A filter assay specific to Eco helix-destabilizing Protein I in crude extracts

A filter assay for Eco helix-destabilizing Protein I is reported and shown by immunological analysis specifically to detect Eco HD Protein I in crude extracts. The assay detects no activity in strains carrying a temperature-sensitive allele for Eco HD Protein I. The assay is linear over a 20-fold range and can reliably detect a nanogram or less of Eco HD Protein I.     

Domain organization and functional analysis of type IIS restriction endonuclease Eco31I

Type IIS restriction endonuclease Eco31I harbors a single HNH active site and cleaves both DNA strands close to its recognition sequence, 5'-GGTCTC(1/5). A two-domain organization of Eco31I was determined by limited proteolysis. Analysis of proteolytic fragments revealed that the N-terminal domain of Eco31I is responsible for the specific DNA binding, while the C-terminal domain contains the HNH nuclease-like active site. Gel-shift and gel-filtration experiments revealed that a monomer of the N-terminal domain of Eco31I is able to bind a single copy of cognate DNA. However, in contrast to other studied type IIS enzymes, the isolated catalytic domain of Eco31I was inactive. Steady-state and transient kinetic analysis of Eco31I reactions was inconsistent with dimerization of Eco31I on DNA. Thus, we propose that Eco31I interacts with individual copies of its recognition sequence in its monomeric form and presumably remains a monomer as it cleaves both strands of double-stranded DNA. The domain organization and reaction mechanism established for Eco31I should be common for a group of evolutionary related type IIS restriction endonucleases Alw26I, BsaI, BsmAI, BsmBI and Esp3I that recognize DNA sequences bearing the common pentanucleotide 5'-GTCTC.     

Mutational analysis of two putative catalytic motifs of the type IV restriction endonuclease Eco57I

The role of two sequence motifs (SM) as putative cleavage catalytic centers (77)PDX(13)EAK (SM I) and (811)PDX(20)DQK (SM II) of type IV restriction endonuclease Eco57I was studied by site-directed mutational analysis. Substitutions within SM I; D78N, D78A, D78K, and E92Q reduced cleavage activity of Eco57I to a level undetectable both in vivo and in vitro. Residual endonucleolytic activity of the E92Q mutant was detected only when the Mg(2+) in the standard reaction mixture was replaced with Mn(2+). The mutants D78N and E92Q retained the ability to interact with DNA specifically. The mutants also retained DNA methylation activity of Eco57I. The properties of the SM I mutants indicate that Asp(78) and Glu(92) residues are essential for cleavage activity of the Eco57I, suggesting that the sequence motif (77)PDX(13)EAK represents the cleavage active site of this endonuclease. Eco57I mutants containing single amino acid substitutions within SM II (D812A, D833N, D833A) revealed only a small or moderate decrease of cleavage activity as compared with wild-type Eco57I, indicating that the SM II motif does not represent the catalytic center of Eco57I. The results, taken together, allow us to conclude that the Eco57I restriction endonuclease has one catalytic center for cleavage of DNA.     

Substrate specificity of restriction endonuclease Eco781

The recognition sequence and cleavage point of restriction endonuclease Eco781 have been determined as 5'-GGCGCC-. There are several known enzymes recognizing the same sequence, although the prototype NarI and isoschizomers NdaI and NunII cleave the substrate to produce 5'-protruding ends, whereas cleavage with isoschizomer BbeI results in 3'-protruding ends. Therefore, restrictase Eco78I, generating flush ends, may be regarded as an enzyme with new specificity among the restriction endonucleases recognizing the 5'-GGCGCC-sequence.     

Purification and properties of the Eco57I restriction endonuclease and methylase--prototypes of a new class (type IV).

The Eco57I restriction endonuclease and methylase were purified to homogeneity from the E.coli RR1 strain carrying the eco57IRM genes on a recombinant plasmid. The molecular weight of the denaturated methylase is 63 kDa. The restriction endonuclease exists in a monomeric form with an apparent molecular weight of 104-108 kDa. R.Eco57I also possesses methylase activity. The methylation activities of both enzymes modify the outer A residue in the target sequence 5'CTGAAG yielding N6-methyladenine. M.Eco57I modifies both strands of the substrate while R.Eco57I modifies only one. Only the methylase enzyme is stimulated by Ca2+. The restriction endonuclease shows an absolute requirement for Mg2+ and is stimulated by AdoMet. ATP has no influence on either activity of the enzymes. The subunit structure and enzymatic properties of the Eco57I enzymes distinguish them from all other restriction-modification enzymes that have been described previously. Therefore, RM.Eco57I may be regarded as a representative of a novel class of restriction-modification systems, and we propose to classify it as type IV.     

A new restriction endonuclease Eco31I recognizing a non-palindromic sequence

A restriction endonuclease with a novel site-specificity has been isolated from the Escherichia coli strain RFL31. The nucleotide sequences around a single Eco31I cut on pBR322 DNA and two cuts of lambda DNA have been compared. A common 5'GAGACC 3'CTCTGG sequence occurs near each cleavage site. Precise mapping of the cleavages in both DNA strands places the cuts five nucleotides to the left of the upper sequence and one nucleotide to the left of the lower sequence. This enabled us to deduce the following recognition and cleavage specificity of Eco31I: 5' GGTCTCN decreases 3' CCAGAGN NNNN increases.     

Identification of a single HNH active site in type IIS restriction endonuclease Eco31I

Type IIS restriction endonuclease Eco31I is a "short-distance cutter", which cleaves DNA strands close to its recognition sequence, 5'-GGTCTC(1/5). Previously, it has been proposed that related endonucleases recognizing a common sequence core GTCTC possess two active sites for cleavage of both strands in the DNA substrate. Here, we present bioinformatic identification and experimental evidence for a single nuclease active site. We identified a short region of homology between Eco31I and HNH nucleases, constructed a three-dimensional model of the putative catalytic domain and validated our predictions by random and site-specific mutagenesis. The restriction mechanism of Eco31I is suggested by analogy to the mechanisms of phage T4 endonuclease VII and homing endonuclease I-PpoI. We propose that residues D311 and N334 coordinate the cofactor. H312 acts as a general base-activating water molecule for the nucleophilic attack. K337 together with R340 and D345 are located in close proximity to the active center and are essential for correct folding of catalytic motif, while D345 together with R264 and D273 could be directly involved in DNA binding. We also predict that the Eco31I catalytic domain contains a putative Zn-binding site, which is essential for its structural integrity. Our results suggest that the HNH-like active site is involved in the cleavage of both strands in the DNA substrate. On the other hand, analysis of site-specific mutants in the region, previously suggested to harbor the second active site, revealed its irrelevance to the nuclease activity. Thus, our data argue against the earlier prediction and indicate the presence of a single conserved active site in type IIS restriction endonucleases that recognize common sequence core GTCTC.     

On the possibility of localizing in situ Mus musculus and Drosophila virilus satellite DNAs by Alu I and Eco RI restriction endonucleases

Mus musculus and Drosophila virilus metaphase chromosomes have been treated with Alu I or Eco RI restriction endonucleases and, to ascertain possible selective digestion, subsequently stained with the DNA-specific dye ethidium bromide. The correlation between our findings and previously known cytological and biochemical data allowed us to show that Alu I is an enzyme capable of cytologically detecting repetitive DNAs, while Eco RI is unable to do so. This would be a consequence of the fact that Alu I extensively digests and extracts all chromosomal DNA except that localized in those regions where the presence of satellite DNAs has previously demonstrated. Eco RI, on the contrary, is not capable of doing so and its activity seems to be obstructed by alcohol:acid fixation procedures.     

Crystallization and preliminary crystallographic studies of a bifunctional restriction endonuclease Eco57I

Restriction endonuclease Eco57I from Escherichia coli recognizes asymmetric DNA sequence 5'-CTGAAG and has both restriction (DNA cleavage a short distance away from the recognition site) and modification (methylation) activities residing in a single polypeptide chain. Single crystals of wild-type Eco57I ternary complexes with double-stranded DNA and sinefungin, a stimulator of endonuclease activity, were obtained by the vapor diffusion technique and characterized crystallographically for different variants of the DNA component. The best data for the complex with 25-mer DNA were collected to 4.2-A resolution at 100 K using synchrotron radiation. The crystals are orthorhombic, space group P2(1)2(1)2, with a=164.3, b=293.0, c=71.1 A, and contain two to four copies of the protein in the asymmetric unit.     

Mapping and cloning of Eco RI-fragments of bacteriophage T5+ DNA.

The Eco RI-fragments of bacteriophage T5 DNA were mapped using a technique which involves primarily length measurements of molecules observed in the electron microscope. Since Eco RI cleavage generates termini with 4-nucleotide long cohesive ends, fragments of complete and partial Eco RI digests were covalently circularized with DNA ligase at dilute DNA concentrations before measuring relative to internal length standards. This established the order of the internal Eco RI fragments. The two external Eco RI fragments, which had only one Eco RI terminus, were positioned relative to the internal fragments by identifying the location of some of the naturally-occurring nicks in partially denatured linear Eco RI fragments. An attempt was made to clone each of the internal Eco RI-fragments of T5 DNA via transformation into E. coli after ligation in vitro with the plasmid pMB 9. Only one fragment could be cloned and this fragment did not specify any new polypeptides in minicells of either the E. coli EK1 host, X1411, or the EK 2 host, X1776.     

Alw26I, Eco31I and Esp3I--type IIs methyltransferases modifying cytosine and adenine in complementary strands of the target DNA.

The specificity of three DNA methyltransferases M.Alw26I, M.Eco31I and M.Esp3I, isolated from Acinetobacter Iwoffi RFL26, Escherichia coli RFL31 and Hafnia alvei RFL3+, respectively, was determined. All the enzymes methylate both strands of asymmetric recognition sites yielding m5C in the top-strand and m6A in the bottom-strand, as below: 5'-GTm5CTC 5'-GGTm5CTC 5'-CGTm5CTC 3'-Cm6AGAG 3'-CCm6AGAG 3'-GCm6AGAG (M.Alw26I) (M.Eco31I) (M.Esp3I) They are the first members of type IIs methyltransferases that modify different types of nucleotides in the recognition sequence.     

The DNA translocation and ATPase activities of restriction-deficient mutants of Eco KI.

Eco KI, a type I restriction enzyme, specifies DNA methyltransferase, ATPase, endonuclease and DNA translocation activities. One subunit (HsdR) of the oligomeric enzyme contributes to those activities essential for restriction. These activities involve ATP-dependent DNA translocation and DNA cleavage. Mutations that change amino acids within recognisable motifs in HsdR impair restriction. We have used an in vivo assay to monitor the effect of these mutations on DNA translocation. The assay follows the Eco KI-dependent entry of phage T7 DNA from the phage particle into the host cell. Earlier experiments have shown that mutations within the seven motifs characteristic of the DEAD-box family of proteins that comprise known or putative helicases severely impair the ATPase activity of purified enzymes. We find that the mutations abolish DNA translocation in vivo. This provides evidence that these motifs are relevant to the coupling of ATP hydrolysis to DNA translocation. Mutations that identify an endonuclease motif similar to that found at the active site of type II restriction enzymes and other nucleases have been shown to abolish DNA nicking activity. When conservative changes are made at these residues, the enzymes lack nuclease activity but retain the ability to hydrolyse ATP and to translocate DNA at wild-type levels. It has been speculated that nicking may be necessary to resolve the topological problems associated with DNA translocation by type I restriction and modification systems. Our experiments show that loss of the nicking activity associated with the endonuclease motif of Eco KI has no effect on ATPase activity in vitro or DNA translocation of the T7 genome in vivo.     

Cdk1-dependent destruction of Eco1 prevents cohesion establishment after S phase

Accurate genome segregation depends on cohesion mechanisms that link duplicated sister chromatids, thereby allowing their tension-dependent biorientation in metaphase. In Saccharomyces cerevisiae, cohesion is established during DNA replication when Eco1 acetylates the cohesin subunit Smc3. Cohesion establishment is restricted to S phase of the cell cycle, but the molecular basis of this regulation is unknown. Here, we show that Eco1 is negatively regulated by the protein kinase Cdk1. Phosphorylation of Eco1 after S phase targets it to SCF(Cdc4) for ubiquitination and subsequent degradation. A nonphosphorylatable mutant of Eco1 establishes cohesion after DNA replication, suggesting that Cdk1-dependent phosphorylation of Eco1 is a key factor limiting establishment to S phase. We also show that deregulation of Eco1 results in chromosome separation defects in anaphase. We conclude that this regulatory mechanism helps optimize the level of sister chromatid cohesion, ensuring a robust and efficient anaphase.     

Eco1 is important for DNA damage repair in S. cerevisiae

The cohesin network has an essential role in chromosome segregation, but also plays a role in DNA damage repair. Eco1 is an acetyltransferase that targets subunits of the cohesin complex and is involved in both the chromosome segregation and DNA damage repair roles of the network. Using budding yeast as a model system, we find that mutations in Eco1, including a genocopy of a human Roberts syndrome allele, do not cause gross defects in chromosome cohesion. We examined how mitotic and meiotic DNA damage repair is affected by mutations in Eco1. Strains containing mutations in Eco1 are sensitive to DNA damaging agents that cause double-strand breaks, such as Xrays and bleomycin. While meiotic crossing over is relatively unaffected in strains containing the Roberts mutation, reciprocal mitotic crossovers occur with extremely low frequency in this mutant background. Our results suggest that Eco1 promotes the reciprocal exchange of chromosome arms and maintenance of heterozygosity during mitosis.     

Methylation of Eco RI (GAm'ATTC) sequences of bacterial DNA

The methylation of Eco RI (GAm6 ATTC) sequences of DNA of bacteria related to the main branches of their phylogenetic dendrogramme, was studied. It was found that methylation of Eco RI sites is observed in bacteria Caulobacter and in Thermus aquaticus. This finding can be substantiated by the resistance of these DNAs to Eco RI restrictase as well as by the fact that Bam HI fragments of these DNAs cloned within the composition of the vector plasmid pUC8 in E. coli cells contain GAATTC sites.     

Eco1 is important for DNA damage repair in S. cerevisiae

The cohesin network has an essential role in chromosome segregation, but also plays a role in DNA damage repair. Eco1 is an acetyltransferase that targets subunits of the cohesin complex and is involved in both the chromosome segregation and DNA damage repair roles of the network. Using budding yeast as a model system, we find that mutations in Eco1, including a genocopy of a human Roberts syndrome allele, do not cause gross defects in chromosome cohesion. We examined how mitotic and meiotic DNA damage repair is affected by mutations in Eco1. Strains containing mutations in Eco1 are sensitive to DNA damaging agents that cause double-strand breaks, such as X-rays and bleomycin. While meiotic crossing over is relatively unaffected in strains containing the Roberts mutation, reciprocal mitotic crossovers occur with extremely low frequency in this mutant background. Our results suggest that Eco1 promotes the reciprocal exchange of chromosome arms and maintenance of heterozygosity during mitosis.Key words: cohesin, recombination, double-strand break, acetyltransferase, Roberts syndrome     

Distribution of 5-methylcytosine in phage lambda genome methylated by DNA methylase Eco RII

The distribution of 5-methylcytosine in Eco RI-Bam HI fragments of phage lambda DNA in vitro methylated by Eco RII methylase has been studied. The general picture of distribution of methylated sites in phage lambda DNA is slightly different from the statistical distribution. However, the sites have been found, where the distribution of 5-methylcytosine is not accidental. A complete absence of 5-methylcytosine in the J-fragment, a genome lambda area essential for site-specific recombination, has been found. The absence of Eco RII is supposed to be the best protection of this area of phage genome from the increased mutagenesis, characteristic for nucleotide sequences methylated by DNA-methylated Eco RII and Eco RII type.