Rare-cutting Restriction Enzymes Research Articles

Break-induced replication drives large-scale genomic amplifications in cancer cells.

DNA double-strand breaks (DSBs) are highly toxic lesions that underly the efficacy of ionizing radiation (IR) and a large number of cytotoxic chemotherapies 1-3 . Yet, abnormal repair of DSBs is associated with genomic instability and may contribute to cancer heterogeneity and tumour evolution. Here, we show that DSBs induced by IR, by DSB-inducing chemotherapeutics, or by the expression of a rare-cutting restriction endonuclease induce large-scale genomic amplification in human cancer cells. Importantly, the extent of DSB-induced genomic amplification (DIGA) in a panel of melanoma cell lines correlated with the degree of cytotoxicity elicited by IR, suggesting that DIGA contributes significantly to DSB-induced cancer cell lethality. DIGA, which is mediated through conservative DNA synthesis, does not require origin re-licensing, and is enhanced by the depletion or deletion of the methyltransferases SET8 and SUV4-20H1, which function sequentially to mono- and di-methylate histone H4 lysine 20 (H4K20) at DSBs to facilitate the recruitment of 53BP1-RIF1 and its downstream effector shieldin complex to DSBs to prevent hyper-resection 4-11 . Consistently, DIGA was enhanced in cells lacking 53BP1 or RIF1, or in cells that lacked components of the shieldin complex or of other factors that help recruit 53BP1 to DSBs. Mechanistically, DIGA requires MRE11/CtIP and EXO1, factors that promote resection and hyper-resection at DSBs, and is dependent on the catalytic activity of the RAD51 recombinase. Furthermore, deletion or depletion of POLD3, POLD4, or RAD52, proteins involved in break-induced replication (BIR), significantly inhibited DIGA, suggesting that DIGA is mediated through a RAD51-dependent BIR-like process. DIGA induction was maximal if the cells encountered DSBs in early and mid S-phase, whereas cells competent for homologous recombination (in late S and G2) exhibited less DIGA induction. We propose that unshielded, hyper-resected ends of DSBs may nucleate a replication-like intermediate that enables cytotoxic long-range genomic DNA amplification mediated through BIR.

Pulsed-Field Gel Electrophoresis Typing of Staphylococcus aureus Strains.

Pulsed-field gel electrophoresis (PFGE) is one of the most effective genotyping methods for the study of genetic relatedness of microorganisms as well as for epidemiologic investigation of outbreaks caused by pathogenic bacteria, including Staphylococcus aureus. The technique relies on the size patterns of large DNA fragments generated by the cleavage of intact bacterial chromosomes with a rare cutting restriction enzyme, and subsequently resolved by pulsed-field electrophoresis with periodic changes of the orientation of the electrical field across the gel. The high discriminatory power, improved reproducibility by standardization of experimental protocols and data analysis guidelines, and establishment of a national PFGE database of S. aureus profiles have made it a valuable means for global tracking of S. aureus infection sources and determination of genetic relatedness of the strains.

Plant X-tender: An extension of the AssemblX system for the assembly and expression of multigene constructs in plants.

Cloning multiple DNA fragments for delivery of several genes of interest into the plant genome is one of the main technological challenges in plant synthetic biology. Despite several modular assembly methods developed in recent years, the plant biotechnology community has not widely adopted them yet, probably due to the lack of appropriate vectors and software tools. Here we present Plant X-tender, an extension of the highly efficient, scar-free and sequence-independent multigene assembly strategy AssemblX, based on overlap-depended cloning methods and rare-cutting restriction enzymes. Plant X-tender consists of a set of plant expression vectors and the protocols for most efficient cloning into the novel vector set needed for plant expression and thus introduces advantages of AssemblX into plant synthetic biology. The novel vector set covers different backbones and selection markers to allow full design flexibility. We have included ccdB counterselection, thereby allowing the transfer of multigene constructs into the novel vector set in a straightforward and highly efficient way. Vectors are available as empty backbones and are fully flexible regarding the orientation of expression cassettes and addition of linkers between them, if required. We optimised the assembly and subcloning protocol by testing different scar-less assembly approaches: the noncommercial SLiCE and TAR methods and the commercial Gibson assembly and NEBuilder HiFi DNA assembly kits. Plant X-tender was applicable even in combination with low efficient homemade chemically competent or electrocompetent Escherichia coli. We have further validated the developed procedure for plant protein expression by cloning two cassettes into the newly developed vectors and subsequently transferred them to Nicotiana benthamiana in a transient expression setup. Thereby we show that multigene constructs can be delivered into plant cells in a streamlined and highly efficient way. Our results will support faster introduction of synthetic biology into plant science.

AssemblX: a user-friendly toolkit for rapid and reliable multi-gene assemblies.

The assembly of large DNA constructs coding for entire pathways poses a major challenge in the field of synthetic biology. Here, we present AssemblX, a novel, user-friendly and highly efficient multi-gene assembly strategy. The software-assisted AssemblX process allows even unexperienced users to rapidly design, build and test DNA constructs with currently up to 25 functional units, from 75 or more subunits. At the gene level, AssemblX uses scar-free, overlap-based and sequence-independent methods, allowing the unrestricted design of transcriptional units without laborious parts domestication. The assembly into multi-gene modules is enabled via a standardized, highly efficient, polymerase chain reaction-free and virtually sequence-independent scheme, which relies on rare cutting restriction enzymes and optimized adapter sequences. Selection and marker switching strategies render the whole process reliable, rapid and very effective. The assembly product can be easily transferred to any desired expression host, making AssemblX useful for researchers from various fields.

Less is more: extreme genome complexity reduction with ddRAD using Ion Torrent semiconductor technology.

Massively parallel sequencing a small proportion of the whole genome at high coverage enables answering a wide range of questions from molecular evolution and evolutionary biology to animal and plant breeding and forensics. In this study, we describe the development of restriction-site associated DNA (RAD) sequencing approach for Ion Torrent PGM platform. Our protocol results in extreme genome complexity reduction using two rare-cutting restriction enzymes and strict size selection of the library allowing sequencing of a relatively small number of genomic fragments with high sequencing depth. We applied this approach to a common freshwater fish species, the Eurasian perch (Perca fluviatilis L.), and generated over 2.2 MB of novel sequence data consisting of ~17,000 contigs, identified 1259 single nucleotide polymorphisms (SNPs). We also estimated genetic differentiation between the DNA pools from freshwater (Lake Peipus) and brackish water (the Baltic Sea) populations and identified SNPs with the strongest signal of differentiation that could be used for robust individual assignment in the future. This work represents an important step towards developing genomic resources and genetic tools for the Eurasian perch. We expect that our ddRAD sequencing protocol for semiconductor sequencing technology will be useful alternative for currently available RAD protocols.

The use of pulsed-field gel electrophoresis for genotyping of Clostridium difficile.

Genotyping approaches are important for tracking infectious agents and can be used for various purposes. Pulsed-Field Gel Electrophoresis (PFGE) is among the highly discriminatory genotyping approaches commonly used for characterizing Clostridium difficile. Other genotyping methods used for C. difficile include Ribotyping, Restriction Endonuclease Assay (REA), Multilocus Variable Number Tandem Repeats (VNTR) Assay, and others. PFGE has a high discriminatory power, high reproducibility, and typeability. We utilized PFGE for typing C. difficile isolates of porcine and human origin. We used a macrorestriction fragment analysis of an intact genomic DNA using SmaI, a rare cutting restriction endonuclease. Using a Contour-Clamped Homogeneous Electric Field (CHEF) system with running conditions of 120° angle; initial switch time of 5 s; final switch time of 40 s with a run time of 18 h in a low-melting temperature agarose (Seakem Gold); and 0.5× TBE circulated in the CHEF system at 6 V/cm [CDC (2014) Pulsenet. http://www.cdc.gov/pulsenet/index.html . Accessed 22 Aug 2014] supported by 14 °C cooling module, we were able to separate very large DNA fragments (up to 2 Mb).

Pulsed-field gel electrophoresis of Staphylococcus aureus.

For many bacterial pathogens, including Staphylococcus aureus, pulsed-field gel electrophoresis (PFGE) is a molecular typing method widely used in surveillance and epidemiological investigations. The general principle of PFGE involves creating large DNA fragments from intact bacterial chromosomes using rare cutting restriction endonucleases. These large DNA fragments are successfully separated in an agarose gel by alternating the direction of the electrical fields over a prolonged period of time. The resulting DNA banding patterns in the gel create a "DNA fingerprint," which can then be used to discriminate clonal relatedness of isolates based on set interpretation guidelines. Standardization of protocols has greatly enhanced the reproducibility of PFGE between labs, enabling national surveillance and further molecular epidemiological studies of S. aureus.

Pulsed-field gel electrophoresis for Leuconostoc mesenteroides and L. pseudomesenteroides.

Pulsed-field gel electrophoresis (PFGE) is a technique using alternating electric fields to help the separation of high molecular weight DNA fragments with a high resolution. This method consists of the digestion of bacterial chromosomal DNA with rare-cutting restriction enzymes and of applying an alternating electrical current between spatially distinct pairs of electrodes. DNA molecules migrate at different speeds according to the size of the fragment. This method is considered as the "gold standard" for genotyping, genetic fingerprinting, epidemiological studies, genome size estimation, and studying radiation-induced DNA damage and repair.

Pulsed field gel electrophoresis for dairy propionibacteria.

Pulsed field gel electrophoresis (PFGE) is a technique using alternating electric fields to migrate high molecular weight DNA fragments with a high resolution. This method consists of the digestion of bacterial chromosomal DNA with rare-cutting restriction enzymes and in applying an alternating electrical current between spatially distinct pairs of electrodes. DNA molecules migrate at different speeds according to the size of the fragments. Among other things, this technique is considered as the "gold standard" for genotyping, genetic fingerprinting, epidemiological studies, genome size estimation, and studying radiation-induced DNA damage and repair. This chapter describes a PFGE method that can be used to differentiate dairy propionibacteria.

Pulsed-field gel electrophoresis for Listeria monocytogenes.

Pulsed-Field Gel Electrophoresis (PFGE) is a molecular subtyping method with high discriminatory power, reproducibility, and epidemiological concordance for the subtyping of Listeria monocytogenes and other bacteria. PFGE uses rare-cutting restriction enzymes (macrorestriction) that cut the genomic DNA, usually resulting in 6-25 DNA fragments ranging between 30 and 600 kb. Bacterial cells are immobilized in agarose plugs and subsequently lysed to release genomic DNA, which is then subjected to DNA digestion. AscI and ApaI restriction enzymes are typically used for L. monocytogenes. Electrophoresis using an alternating electric field direction results in a DNA banding pattern, or fingerprint, which is used to classify isolates into different pulsotypes. PFGE is currently the CDC's gold standard method for epidemiological studies in foodborne outbreaks.

Genotyping and its epidemiological significance on Yunnan Yersinia pestis under Fse I enzyme digestion method

To create the fingerprint library of Yunnan Yersinia pestis by pulse field gel electrophoresis(PFGE)with Fse I enzyme digestion method and to study its epidemiological significance. We used rare cutting restriction enzyme Fse? to digest Yunnan Yersinia pestis strains that were isolated from foci including Rattus flavipectus Plague Focus,Apodemus chevrieri-Eothenomys miletus Plague Focus and Yulong Plague Focus. Fingerprints to Bionumerics software were used for cluster analysis. 30 tested strains were divided into 16 genotypes with the similarity value as 79.8%-100.0% . 16 genotypes wee formed into 4 clusters, with one consisted of only EV76 while the other three belonged to Rattus flavipectus,Apodemus chevrieri- Eothenomys miletus and Yulong clusters, respectively. PFGE genotypes of Yunnan Yersinia pestis accorded with its ecotypes and biovars, with clustered regional features. The strains isolated from Yulong showed an unique PFGE type, indicating a new clone group was identified.

Genotyping methods.

Genotyping allows for the identification of bacterial isolates to the strain level and provides basic information about the evolutionary biology, population biology, taxonomy, ecology, and genetics of bacteria. Depending on the underlying question and available resources, Pseudomonas aeruginosa strains may be typed by anonymous fingerprinting techniques or electronically portable sequence-based typing methods such as multiple locus variable number tandem repeat (VNTR) analysis (MLVA), multilocus sequence typing, or oligonucleotide microarray. Macrorestriction fragment pattern analysis is a genotyping method that is globally applicable to all bacteria and hence has been and still is the reference method for strain typing in bacteriology. Agarose-embedded chromosomal DNA is cleaved with a rare-cutting restriction endonuclease and the generated 20-70 fragments are then separated by pulsed-field gel electrophoresis. The chapter provides a detailed step-by-step manual for SpeI genome fingerprinting of Pseudomonas chromosomes that has been optimized for SpeI fragment pattern analysis of P. aeruginosa.

Dickeya solani sp. nov., a pectinolytic plant-pathogenic bacterium isolated from potato (Solanum tuberosum)

Pectinolytic bacteria have been recently isolated from diseased potato plants exhibiting blackleg and slow wilt symptoms found in a number of European countries and Israel. These Gram-reaction-negative, motile, rods were identified as belonging to the genus Dickeya, previously the Pectobacterium chrysanthemi complex (Erwinia chrysanthemi), on the basis of production of a PCR product with the pelADE primers, 16S rRNA gene sequence analysis, fatty acid methyl esterase analysis, the production of phosphatases and the ability to produce indole and acids from α-methylglucoside. Differential physiological assays used previously to differentiate between strains of E. chrysanthemi, showed that these isolates belonged to biovar 3. Eight of the isolates, seven from potato and one from hyacinth, were analysed together with 21 reference strains representing all currently recognized taxa within the genus Dickeya. The novel isolates formed a distinct genetic clade in multilocus sequence analysis (MLSA) using concatenated sequences of the intergenic spacer (IGS), as well as dnaX, recA, dnaN, fusA, gapA, purA, rplB, rpoS and gyrA. Characterization by whole-cell MALDI-TOF mass spectrometry, pulsed field gel electrophoresis after digestion of whole-genome DNA with rare-cutting restriction enzymes, average nucleotide identity analysis and DNA-DNA hybridization studies, showed that although related to Dickeya dadantii, these isolates represent a novel species within the genus Dickeya, for which the name Dickeya solani sp. nov. (type strain IPO 2222(T) = LMG25993(T) = NCPPB4479(T)) is proposed.

Characterization of Mycoplasma mycoides subsp. capri isolates by SDS-PAGE, immunoblotting and PFGE

The purpose of this study was to analyze a bank of 35 Mycoplasma mycoides subsp. capri (Mmc) isolates collected from contagious agalactia outbreaks during a 9-year period (2004–2012) in Sardinia, Italy. Isolates were characterized using genotyping and phenotyping methods. For each isolate, pulsed field gel electrophoresis (PFGE) DNA profile was determined. The PFGE study, performed with the rare-cutting restriction enzyme BnlI, showed a high degree of genetic heterogeneity. Our findings show the value of PFGE analysis for revealing the genetic diversity of isolates and its usefulness for epidemiology investigations. In addition, the whole cell protein profiles of each isolate were studied by Coomassie Blue-stained sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot (IB). After IB with pooled serum from naturally infected goats, two immunogenic proteins of about 90 and 60kDa were identified in all isolates analyzed. These proteins may have potential for the development of serological diagnostic tests.

From Agrobacterium to viral vectors: genome modification of plant cells by rare cutting restriction enzymes

Researchers and biotechnologists require methods to accurately modify the genome of higher eukaryotic cells. Such modifications include, but are not limited to, site-specific mutagenesis, site-specific insertion of foreign DNA, and replacement and deletion of native sequences. Accurate genome modifications in plant species have been rather limited, with only a handful of plant species and genes being modified through the use of early genome-editing techniques. The development of rare-cutting restriction enzymes as a tool for the induction of site-specific genomic double-strand breaks and their introduction as a reliable tool for genome modification in animals, animal cells and human cell lines have paved the way for the adaptation of rare-cutting restriction enzymes to genome editing in plant cells. Indeed, the number of plant species and genes which have been successfully edited using zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and engineered homing endonucleases is on the rise. In our review, we discuss the basics of rare-cutting restriction enzyme-mediated genome-editing technology with an emphasis on its application in plant species.

Small Molecule Screening for Novel Double-strand Break Repair Inhibitors using High-throughput Flow Cytometry

The two major double-strand break (DSB) repair pathways, homologous recombination (HR), and non-homologous end joining (NHEJ) play a critical role in the response to ionizing radiation (IR). Furthermore, it is well established that disrupting the balance between NHEJ and HR induces significant radiosensitivity. The rare cutting restriction endonuclease, I-SceI, is utilized commonly to assess intrachromosomal DSB repair in mammalian cells. We sought to develop an I-SceI-based assay and high-throughput screening platform to screen for drugs which modulate DSB repair.

Strain-specific detection by pulsed-field gel electrophoresis of Lactobacillus gasseri TMC0356 in human feces after oral administration of these organisms

The present study aimed to develop an innovative, strain-specific means of identifying the probiotic Lactobacillus gasseri TMC0356 and to determine whether orally administered TMC0356 could be recovered from the human intestine. High molecular weight genomic DNA was isolated from TMC0356 and 14 reference strains of L. gasseri, including the type strain. The DNA samples were digested with the selected rare-cutting restriction endonucleases SmaI, SacII and ApaI and the resulting fragments separated by pulsed-field gel electrophoresis (PFGE) in a size range between 20 to 290 kb. TMC0356 could be distinguished from the other L. gasseri strains on the basis of the SmaI and SacII macrorestriction patterns. Furthermore, L. gasseri strains isolated from the feces of subjects who had ingested TMC0356 were identical to TMC0356 in the SmaI, SacII and ApaI macrorestriction fragments of digested DNA. These results suggest that PFGE of genomic DNA digested with SmaI, SacII, could be a practical means of identification of TMC0356. Furthermore, these results indicate that ingested TMC0356 can survive in, and colonize, the human intestine.

A whole genome DArT assay to assess germplasm collection diversity in common beans

A Diversity Arrays Technology (DArT) marker system was applied for the first time to common beans (Phaseolus vulgaris L.) and tested on 89 accessions from the IAC and CIAT common bean breeding programs, as well as on landraces from the FAO collection. Seven frequently used restriction endonucleases were tested in combination with a rare-cutting restriction enzyme, PstI, to evaluate their suitability for DArT technology. Two restriction enzyme combinations (PstI/BstNI and PstI/TaqI) were selected to evaluate polymorphisms and PstI/BstNI, which yielded the most polymorphisms, was used to construct the final array. Genotyping was done by labelling the genomic representations with the fluorescent nucleotides cy3-dUTP and cy5-dUTP. The poly-linker fragment was labelled with 6-FAM and used as a control treatment and standard to determine the amount of DNA spotted on the array for each clone. DArTsoft version 7.3 software was used to analyse, identify and score polymorphic markers. Arrays containing individual fragments from these representations generated DArT fingerprints with a genotype call rate of 97.1% and a scoring reproducibility of at least 99.9%. A total of 2,501 polymorphic markers were found. Neighbour-joining distance matrices were used to create dispersion graphs that distinguished the two major gene pools of common beans and classified the accessions as either Andean or Mesoamerican. A principal coordinate analysis of the DArT marker results explained 82% of the total data variation. These results show that the DArT platform was accurate for studying the genetic diversity of the common bean and efficient for the large-scale detection of polymorphisms. These properties make marker technology a choice for future experiments. This is the first report to describe the use of DArT technology for genotyping the common bean.

Cell Cycle-Dependent Induction of Homologous Recombination by a Tightly Regulated I-SceI Fusion Protein

Double-strand break repair is executed by two major repair pathways: non-homologous end joining (NHEJ) and homologous recombination (HR). Whereas NHEJ contributes to the repair of ionizing radiation (IR)-induced double strand breaks (DSBs) throughout the cell cycle, HR acts predominantly during the S and G2 phases of the cell cycle. The rare-cutting restriction endonuclease, I-SceI, is in common use to study the repair of site-specific chromosomal DSBs in vertebrate cells. To facilitate analysis of I-SceI-induced DSB repair, we have developed a stably expressed I-SceI fusion protein that enables precise temporal control of I-SceI activation, and correspondingly tight control of the timing of onset of site-specific chromosome breakage. I-SceI-induced HR showed a strong, positive linear correlation with the percentage of cells in S phase, and was negatively correlated with the G1 fraction. Acute depletion of BRCA1, a key regulator of HR, disrupted the relationship between S phase fraction and I-SceI-induced HR, consistent with the hypothesis that BRCA1 regulates HR during S phase.

Expression, purification and characterization of cloning-grade zinc finger nuclease

The limited number of naturally occurring rare-cutting restriction enzymes and the slow and tedious engineering of existing restriction enzymes for novel specificities have prompted the design of new strategies for the development of restriction enzymes with specificities for long DNA sequences. One possibility is using zinc finger nucleases (ZFNs)—synthetic restriction enzymes that are custom-designed to target and cleave long DNA sequences and which have been recently shown useful for DNA cloning. Here we report on the purification and biochemical analysis of ZFN-10, a custom-made ZFN. We show that Ni-affinity and gel-filtration purification methods are sufficient to produce a cloning-grade enzyme. We show that ZFN-10 can function as an accurate and reliable ZFN using the same reagents and protocols used for naturally occurring and commercially available recombinant restriction enzymes. We also show that ZFN-10 tolerates a set of target-site substitutions which can be predicted from the specificities of recognition helices incorporated into the structure of its DNA-binding domain. The relative simplicity of ZFN-10 design, expression, purification and analysis suggests that novel ZFNs can potentially be designed and applied for various recombinant DNA applications.