The restriction endonuclease R.NmeDI from Neisseria meningitidis that recognizes a palindromic sequence and cuts the DNA on both sides of the recognition sequence

The restriction endonuclease Type II R.NmeDI from Neisseria meningitidis 2120 (serogroup C, ST-11 complex) was characterized. The cloned nmeDIR gene was expressed in Escherichia coli cells, and the endonucleolytic and restriction activities of R.NmeDI were then observed in vitro and in vivo. The nmeDIR gene consists of 1056 bp coding 351 aa protein with a calculated molecular weight of M(r) = 39 000 ± 1000 Da. The R.NmeDI enzyme was purified to apparent homogeneity following overexpression, using metal affinity chromatography. This enzyme recognizes a palindrome sequence and cleaves double-stranded DNA upstream and downstream of its recognition sequence (12/7) RCCGGY (7/12) (R = A/G, Y = C/T) cutting out a 25-bp fragment. R.NmeDI cleaves in two steps. The enzyme cleaves the first strand randomly on either side of the recognition sequence generating an intermediate, and the second cleavage occurs more slowly and results in the production of a final reaction product. The R.NmeDI endonuclease requires two recognition sequences for effective cleavage. The tetramer is an active form of the R.NmeDI enzyme

DNA uptake sequences in 'Neisseria gonorrhoeae' as intrinsic transcriptional terminators and markers of horizontal gene transfer

DNA uptake sequences are widespread throughout the Neisseria gonorrhoeae genome. These short, conserved sequences facilitate the exchange of endogenous DNA between members of the genus Neisseria. Often the DNA uptake sequences are present as inverted repeats that are able to form hairpin structures. It has been suggested previously that DNA uptake sequence inverted repeats present 3′ of genes play a role in rho-independent termination and attenuation. However, there is conflicting experimental evidence to support this role. The aim of this study was to determine the role of DNA uptake sequences in transcriptional termination. Both bioinformatics predictions, conducted using TransTermHP, and experimental evidence, from RNA-seq data, were used to determine which inverted repeat DNA uptake sequences are transcriptional terminators and in which direction. Here we show that DNA uptake sequences in the inverted repeat configuration occur in N. gonorrhoeae both where the DNA uptake sequence precedes the inverted version of the sequence and also, albeit less frequently, in reverse order. Due to their symmetrical configuration, inverted repeat DNA uptake sequences can potentially act as bi-directional terminators, therefore affecting transcription on both DNA strands. This work also provides evidence that gaps in DNA uptake sequence density in the gonococcal genome coincide with areas of DNA that are foreign in origin, such as prophage. This study differentiates for the first time, to our knowledge, between DNA uptake sequences that form intrinsic transcriptional terminators and those that do not, providing characteristic features within the flanking inverted repeat that can be identified

Generation of one control and four iPSCs clones from patients with Emery-Dreifuss muscular dystrophy type 1.

Abstract Emery-Dreifuss muscular dystrophy type 1 (EDMD1) is a rare genetic disease caused by mutations in the EMD gene coding for a nuclear envelope protein emerin. We generated and characterized induced pluripotent stem cells (iPSCs) from two EDMD1 patients bearing a mutation c.del153C and from one healthy donor. That mutation leads to generation of premature STOP codon. Established iPSCs are very valuable tool for disease pathogenesis investigation and for the development of new therapeutic methods after differentiation to cardiac or muscle cells. Obtained iPSCs show the proper morphology, pluripotency markers expression, normal karyotype and potential to differentiate into three germ layers

Characterization of the dsDNA prophage sequences in the genome of Neisseria gonorrhoeae and visualization of productive bacteriophage

<p>Abstract</p> <p>Background</p> <p>Bioinformatic analysis of the genome sequence of <it>Neisseria gonorrhoeae </it>revealed the presence of nine probable prophage islands. The distribution, conservation and function of many of these sequences, and their ability to produce bacteriophage particles are unknown.</p> <p>Results</p> <p>Our analysis of the genomic sequence of FA1090 identified five genomic regions (NgoΦ1 – 5) that are related to dsDNA lysogenic phage. The genetic content of the dsDNA prophage sequences were examined in detail and found to contain blocks of genes encoding for proteins homologous to proteins responsible for phage DNA replication, structural proteins and proteins responsible for phage assembly. The DNA sequences from NgoΦ1, NgoΦ2 and NgoΦ3 contain some significant regions of identity. A unique region of NgoΦ2 showed very high similarity with the <it>Pseudomonas aeruginosa </it>generalized transducing phage F116. Comparative analysis at the nucleotide and protein levels suggests that the sequences of NgoΦ1 and NgoΦ2 encode functionally active phages, while NgoΦ3, NgoΦ4 and NgoΦ5 encode incomplete genomes. Expression of the NgoΦ1 and NgoΦ2 repressors in <it>Escherichia coli </it>inhibit the growth of <it>E. coli </it>and the propagation of phage λ. The NgoΦ2 repressor was able to inhibit transcription of <it>N. gonorrhoeae </it>genes and <it>Haemophilus influenzae </it>HP1 phage promoters. The holin gene of NgoΦ1 (identical to that encoded by NgoΦ2), when expressed in <it>E. coli</it>, could serve as substitute for the phage λ <it>s </it>gene. We were able to detect the presence of the DNA derived from NgoΦ1 in the cultures of <it>N. gonorrhoeae</it>. Electron microscopy analysis of culture supernatants revealed the presence of multiple forms of bacteriophage particles.</p> <p>Conclusion</p> <p>These data suggest that the genes similar to dsDNA lysogenic phage present in the gonococcus are generally conserved in this pathogen and that they are able to regulate the expression of other neisserial genes. Since phage particles were only present in culture supernatants after induction with mitomycin C, it indicates that the gonococcus also regulates the expression of bacteriophage genes.</p

Type III restriction-modification enzymes: a historical perspective

Restriction endonucleases interact with DNA at specific sites leading to cleavage of DNA. Bacterial DNA is protected from restriction endonuclease cleavage by modifying the DNA using a DNA methyltransferase. Based on their molecular structure, sequence recognition, cleavage position and cofactor requirements, restriction-modification (R-M) systems are classified into four groups. Type III R-M enzymes need to interact with two separate unmethylated DNA sequences in inversely repeated head-to-head orientations for efficient cleavage to occur at a defined location (25-27 bp downstream of one of the recognition sites). Like the Type I R-M enzymes, Type III R-M enzymes possess a sequence-specific ATPase activity for DNA cleavage. ATP hydrolysis is required for the long-distance communication between the sites before cleavage. Different models, based on 1D diffusion and/or 3D-DNA looping, exist to explain how the long-distance interaction between the two recognition sites takes place. Type III R-M systems are found in most sequenced bacteria. Genome sequencing of many pathogenic bacteria also shows the presence of a number of phase-variable Type III R-M systems, which play a role in virulence. A growing number of these enzymes are being subjected to biochemical and genetic studies, which, when combined with ongoing structural analyses, promise to provide details for mechanisms of DNA recognition and catalysis

Novel non-specific DNA adenine methyltransferases

The mom gene of bacteriophage Mu encodes an enzyme that converts adenine to N6-(1-acetamido)-adenine in the phage DNA and thereby protects the viral genome from cleavage by a wide variety of restriction endonucleases. Mu-like prophage sequences present in Haemophilus influenzae Rd (FluMu), Neisseria meningitidis type A strain Z2491 (Pnme1) and H. influenzae biotype aegyptius ATCC 11116 do not possess a Mom-encoding gene. Instead, at the position occupied by mom in Mu they carry an unrelated gene that encodes a protein with homology to DNA adenine N6-methyltransferases (hin1523, nma1821, hia5, respectively). Products of the hin1523, hia5 and nma1821 genes modify adenine residues to N6-methyladenine, both in vitro and in vivo. All of these enzymes catalyzed extensive DNA methylation; most notably the Hia5 protein caused the methylation of 61% of the adenines in λ DNA. Kinetic analysis of oligonucleotide methylation suggests that all adenine residues in DNA, with the possible exception of poly(A)-tracts, constitute substrates for the Hia5 and Hin1523 enzymes. Their potential ‘sequence specificity’ could be summarized as AB or BA (where B = C, G or T). Plasmid DNA isolated from Escherichia coli cells overexpressing these novel DNA methyltransferases was resistant to cleavage by many restriction enzymes sensitive to adenine methylation

S-Adenosyl homocysteine and DNA ends stimulate promiscuous nuclease activities in the Type III restriction endonuclease EcoPI

In the absence of the methyl donor S-adenosyl methionine and under certain permissive reaction conditions, EcoPI shows non-specific endonuclease activity. We show here that the cofactor analogue S-adenosyl homocysteine promotes this promiscuous DNA cleavage. Additionally, an extensive exonuclease-like processing of the DNA is also observed that can even result in digestion of non-specific DNA in trans. We suggest a model for how DNA communication events initiating from non-specific sites, and in particular free DNA ends, could produce the observed cleavage patterns

Concerted action at eight phosphodiester bonds by the BcgI restriction endonuclease

The BcgI endonuclease exemplifies a subset of restriction enzymes, the Type IIB class, which make two double-strand breaks (DSBs) at each copy of their recognition sequence, one either side of the site, to excise the sequence from the remainder of the DNA. In this study, we show that BcgI is essentially inactive when bound to a single site and that to cleave a DNA with one copy of its recognition sequence, it has to act in trans, bridging two separate DNA molecules. We also show that BcgI makes the two DSBs at an individual site in a highly concerted manner. Intermediates cut on one side of the site do not accumulate during the course of the reaction: instead, the DNA is converted straight to the final products cut on both sides. On DNA with two sites, BcgI bridges the sites in cis and then generally proceeds to cut both strands on both sides of both sites without leaving the DNA. The BcgI restriction enzyme can thus excise two DNA segments together, by cleaving eight phosphodiester bonds within a single-DNA binding event

Fused eco29kIR- and M genes coding for a fully functional hybrid polypeptide as a model of molecular evolution of restriction-modification systems

<p>Abstract</p> <p>Background</p> <p>The discovery of restriction endonucleases and modification DNA methyltransferases, key instruments of genetic engineering, opened a new era of molecular biology through development of the recombinant DNA technology. Today, the number of potential proteins assigned to type II restriction enzymes alone is beyond 6000, which probably reflects the high diversity of evolutionary pathways. Here we present experimental evidence that a new type IIC restriction and modification enzymes carrying both activities in a single polypeptide could result from fusion of the appropriate genes from preexisting bipartite restriction-modification systems.</p> <p>Results</p> <p>Fusion of <it>eco29kIR </it>and <it>M </it>ORFs gave a novel gene encoding for a fully functional hybrid polypeptide that carried both restriction endonuclease and DNA methyltransferase activities. It has been placed into a subclass of type II restriction and modification enzymes - type IIC. Its MTase activity, 80% that of the M.Eco29kI enzyme, remained almost unchanged, while its REase activity decreased by three times, concurrently with changed reaction optima, which presumably can be caused by increased steric hindrance in interaction with the substrate. <it>In vitro </it>the enzyme preferentially cuts DNA, with only a low level of DNA modification detected. <it>In vivo </it>new RMS can provide a 10²-fold less protection of host cells against phage invasion.</p> <p>Conclusions</p> <p>We propose a molecular mechanism of appearing of type IIC restriction-modification and M.SsoII-related enzymes, as well as other multifunctional proteins. As shown, gene fusion could play an important role in evolution of restriction-modification systems and be responsible for the enzyme subclass interconversion. Based on the proposed approach, hundreds of new type IIC enzymes can be generated using head-to-tail oriented type I, II, and III restriction and modification genes. These bifunctional polypeptides can serve a basis for enzymes with altered recognition specificities. Lastly, this study demonstrates that protein fusion may change biochemical properties of the involved enzymes, thus giving a starting point for their further evolutionary divergence.</p