lineage ALL compared to B lineage ALL (P < 0.01). Each of six ALL patients who were investigated at initial diagnosis and at relapse showed alterations in DNA methylation between the two stages. These six cases were also investigated by Southern blot analysis with methylcytosine-sensitive restriction enzymes and this method showed an increase in DNA methylation in only four of the six cases. The DNA sequencing method thus appears to be better suited to assess alterations of DNA methylation than Southern blot analysis. There are marked regional differences in the frequency of methylation of individual CpG sites and in the frequency of alterations between the two stages. Our results show that alterations in DNA methylation continue to occur from the initial stage to the relapse stage of ALL, suggesting that aberrant DNA methylation may play a role in tumor progression.     

A novel system for the rapid generation of precise DNA deletions

To generate DNA deletions, a tandem array of class IIS restriction enzyme recognition sites was cloned into a plasmid. The recognition sites were arranged so that each enzyme cleaves at a different site within an adjacent target sequence. Digestion with both enzymes followed by end repair and ligation resulted in the deletion of DNA between the two sites of cleavage. Because both recognition sites are preserved following deletion, it was found that sequential deletions could be generated using cycles of restriction enzyme digestion, end repair and ligation. Therefore, this system represents a valuable tool in the definition of functional DNA sequences.     

Crystal structure of restriction endonuclease BglI bound to its interrupted DNA recognition sequence

The crystal structure of the type II restriction endonuclease BglI bound to DNA containing its specific recognition sequence has been determined at 2.2 A resolution. This is the first structure of a restriction endonuclease that recognizes and cleaves an interrupted DNA sequence, producing 3' overhanging ends. BglI is a homodimer that binds its specific DNA sequence with the minor groove facing the protein. Parts of the enzyme reach into both the major and minor grooves to contact the edges of the bases within the recognition half-sites. The arrangement of active site residues is strikingly similar to other restriction endonucleases, but the co-ordination of two calcium ions at the active site gives new insight into the catalytic mechanism. Surprisingly, the core of a BglI subunit displays a striking similarity to subunits of EcoRV and PvuII, but the dimer structure is dramatically different. The BglI-DNA complex demonstrates, for the first time, that a conserved subunit fold can dimerize in more than one way, resulting in different DNA cleavage patterns.     

Concurrent replication and methylation at mammalian origins of replication

Observations made with Escherichia coli have suggested that a lag between replication and methylation regulates initiation of replication. To address the question of whether a similar mechanism operates in mammalian cells, we have determined the temporal relationship between initiation of replication and methylation in mammalian cells both at a comprehensive level and at specific sites. First, newly synthesized DNA containing origins of replication was isolated from primate-transformed and primary cell lines (HeLa cells, primary human fibroblasts, African green monkey kidney fibroblasts [CV-1], and primary African green monkey kidney cells) by the nascent-strand extrusion method followed by sucrose gradient sedimentation. By a modified nearest-neighbor analysis, the levels of cytosine methylation residing in all four possible dinucleotide sequences of both nascent and genomic DNAs were determined. The levels of cytosine methylation observed in the nascent and genomic DNAs were equivalent, suggesting that DNA replication and methylation are concomitant events. Okazaki fragments were also demonstrated to be methylated, suggesting that the rapid kinetics of methylation is a feature of both the leading and the lagging strands of nascent DNA. However, in contrast to previous observations, neither nascent nor genomic DNA contained detectable levels of methylated cytosines at dinucleotide contexts other than CpG (i.e., CpA, CpC, and CpT are not methylated). The nearest-neighbor analysis also shows that cancer cell lines are hypermethylated in both nascent and genomic DNAs relative to the primary cell lines. The extent of methylation in nascent and genomic DNAs at specific sites was determined as well by bisulfite mapping of CpG sites at the lamin B2, c-myc, and beta-globin origins of replication. The methylation patterns of genomic and nascent clones are the same, confirming the hypothesis that methylation occurs concurrently with replication. Interestingly, the c-myc origin was found to be unmethylated in all clones tested. These results show that, like genes, different origins of replication exhibit different patterns of methylation. In summary, our results demonstrate tight coordination of DNA methylation and replication, which is consistent with recent observations showing that DNA methyltransferase is associated with proliferating cell nuclear antigen in the replication fork.     

High resolution footprinting of a type I methyltransferase reveals a large structural distortion within the DNA recognition site

The type I DNA methyltransferase M.EcoR124I is a multi-subunit enzyme that binds to the sequence GAAN6RTCG, transferring a methyl group from S-adenosyl methionine to a specific adenine on each DNA strand. We have investigated the protein-DNA interactions in the complex by DNase I and hydroxyl radical footprinting. The DNase I footprint is unusually large: the protein protects the DNA on both strands for at least two complete turns of the helix, indicating that the enzyme completely encloses the DNA in the complex. The higher resolution hydroxyl radical probe shows a smaller, but still extensive, 18 bp footprint encompassing the recognition site. Within this region, however, there is a remarkably hyper-reactive site on each strand. The two sites of enhanced cleavage are co-incident with the two adenines that are the target bases for methylation, showing that the DNA is both accessible and highly distorted at these sites. The hydroxyl radical footprint is unaffected by the presence of the cofactor S-adenosyl methionine, showing that the distorted DNA structure induced by M.EcoR124I is formed during the initial DNA binding reaction and not as a transient intermediate in the reaction pathway.     

Effects of DNA methylation on topoisomerase I and II cleavage activities

DNA methylation is deregulated during oncogenesis. Since several major anti-cancer drugs act on topoisomerases, we investigated the effects of cytosine methylation on topoisomerase cleavage activities. Both topoisomerase I and II cleavage patterns were modified by CpG methylation in c-myc gene DNA fragments. Topoisomerase II changes, mainly cleavage reduction, occurred for methylation sites within 7 base pairs from the topoisomerase II breaks and were different for VM-26 and azatoxin. For topoisomerase I, cleavage enhancement as well as suppression were observed. Using synthetic methylated oligonucleotides, we show that hemimethylation is sufficient to alter topoisomerase I activity. Cytosine methylation on the scissile strand within the topoisomerase I consensus sequence had strong effects. Cleavage was stimulated by methylation at position -4 and was strongly inhibited by methylation at position -3 (with position -1 being the enzyme-linked nucleotide). This inhibitory effect was attributed to the presence of a methyl group in the major groove, since the transition uracil to thymine also inhibited cleavage. Altogether these results suggest an interaction of topoisomerase I with the DNA major grove at positions -3 and -4. In addition, DNA methylation may have profound effects on the activity of topoisomerases and may alter the distribution of cleavage sites produced by anticancer drugs in chromatin.     

Recombination by resolvase to analyse DNA communications by the SfiI restriction endonuclease

The SfiI endonuclease differs from other type II restriction enzymes by cleaving DNA concertedly at two copies of its recognition site, its optimal activity being with two sites on the same DNA molecule. The nature of this communication event between distant DNA sites was analysed on plasmids with recognition sites for SfiI interspersed with recombination sites for resolvase. These were converted by resolvase to catenanes carrying one SfiI site on each ring. The catenanes were cleaved by SfiI almost as readily as a single ring with two sites, in contrast to the slow reactions on DNA rings with one SfiI site. Interactions between SfiI sites on the same DNA therefore cannot follow the DNA contour and, instead, must stem from their physical proximity. In buffer lacking Mg2+, where SfiI is inactive while resolvase is active, the addition of SfiI to a plasmid with target sites for both proteins blocked recombination by resolvase, due to the restriction enzyme bridging its sites and thus isolating the sites for resolvase into separate loops. The extent of DNA looping by SfiI matched its extent of DNA cleavage in the presence of Mg2+.     

Cooperative binding properties of restriction endonuclease EcoRII with DNA recognition sites

EcoRII is a member of the expanding group of type IIe restriction endonucleases that share the distinguishing feature of requiring cooperativity between two recognition sites in their substrate DNA. To determine the stoichiometry of the active DNA-enzyme complex and the mode of cooperative interaction, we have investigated the dependence of EcoRII cleavage on the concentration of EcoRII dimers. Maximal restriction was observed at dimer/site ratios of 0.25 and 0. 5. The molecular weight of the DNA-enzyme complex eluted from a gel filtration column also corresponds to a dimeric enzyme structure bound to two substrate sites. We conclude that one EcoRII dimer is sufficient to interact cooperatively with two DNA recognition sites. A Lac repressor "barrier" bound between two normally reactive EcoRII sites did not inhibit restriction endonuclease activity, indicating that cooperativity between EcoRII sites is achieved by bending or looping of the intervening DNA stretch. Comparative cleavage of linear substrates with differently spaced interacting sites revealed an inverse correlation between cleavage rate and site distance. At the optimal distance of one helical turn, EcoRII cleavage is independent of the orientation of the recognition sequence in the DNA double strand.     

Cytosine methylation enhances DNA damage produced by groove binding and intercalating enediynes: studies with esperamicins A1 and C

Methylation of the C5 position of cytosine in CG dinucleotides represents an important element in the control of gene expression in eukaryotic cells. This major groove modification of DNA causes changes in DNA conformation that are recognized by DNA-binding proteins and DNA-damaging chemicals. We have observed that CG methylation affects the DNA damage produced by the enediyne esperamicin A1 and its analog lacking the intercalating anthranilate, esperamicin C. Fragments of the human phosphoglycerate kinase gene (PGK1) and the plasmid pUC19 were methylated with SssI methylase and subjected to damage by esperamicins A1 and C. Damage produced by esperamicin A1 was enhanced 1.5-2-fold near a single CG sequence at position -101 in PGK1 and in a region containing several methylated CG dinucleotides between positions -120 and -131. Esperamicin C-induced damage was enhanced to a similar degree in PGK1 but only at the site that contained multiple CG dinucleotides. There was enhancement of damage for both drugs in the pUC19 fragment at several sites near CG sequences. Analysis of the chemistry of esperamicin-induced DNA damage suggests that cytosine methylation does not affect the identity of drug-abstracted hydrogen atoms. The damage chemistry was also used to identify the DNA binding orientation of the esperamicins. The anthranilate of esperamicin A1 is predicted to intercalate in a CT step four base pairs in a 3'-direction to the CG sequence at -101 in PGK1 and in a CG dinucleotide at the site containing multiple CGs (positions -120 to -131). These observations are consistent with other studies that indicate a long range effect of cytosine methylation on DNA conformation.     

cis-Acting signal for inheritance of imprinted DNA methylation patterns in the preimplantation mouse embryo

The inheritance of gametic methylation patterns is a critical event in the imprinting of genes. In the case of the imprinted RSVIgmyc transgene, the methylation pattern in the unfertilized egg is maintained by the early mouse embryo, whereas the sperm's methylation pattern is lost in the early embryo. To investigate the cis-acting requirements for this preimplantation stage of genomic imprinting, we examined the fate of different RSVIgmyc methylation patterns, preimposed on RSVIgmyc and introduced into the mouse zygote by pronuclear injection. RSVIgmyc methylation patterns with a low percentage of methylated CpG dinucleotides, generated by using bacterial cytosine methylases with four-base recognition sequences, were lost in the early embryo. In contrast, methylation was maintained when all CpG dinucleotides were methylated with the bacterial SssI (CpG) methylase. This singular maintenance of RSVIgmyc methylation preimposed with SssI methylase appears to be specific to the early, undifferentiated embryo; differentiated NIH 3T3 fibroblasts transfected with methylated versions of RSVIgmyc maintained all methylation patterns, independent of the level of preimposed methylation. The methylation pattern of the RSVIgmyc allele in adult founder transgenic mice that was produced by pronuclear injection of an SssI-methylated construct could not be distinguished from the maternal RSVIgmyc methylation pattern. Thus, a highly methylated allele in adult mice, normally generated by transmission of RSVIgmyc through the female germ line, was also produced in founder transgenic mice by bypassing gametogenesis and introducing a highly methylated RSVIgmyc into the mouse zygote. These results suggest that RSVIgmyc methylation itself is a cis-acting signal for the preimplantation maintenance of the oocyte's methylation pattern and, therefore, a cis-acting signal for RSVIgmyc imprinting. Furthermore, our inability to identify a sequence element within RSVIgmyc that was absolutely required for its imprinting suggests that the extent of RSVIgmyc methylation, rather than a particular pattern of methylation, is the principal feature of this imprinting signal.