Arvydas Lubys

Novel Subtype of Type IIs Restriction Enzymes BfiI ENDONUCLEASE EXHIBITS SIMILARITIES TO THE EDTA-RESISTANT NUCLEASE Nuc OF SALMONELLA TYPHIMURIUM

The type IIs restriction enzyme BfiI recognizes the non-palindromic nucleotide sequence 5′-ACTGGG-3′ and cleaves complementary DNA strands 5/4 nucleotides downstream of the recognition sequence. The genes coding for the BfiI restriction-modification (R-M) system were cloned/sequenced and biochemical characterization of BfiI restriction enzyme was performed. The BfiI R-M system contained three proteins: two N4-methylcytosine methyltransferases and a restriction enzyme. Sequencing of bisulfite-treated methylated DNA indicated that each methyltransferase modifies cytosines on opposite strands of the recognition sequence. The N-terminal part of the BfiI restriction enzyme amino acid sequence revealed intriguing similarities to an EDTA-resistant nuclease of Salmonella typhimurium. Biochemical analyses demonstrated that BfiI, like the nuclease of S. typhimurium, cleaves DNA in the absence of Mg2+ ions and hydrolyzes an artificial substrate bis(p-nitrophenyl) phosphate. However, unlike the nonspecific S. typhimurium nuclease, BfiI restriction enzyme cleaves DNA specifically. We propose that the DNA-binding specificity of BfiI stems from the C-terminal part of the protein. The catalytic N-terminal subdomain ofBfiI radically differs from that of type II restriction enzymes and is presumably similar to the EDTA-resistant nonspecific nuclease of S. typhimurium; therefore, BfiI did not require metal ions for catalysis. We suggest that BfiI represents a novel subclass of type IIs restriction enzymes that differs from the archetypal FokI endonuclease by the fold of its cleavage domain, the domain location, and reaction mechanism.

Identification of a new subfamily of HNH nucleases and experimental characterization of a representative member, HphI restriction endonuclease

The restriction endonuclease (REase) R. HphI is a Type IIS enzyme that recognizes the asymmetric target DNA sequence 5′‐GGTGA‐3′ and in the presence of Mg2+ hydrolyzes phosphodiester bonds in both strands of the DNA at a distance of 8 nucleotides towards the 3′ side of the target, producing a 1 nucleotide 3′‐staggered cut in an unspecified sequence at this position. REases are typically ORFans that exhibit little similarity to each other and to any proteins in the database. However, bioinformatics analyses revealed that R.HphI is a member of a relatively big sequence family with a conserved C‐terminal domain and a variable N‐terminal domain. We predict that the C‐terminal domains of proteins from this family correspond to the nuclease domain of the HNH superfamily rather than to the most common PD‐(D/E)XK superfamily of nucleases. We constructed a three‐dimensional model of the R.HphI catalytic domain and validated our predictions by site‐directed mutagenesis and studies of DNA‐binding and catalytic activities of the mutant proteins. We also analyzed the genomic neighborhood of R.HphI homologs and found that putative nucleases accompanied by a DNA methyltransferase (i.e. predicted REases) do not form a single group on a phylogenetic tree, but are dispersed among free‐standing putative nucleases. This suggests that nucleases from the HNH superfamily were independently recruited to become REases in the context of RM systems multiple times in the evolution and that members of the HNH superfamily may be much more frequent among the so far unassigned REase sequences than previously thought. Proteins 2006.

Mva1269I: A Monomeric Type IIS Restriction Endonuclease from Micrococcus Varians with Two EcoRI- and FokI-like Catalytic Domains

Type II restriction endonuclease Mva1269I recognizes an asymmetric DNA sequence 5′-GAATGCN↓-3′/5′-NG↓CATTC-3′ and cuts top and bottom DNA strands at positions, indicated by the “↓” symbol. Most restriction endonucleases require dimerization to cleave both strands of DNA. We found that Mva1269I is a monomer both in solution and upon binding of cognate DNA. Protein fold-recognition analysis revealed that Mva1269I comprises two “PD-(D/E)XK” domains. The N-terminal domain is related to the 5′-GAATTC-3′-specific restriction endonuclease EcoRI, whereas the C-terminal one resembles the nonspecific nuclease domain of restriction endonuclease FokI. Inactivation of the C-terminal catalytic site transformed Mva1269I into a very active bottom strand-nicking enzyme, whereas mutants in the N-terminal domain nicked the top strand, but only at elevated enzyme concentrations. We found that the cleavage of the bottom strand is a prerequisite for the cleavage of the top strand. We suggest that Mva1269I evolved the ability to recognize and to cleave its asymmetrical target by a fusion of an EcoRI-like domain, which incises the bottom strand within the target, and a FokI-like domain that completes the cleavage within the nonspecific region outside the target sequence. Our results have implications for the molecular evolution of restriction endonucleases, as well as for perspectives of engineering new restriction and nicking enzymes with asymmetric target sites.

Improved genome recovery and integrated cell-size analyses of individual uncultured microbial cells and viral particles

Microbial single-cell genomics can be used to provide insights into the metabolic potential, interactions, and evolution of uncultured microorganisms. Here we present WGA-X, a method based on multiple displacement amplification of DNA that utilizes a thermostable mutant of the phi29 polymerase. WGA-X enhances genome recovery from individual microbial cells and viral particles while maintaining ease of use and scalability. The greatest improvements are observed when amplifying high G+C content templates, such as those belonging to the predominant bacteria in agricultural soils. By integrating WGA-X with calibrated index-cell sorting and high-throughput genomic sequencing, we are able to analyze genomic sequences and cell sizes of hundreds of individual, uncultured bacteria, archaea, protists, and viral particles, obtained directly from marine and soil samples, in a single experiment. This approach may find diverse applications in microbiology and in biomedical and forensic studies of humans and other multicellular organisms.Single-cell genomics can be used to study uncultured microorganisms. Here, Stepanauskas et al. present a method combining improved multiple displacement amplification and FACS, to obtain genomic sequences and cell size information from uncultivated microbial cells and viral particles in environmental samples.

Targeted gene editing by transfection of in vitro reconstituted Streptococcus thermophilus Cas9 nuclease complex.

Cas9 protein of the Type II CRISPRCas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated) bacterial adaptive immune system emerged recently as a promising tool for genome editing in human and other eukaryotic cells. Cas9 binds a dual crRNA (CRISPR RNA)-tracrRNA (transactivating RNA) molecule, or an artificial single-guide RNA (sgRNA) into a functional complex that acts as an RNAdirected DNA endonuclease. It locates and binds to the target site guided by crRNA (sgRNA) while the Cas9 protein cuts DNA generating a double strand break (DSB) within the target sequence. In eukaryotic cells, DSB is repaired by “error prone” non-homologous end joining (NHEJ) or by homology directed repair (HDR) mechanisms resulting in the genome modification or insertion of new genetic information. Cas9 of Streptococcus pyogenes (SpCas9) is currently used as a model system for genome editing applications. Typically, the DNA expression cassettes encoding nucleus-targeted codon-optimized Cas9 protein and sgRNAs are transfected into the cells. The efficiency of DNA cleavage by plasmid-delivered Cas9 in eukaryotic cells depends on multiple factors, including expression vector design, transfection efficiency, cell type, recovery yield of functional Cas9 complex, and usually requires optimization of a set of experimental conditions. Cas9 delivery by plasmid transfection is still difficult to achieve for some hard-to-transform cell lines including human primary cells and pluripotent stem cells. Moreover, plasmid transfection occasionally results in undesirable integration of vector plasmid into the genome and is often inefficient and stressful to cells. Here we report an alternative way for the Cas9-mediated genome modification in eukaryotic cells (Fig. 1A) by chemical transfection of in vitro reconstituted functionally active Cas9-crRNA-tracrRNA complex of Streptococcus thermophilus (StCas9) CRISPR3-Cas system. The StCas9 protein bearing the nuclear localization signal (NLS) and 6xHis tag was purified from E.coli, and the StCas9 complex was reconstituted in vitro as described by Karvelis et al. To enable the delivery of reconstituted StCas9 complex into CHO-K1 cells, transfection experiments were performed using a protein delivery agent TurboFect. Alternatively, other transfection reagents like Lipofectamine 2000 or 3000 can be used to transfect Cas9 complexes into cells (data not shown). StCas9 localization was monitored using mouse polyclonal antiCas9 antibodies along with FITC-labeled secondary antibodies; crRNA-tracrRNA duplex was detected via 30-biotin labeled tracrRNA using streptavidin-coupled Qdots 585 nm. Both StCas9 and tracrRNA were observed in the perinuclear region and within the nucleus indicating that in vitro pre-assembled StCas9 complexes can be efficiently delivered into mammalian cells for targeted genome modification (Fig. S1). To monitor the DNA cleavage activity of transfected Cas9 complexes in mammalian cells, we constructed a dual reporter cassette bearing Red Fluorescent Protein (RFP) and enhanced Green Fluorescent Protein (eGFP) genes (Fig. 1B). eGFP gene contains 2 sites, T1 and T2, targeted by 2 different StCas9 complexes. The I-CreI nuclease target site was also engineered into the cassette. In the absence of Cas9, eGFP fluorescence should be observed following intron processing in vivo. Cas9 facilitated DSB at T1 or T2

Fluoride-Cleavable, Fluorescently Labelled Reversible Terminators: Synthesis and Use in Primer Extension

Fluorescent 2′-deoxynucleotides containing a protecting group at the 3′-O-position are reversible terminators that enable array-based DNA sequencing-by-synthesis (SBS) approaches. Herein, we describe the synthesis and full characterisation of four reversible terminators bearing a 3′-blocking moiety and a linker-dye system that is removable under the same fluoride-based treatment. Each nucleotide analogue has a different fluorophore attached to the base through a fluoride-cleavable linker and a 2-cyanoethyl moiety as the 3′-blocking group, which can be removed by using a fluoride treatment as well. Furthermore, we identified a DNA polymerase, namely, RevertAid M-MuLV reverse transcriptase, which can incorporate the four modified reversible terminators. The synthesised nucleotides and the optimised DNA polymerase were used on CodeLink slides spotted with hairpin oligonucleotides to demonstrate their potential in a cyclic reversible terminating approach.

Three-stage biochemical selection: cloning of prototype class IIS/IIC/IIG restriction endonuclease-methyltransferase TsoI from the thermophile Thermus scotoductus

BackgroundIn continuing our research into the new family of bifunctional restriction endonucleases (REases), we describe the cloning of the tsoIRM gene. Currently, the family includes six thermostable enzymes: TaqII, Tth111II, TthHB27I, TspGWI, TspDTI, TsoI, isolated from various Thermus sp. and two thermolabile enzymes: RpaI and CchII, isolated from mesophilic bacteria Rhodopseudomonas palustris and Chlorobium chlorochromatii, respectively. The enzymes have several properties in common. They are large proteins (molecular size app. 120 kDa), coded by fused genes, with the REase and methyltransferase (MTase) in a single polypeptide, where both activities are affected by S-adenosylmethionine (SAM). They recognize similar asymmetric cognate sites and cleave at a distance of 11/9 nt from the recognition site. Thus far, we have cloned and characterised TaqII, Tth111II, TthHB27I, TspGWI and TspDTI.ResultsTsoI REase, which originate from thermophilic Thermus scotoductus RFL4 (T. scotoductus), was cloned in Escherichia coli (E. coli) using two rounds of biochemical selection of the T. scotoductus genomic library for the TsoI methylation phenotype. DNA sequencing of restriction-resistant clones revealed the common open reading frame (ORF) of 3348 bp, coding for a large polypeptide of 1116 aminoacid (aa) residues, which exhibited a high level of similarity to Tth111II (50% identity, 60% similarity). The ORF was PCR-amplified, subcloned into a pET21 derivative under the control of a T7 promoter and was subjected to the third round of biochemical selection in order to isolate error-free clones. Induction experiments resulted in synthesis of an app. 125 kDa protein, exhibiting TsoI-specific DNA cleavage. Also, the wild-type (wt) protein was purified and reaction optima were determined.ConclusionsPreviously we identified and cloned the Thermus family RM genes using a specially developed method based on partial proteolysis of thermostable REases. In the case of TsoI the classic biochemical selection method was successful, probably because of the substantially lower optimal reaction temperature of TsoI (app. 10-15°C). That allowed for sufficient MTase activity in vivo in recombinant E. coli. Interestingly, TsoI originates from bacteria with a high optimum growth temperature of 67°C, which indicates that not all bacterial enzymes match an organism’s thermophilic nature, and yet remain functional cell components. Besides basic research advances, the cloning and characterisation of the new prototype REase from the Thermus sp. family enzymes is also of practical importance in gene manipulation technology, as it extends the range of available DNA cleavage specificities.

Draft Genome Sequence of the Cyanobacterium Aphanizomenon flos-aquae Strain 2012/KM1/D3, Isolated from the Curonian Lagoon (Baltic Sea)

ABSTRACT We report here the de novo genome assembly of a cyanobacterium, Aphanizomenon flos-aquae strain 2012/KM1/D3, a harmful bloom-forming species in temperate aquatic ecosystems. The genome is 5.7 Mb with a G+C content of 38.2%, and it is enriched mostly with genes involved in amino acid and carbohydrate metabolism.

Bioinformatic and partial functional analysis of pEspA and pEspB, two plasmids from Exiguobacterium arabatum sp. nov. RFL1109.

The complete nucleotide sequences of two plasmids from Exiguobacterium arabatum sp. nov. RFL1109, pEspA (4563bp) and pEspB (38,945bp), have been determined. Five ORFs were identified in the pEspA plasmid, and putative functions were assigned to two of them. Using deletion mapping approach, the Rep-independent replication region of pEspA, which functions in Bacillus subtilis, was localized within a 0.6kb DNA region. Analysis of the pEspB sequence revealed 42 ORFs. From these, function of two genes encoding enzymes of the Lsp1109I restriction-modification system was confirmed experimentally, while putative functions of another 18 ORFs were suggested based on comparative analysis. Three functional regions have been proposed for the pEspB plasmid: the putative conjugative transfer region, the region involved in plasmid replication and maintenance, and the region responsible for transposition of the IS21 family-like transposable elements.

Random gene dissection : a tool for the investigation of protein structural organization

To investigate the domain structure of proteins and the function of individual domains, proteins are usually subjected to limited proteolysis, followed by isolation of protein fragments and determination of their functions. We have developed an approach we call random gene dissection (RGD) for the identification of functional protein domains and their interdomain regions as well as their in vivo complementing fragments. The approach was tested on a two-domain protein, the type IIS restriction endonuclease BfiI. The collection of BfiI insertional mutants was screened for those that are endonucleolytically active and thus induce the SOS DNA repair response. Sixteen isolated mutants of the wild-type specificity contained insertions that were dispersed in a relatively large region of the target recognition domain. They split the gene into two complementing parts that separately were unable to induce the SOS DNA repair response. In contrast, all 19 mutants of relaxed specificity contained the cassette inserted into a very narrow interdomain region that connects BfiI domains responsible for DNA recognition and for cleavage. As expected, only the N-terminal fragment of BfiI was required to induce SOS response. Our results demonstrate that RGD can be used as a general method to identify complementing fragments and functional domains in enzymes.