Restriction Endonuclease Activity Research Articles

Action of restriction endonucleases on the DNA and chromosomes of Muntiacus muntjak.

The restriction enzymes EcoR1, Hpa II and Hae III have been used to cleave nuclear DNA of Muntiacus muntjak. Each enzyme gives rise to a different pattern. Each pattern is characterized by the presence or absence of bands and by the type of distribution of most of the sequences cleaved. Hpa II and Hae III cleave DNA segments which are CCGG and GGCC respectively but they produce quite different patterns with the same DNA. Hpa II builds a gradient of large molecules concentrated mainly between 11.0times103 and 600 base pairs whereas Hae III forms a smear of small molecules concentrated mainly between 3.4times103 and 200 base pairs. This difference in pattern is characteristic of the DNA of over 30 eukaryotic species so far investigated. The action of Hae III on fixed metaphase chromosomes of M. muntjak leads to the appearance of a “hairy” structure and a chromomere pattern which is not present in the controls and which does not show up after EcoR1 treatment. The effect of the enzyme on the chromosomes is not understood at present.

Exploring The Spatiotemporaloral Dynamics of DNA Binding and Cleavage by Restriction Endonucleaes

Using restriction endonucleases to catalyze the double-stranded DNA (dsDNA) breakage at certain recognition sequences is an important molecular biology technique. The restriction endonucleases constitute an important defense mechanism of bacteria against viral attacks; this mechanism is to destroy invading foreign DNA molecules via cleaving a specific site (phosphodiester bond) of a dsDNA. By cleaving recognition sites on dsDNA with extraordinary specificity can lead to the DNA double strand breaks (dsb). We presented a novel single-molecule approach to investigate the interaction between DNA and restriction endonucleases, including DNA recognition and cleavage. To elucidate how fast restriction endonucleases recognize and cleavage DNA sequence, we constructed a high resolution dual-beam laser tweezers system to manipulate single DNA molecule, together with the site-specific restriction enzymes, namely, EcoRI (one-site endonuclease) and Cfr9I (two-site endonuclease), conjugated to nanometer-sized fluorescence particle. Because most endonucleases work in the presence of magnesium ions, we will apply optically based reaction mechanism to control and synchronize the restriction endonuclease activity in this study. Furthermore, both laser tweezers and fluorescence particle imaging will be used to probe whether the DNA double strand breaks occurred due to the molecular cutting. Hence, this single-molecule approach allows us to directly observe and visualize the spatiotemporal dynamics of DNA binding and cleavage by restriction endonucleases, and can be further applied to determine the DNA cleavage rate due to the presence of EcoRI and Cfr9I. Finally, we extend this approach, together with the light-induced molecular cutting, to investigate the DNA binding and cleavage by restriction endonucleases under tension at different temperatures.

Unfixed and fixed human chromosomes show different staining patterns after restriction endonuclease digestion

Restriction endonucleases (REs) have been widely used to produce banding patterns on chromosomes, but it remains uncertain to what extent the patterns are due to the sequence specificity of the enzymes, and to what extent chromatin structure influences the pattern of digestion. To throw light on this question, we have digested with restriction endonucleases unfixed chromosomes prepared in two different ways (isolated, and whole metaphase cells spread with a cytocentrifuge) and compared the results with those obtained on conventionally fixed chromosomes. Unfixed isolated chromosomes are easily destroyed by REs; after fixation with cold methanol, which produced minimal alteration to the chromatin structure, the chromosomes are resistant to the action of REs, and conventional methanol-acetic acid fixation is required to permit the induction of banding patterns by REs. Unfixed cytocentrifuge preparations, in which the chromosomes are still surrounded by cytoplasm, are much more resistant to the action of REs, and again banding patterns were only induced after methanol-acetic acid fixation. We conclude that the action of restriction endonucleases on chromosomes is strongly influenced by chromatin organisation, and that methanol-acetic acid fixation is required to permit the induction of conventional banding patterns on chromosomes.

Continuous monitoring of restriction endonuclease cleavage activity by universal molecular beacon light quenching coupled with real-time polymerase chain reaction

We describe a method for sensitive monitoring of restriction endonuclease kinetics and activity by use of a universal molecular beacon (U-MB) coupled with real-time polymerase chain reaction (PCR). The method is used to monitor the progress of DNA cleavage in a sealed reaction tube and offers more accurate and high-throughput detection. The template has a universal tail hybridized with the U-MB and the remaining sequence is complementary to one of the restriction endonuclease digestion products. The U-MB is replaced by the extension of digested product and the fluorescence quenches. With this concept, one universal fluorescence probe can be used in different enzyme analytical systems. In the work described here, homogenous assays were performed with the restriction endonucleases AluΙ, EcoRΙ, XhoΙ, and SacΙ at smoothly controlled temperature. Cleavage efficiencies were determined, and the potential applications of this method were discussed. Furthermore, the AluΙ and EcoRΙ cleavage reactions were monitored online at varying substrate concentrations at the molecular level, and K m, V max, and K cat values were calculated. The results suggest that U-MB monitoring of restriction endonuclease assays based on real-time PCR will be very useful for high-throughput, sensitive, and precise assays for enzyme activity screening and evolutionary biotechnology analysis.

Real-time monitoring of double-stranded DNA cleavage using molecular beacons

Traditional methods to assay enzymatic cleavage of DNA are discontinuous, time-consuming and laborious. Here, we report a new approach for real-time monitoring of double-stranded DNA cleavage by restriction endonuclease based on nucleic acid ligation using molecular beacon. Upon cleavage of DNA, the cleavage product can be ligated by DNA ligase, which results in a fluorescence enhancement of the molecular beacon. This method permits real-time monitoring of DNA cleavage and makes it easy to characterize the activity of restriction endonuclease and to study the cleavage reaction kinetics.

The Fidelity Index provides a systematic quantitation of star activity of DNA restriction endonucleases.

Restriction endonucleases are the basic tools of molecular biology. Many restriction endonucleases show relaxed sequence recognition, called star activity, as an inherent property under various digestion conditions including the optimal ones. To quantify this property we propose the concept of the Fidelity Index (FI), which is defined as the ratio of the maximum enzyme amount showing no star activity to the minimum amount needed for complete digestion at the cognate recognition site for any particular restriction endonuclease. Fidelity indices for a large number of restriction endonucleases are reported here. The effects of reaction vessel, reaction volume, incubation mode, substrate differences, reaction time, reaction temperature and additional glycerol, DMSO, ethanol and Mn2+ on the FI are also investigated. The FI provides a practical guideline for the use of restriction endonucleases and defines a fundamental property by which restriction endonucleases can be characterized.

Isolation and cloning of IGF-1R gene in bovine

Gene cloning, or molecular cloning, has several different meanings to a molecular biologist. A clone is an exact copy, or replica, of something. Molecular biologists exploit the replicative ability of cultured cells to clone genes. Recent advances in the Restriction enzymes technologies have opened up new opportunities in biotechnology and facilitate the way to isolate any gene from any source of cells. Many types of restriction enzymes cleave DNA away from their recognition site. By using the type III restriction enzyme, we provide evidence to show that an intact recognition site on the cleaved DNA sequesters the restriction. We isolated and cloned the IGF-1 R gene from DNA by subjecting DNA with one pair of restriction enzyme (EcoRI + HindIII) and followed by ligation and transformation with the suitable vector. The pair of restriction enzymes (EcoRI + HindIII) which subjected to DNA template before the ligation process was the only pair which had success to liberate IGF-1R fragment from the transformed vector rather than other two pairs of restriction enzymes (EcoRI + XhoI) or (HindIII + XhoI) although this two pair of restriction enzymes have recognition sites in the vector. Moreover we have studied the effect of DNA template size on the action of the restriction enzymes. Where less DNA size, more efficient of the restriction enzyme action. This study is rapid and useful new technology for isolation, cloning and sequencing the gene of interest to be ready for further investigations as gene transfection and/ or transfer. Key words: Restriction enzymes- DNA technology- IGF-1R- bovine.

In vivo restriction endonuclease activity of the Anabaena PCC 7120 XisA protein in Escherichia coli

Anabaena PCC 7120 genome contains three elements, which get excised out during late stages of heterocyst differentiation by a site-specific recombination process. The XisA protein, which excises the nifD element, shows sequence homology with the integrase family of tyrosine recombinase. The 11 bp target site of XisA C GGAGTAA TCC contains a 3 bp inverted repeat. Here, we report restriction endonuclease activity of XisA by specific loss of plasmids containing single or double target sites. The pMX25 plasmid containing two target sites demonstrated endonuclease activity proportional to excision frequency. Different plasmid substrates containing one base pair mutation in the inverted repeat of the target site were monitored for endonuclease activity. Mutation of A4C retained endonuclease activity, while other modifications lost endonuclease activity. The presence of an additional copy of the target site enhanced endonuclease activity. These results suggest that the XisA protein could be an IIE type of restriction endonuclease in addition to being a recombinase.

The Crystal Structure of λ-Gam Protein Suggests a Model for RecBCD Inhibition

In Escherichia coli, RecBCD processes double-stranded DNA breaks during the initial stages of homologous recombination. RecBCD contains helicase and nuclease activities, and unwinds and digests the blunt-ended DNA until a specific eight-nucleotide sequence, Chi, is encountered. Chi modulates the nuclease activity of RecBCD and results in a resected DNA end, which is a substrate for RecA during subsequent steps in recombination. RecBCD also acts as a defence mechanism against bacteriophage infection by digesting linear viral DNA present during virus replication or resulting from the action of restriction endonucleases. To avoid this fate, bacteriophage lambda encodes the gene Gam whose product is an inhibitor of RecBCD. Gam has been shown to bind to RecBCD and inhibit its helicase and nuclease activities. We show that Gam inhibits RecBCD by preventing it from binding DNA. We have solved the crystal structure of Gam from two different crystal forms. Using the published crystal structure of RecBCD in complex with DNA we suggest models for the molecular mechanism of Gam-mediated inhibition of RecBCD. We also propose that Gam could be a mimetic of single-stranded, and perhaps also double-stranded, DNA.

Inhibition of Restriction Enzyme's DNA Sequence Recognition by PUVA Treatment†¶

Applying various restriction enzymes on a specially designed 1.5 kb DNA fragment revealed that the inhibitory effects of psoralens + UVA irradiation (PUVA) treatment on restriction endonuclease activities are caused by recognition inhibition. In this study restriction enzymes that have a 5′-TpA sequence at the cleaving site (KpnI, XbaI, PmeI and DraI), and the noncleaving site (PacI) in recognition sites, or have two 5′-TpA sequences at the recognition site, and a nonspecific sequence between the recognition and the cleaving sites (BciVI), were inhibited by PUVA treatment. Most of the other restriction enzymes used in this study, which do not have a 5′-TpA sequence at their restriction site, were not inhibited by PUVA treatment, although a 5′-TpA sequence is located adjacent (SmaI) or very close (BamHI, SacI and PstI) to the recognition and cleaving sites for these enzymes. Because SphI, which does not have 5′-TpA at its restriction site, was strongly inhibited by PUVA treatment, the 5′-CpA sequence is suggested to be a new binding site of psoralens after UVA irradiation.

Inhibition of endonuclease cleavage and DNA replication of E. coli plasmid by the antitumor rhodium(II) complex

Binding effect of the antitumor complex rhodium(II) acetate [Rh 2(O 2CCH 3) 4] (Rh1) to the plasmid pUC19 DNA has been studied under different molar ratio of Rh1 compound to base pair of pUC19 DNA ( R f ) and reaction time. The Rh1 binding inhibited the activity of restriction enzyme. The binding effect was monitored using gel electrophoresis. The results indicate that at least one Rh1 binds with the recognition sequence and the binding has no preference between A–T and G–C pairs. At high value of R f = 100, ICP-MS (Inductively Coupled Plasma Mass Spectrometry) measurement confirmed that 46% of Rh1 binds to DNA. PCR amplification of the DNA was also inhibited by the Rh1 binding. The transformation experiment using Escherichia coli suggested that the cell growth was inhibited after binding the Rh1 to the plasmid. These results indicated that DNA synthesis could be inhibited both in vitro and in vivo by the Rh 2(O 2CCH 3) 4 binding.

The restriction fold turns to the dark side: a bacterial homing endonuclease with a PD-(D/E)-XK motif

The homing endonuclease I-Ssp6803I causes the insertion of a group I intron into a bacterial tRNA gene-the only example of an invasive mobile intron within a bacterial genome. Using a computational fold prediction, mutagenic screen and crystal structure determination, we demonstrate that this protein is a tetrameric PD-(D/E)-XK endonuclease - a fold normally used to protect a bacterial genome from invading DNA through the action of restriction endonucleases. I-Ssp6803I uses its tetrameric assembly to promote recognition of a single long target site, whereas restriction endonuclease tetramers facilitate cooperative binding and cleavage of two short sites. The limited use of the PD-(D/E)-XK nucleases by mobile introns stands in contrast to their frequent use of LAGLIDADG and HNH endonucleases - which in turn, are rarely incorporated into restriction/modification systems.

Identification of Klebsiella pneumoniae genes uniquely expressed in a strain virulent using a murine model of bacterial pneumonia

Klebsiella pneumoniae is a gram-negative bacterium of significant clinical importance. This study examines the differential pulmonary host anti-bacterial responses towards two clinical isolates of K. pneumoniae. Intratracheal inoculation with 7×10 4 CFU of strain 43816 induced 100% mortality in C57BL/6J mice within 5 days post infection, whereas infection with 5×10 5 CFU of strain IA565 resulted in 100% survival. Infection with strain 43816 resulted in significant pulmonary and peripheral blood bacterial burden and induction of the chemokines MIP-2, KC and MCP-1 by 24 h post infection. In contrast, IA565-infected mice displayed basal chemokine levels and no detectable bacteria by 24 h post inoculation were isolated from lungs or peripheral blood. These data indicate an apparent lack of pathogenicity of strain IA565. Since little is known about Klebsiella-specific virulence genes, we have utilized PCR-based genomic DNA and cDNA suppressive subtractive hybridization and identified nine DNA sequences unique to the pathogenic strain of K. pneumoniae 43816. These sequences were highly homologous to enteric bacterial genes regulating iron uptake, fimbrial-mediated adhesion, energy production and conversion, transcriptional regulation, signal transduction, restriction endonuclease activity, and membrane transport.

Real-time monitoring of restriction endonuclease activity using molecular beacon

Restriction endonucleases are one of the most important enzymes in molecular biology. These enzymes are essential in recombinant technology, genotyping, mapping, and sequencing of large strands of DNA [1,2]. Because of the biological importance of restriction endonucleases and their wide use, restriction endonuclease activity assays are essential. Traditional methods for assaying activity of restriction endonucleases, such as gel electrophoresis, Wlter binding, high-performance liquid chromatography (HPLC), and enzyme-linked immunosorbent assay (ELISA) [3–6], have been used. All of these methods, however, are discontinuous, time-consuming, and laborious, and they usually require isotope labeling. Several spectroscopic methods using Xuorescence measurements have been developed [7–12]. These assays are continuous. However, up to now all of them have been designed only for doubly labeled DNA substrates with very large Xuorophores that may interfere with recognition and reactivity with certain restriction endonucleases. Furthermore, the potential for achieving high sensitivity through Xuorescent resonance energy transfer (FRET) has not been fully realized. Therefore, it is necessary to develop a sensitive, convenient, and non-isotope-labeled method for assaying restriction endonuclease activity. Since they were Wrst reported in 1996 [13], molecular beacons (MBs) have become a class of molecules widely

Some remarks on the use of TaqI to detect highly repetitive DNA sequences in human chromosomes.

In the attempt to conclude investigation of the action of restriction endonucleases on eukaryote chromosomes, we carried out a series of experiments digesting in situ human metaphase chromosomes with AluI/TaqI followed by Giemsa staining. We focused on the centromeric regions of chromosomes1, 2 and 16 and noted that those areas appeared as intensely stained blocks after AluI digestion, but were dramatically reduced in size or completely destroyed after subsequent TaqI treatment. These results permitted us to draw some conclusions on the highly repetitive DNA composition of these regions, in terms of alphoid and classical satellite DNAs.

Characterisation of the novel restriction endonuclease SuiI from Sulfolobus islandicus

A restriction endonuclease activity from Sulfolobus islandicus REN2H1 was purified by phosphocellulose and cation exchange chromatography. The enzyme cuts DNA at the recognition site GCwGC as could be shown by restriction analysis of plasmids and short synthetic duplex DNA. The cleavage occurs after the first guanosine base and is inhibited by 5-methyl-cytosine methylation. The restriction activity is salt-sensitive and has an optimal activity around 70 degrees C.

BpuAmI: a novel SacI neoschizomer from Bacillus pumilus discovered in an isolate from Amazon Basin, recognizing 5'-GAG<FONT FACE=Symbol>¯</FONT>CTC-3'

A strain of Bacillus pumilus was isolated and identified from water samples collected from a small affluent of the Amazon River. Type II restriction endonuclease activity was detected in these bacteria. The enzyme was purified and the molecular weight of the native protein estimated by gel filtration and SDS-PAGE. The optimum pH, temperature and salt requirements were determined. Quality control assays showed the complete absence of nonspecific nucleases. Restriction cleavage analysis and DNA sequencing of restriction fragments allowed the unequivocal demonstration of 5´GAG¯CTC3´ as the recognition sequence. This enzyme was named BpuAmI and is apparently a neoschizomer of the prototype restriction endonuclease SacI. This is the first report of an isoschizomer and/or neoschizomer of the prototype SacI identified in the genus Bacillus.

Internal Restriction Sites: Quality Assurance Aids in Genotyping

Improvements to restriction fragment length polymorphism (RFLP)-based genotyping assays currently used for detection of mutations responsible for bovine ferrochelatase and myophosphorylase deficiencies, and equine hyperkalemic periodic paralysis (HYPP) are described. Reports of sporadic inhibition of restriction enzyme activity suggest a critical factor in RFLP-based genotyping assays should be assurance that restriction enzymes perform to specification with every sample. The RFLP genotyping assays that use either a mismatched recognition sequence in one or both of the oligonucleotides, or incorporate a second native site within the PCR amplicon, provide the mechanism by which efficiency of restriction enzymes can be assessed with every sample. The outcome is confirmation of the activity of the discriminating enzyme regardless of genotype.

Characterization of the Type III restriction endonuclease PstII from Providencia stuartii

A new Type III restriction endonuclease designated PstII has been purified from Providencia stuartii. PstII recognizes the hexanucleotide sequence 5′-CTGATG(N)25-26/27-28-3′. Endonuclease activity requires a substrate with two copies of the recognition site in head-to-head repeat and is dependent on a low level of ATP hydrolysis (∼40 ATP/site/min). Cleavage occurs at just one of the two sites and results in a staggered cut 25–26 nt downstream of the top strand sequence to generate a two base 5′-protruding end. Methylation of the site occurs on one strand only at the first adenine of 5′-CATCAG-3′. Therefore, PstII has characteristic Type III restriction enzyme activity as exemplified by EcoPI or EcoP15I. Moreover, sequence asymmetry of the PstII recognition site in the T7 genome acts as an historical imprint of Type III restriction activity in vivo. In contrast to other Type I and III enzymes, PstII has a more relaxed nucleotide specificity and can cut DNA with GTP and CTP (but not UTP). We also demonstrate that PstII and EcoP15I cannot interact and cleave a DNA substrate suggesting that Type III enzymes must make specific protein–protein contacts to activate endonuclease activity.

Occurrence of restriction–modification systems in ruminal butyrate-producing bacteria

Thirty-five strains of ruminal bacteria belonging to the former Butyrivibrio fibrisolvens species were screened for the presence of site-specific restriction endonuclease and modification methyltransferase activities. Seven strains possessed endonuclease activities detectable in crude cell extracts. The recognition sequences and optimal reaction conditions for seven of them were determined. Five enzymes were found to be isoschizomers of type II endonucleases (EcoRV, NsiI, AseI (2 x) and SauI), one was type IIS (FokI) and two remained unknown. The optimal reaction buffer was found to be a low ionic strength buffer and all enzymes possessed sufficient activity at 39 °C. The presence of DNA modification among all strains was also determined. Most of the methylation activities correlated with restriction activities, yet some strains possessed unaccompanied modification methyltransferases.