Investigation of site-specific DNA binding with nicking endonuclease Nt.BspD6I at single molecule level by atomic force microscopy

Nicking endonuclease Nt.BspD6I is a heterodimeric restriction endonuclease, one subunit of which exhibits specific nicking activity. It gets bound to double-stranded DNA and makes a break (nick) in one chain at a distance of 4 nucleotides from the binding site. In this work, for visualization of the specific binding and protein landing site, atomic force microscopy was used. In five minutes after incubation of DNA solution with nicking endonuclease, DNA molecules with associated proteins which located at the expected binding site and “shared” the DNA strand into two segments (approximately, 1/3 and 2/3 of length) were observed in the images. In addition, near the binding site the DNA molecule had a height corresponding to a single-stranded DNA molecule, which was in good agreement with single-stranded cleavage by nickase in the course of complex formation.     

Investigation of site-specific DNA binding with nicking endonuclease Nt.BspD6I at single molecule level by atomic force microscopy

Nicking endonuclease Nt.BspD6I is a heterodimeric restriction endonuclease, one subunit of which exhibits specific nicking activity. It gets bound to double-stranded DNA and makes a break (nick) in one chain at a distance of 4 nucleotides from the binding site. In this work, for visualization of the specific binding and protein landing site an atomic force microscopy was used. In five minutes after incubation of DNA solution with nicking endonuclease, the DNA molecules with associated proteins which located at the expected binding site and "shared" DNA strand into two segments (approximately, 1/3 and 2/3 of length) were observed in the images. In addition, near the binding site DNA molecule had a height corresponding to a single-stranded DNA molecule, which was in good agreement with single-stranded cleavage by nickase in the course of complex formation.     

Cloning, production and characterisation of wild type and mutant forms of the R.EcoK endonucleases.

The hsdR, hsdM and hsdS genes coding for R.EcoK restriction endonuclease, both with and without a temperature sensitive mutation (ts-1) in the hsdS gene, were cloned in pBR322 plasmid and introduced into E.coli C3-6. The presence of the hsdSts-1 mutation has no effect on the R-M phenotype of this construct in bacteria grown at 42 degrees C. However, DNA sequencing indicates that the mutation is still present on the pBR322-hsdts-1 operon. The putative temperature-sensitive endonuclease was purified from bacteria carrying this plasmid and the ability to cleave and methylate plasmid DNA was investigated. The mutant endonuclease was found to show temperature-sensitivity for restriction. Modification was dramatically reduced at both the permissive and non-permissive temperatures. The wild type enzyme was found to cleave circular DNA in a manner which strongly suggests that only one endonuclease molecule is required per cleavage event. Circular and linear DNA appear to be cleaved using different mechanisms, and cleavage of linear DNA may require a second endonuclease molecule. The subunit composition of the purified endonucleases was investigated and compared to the level of subunit production in minicells. There is no evidence that HsdR is prevented from assembling with HsdM and HsdSts-1 to produce the mutant endonuclease. The data also suggests that the level of HsdR subunit may be limiting within the cell. We suggest that an excess of HsdM and HsdS may produce the methylase in vivo and that assembly of the endonuclease may be dependent upon the prior production of this methylase.     

Cleavage of lambda DNA by a site-specific endonuclease from Serratia marcescens.

Three sites recognized by SmaI endonuclease, purified from Serratia marcescens SB, have been located on lambda DNA at 0.406, 0.656, and 0.825 fractional lengths from the left end of the DNA molecule.     

Serratia marcescens endonuclease. Properties of the enzyme

Some physico-chemical properties of endonuclease (EC 3.1.4.9) from Serratia marcescens were studied and the amino acid composition of the enzyme was determined. The protein molecule was shown to contain one SH-group and one S-S-bond, which renders it different from the well studied nuclease (EC 3.1.4.7) from Staph. pyogenes. The conditions for reconstitution of the S-S-bond by dithioerythritol for quantitative estimation of cysteine residues of the endonuclease molecule were selected. The N-terminal amino acid was found to be threonine. The UV spectra for the enzyme are typical for proteins; A 0,1% 1cm,280nm is 1.46, epsilon 25 degrees 280nm,pH7,4 is 47292 M-1 cm-1. The sedimentation coefficient in phosphate buffer sW, 20 degrees is 3.4 S, pI is 6.5 and 7.5.     

A general method for inserting specific DNA sequences into cloning vehicles

A general method has been developed to introduce any double-stranded DNA molecule into cloning vehicles at different restriction endonuclease sites. In this method a chemically synthesized decadeoxyribonucleotide duplex, containing a specific restriction endonuclease sequence, is joinlex DNA is cut by the same restriction endonuclease to generate the cohesive ends. It is then inserted into the restriction endonuclease cleavage site of the cloning vehicle. To demonstrate the feasibility of this new method, we have inserted separately the synthetic lac operator DNA at the Bam I and HindIII cleavage sites of the plasmid pMB9 DNA.     

Reductive methylation of the amino terminus of endonuclease V eradicates catalytic activities. Evidence for an essential role of the amino terminus in the chemical mechanisms of catalysis

Endonuclease V, a pyrimidine dimer-specific DNA repair enzyme, was chemically modified by reductive methylation, a technique that specifically methylates primary amino groups. Upon reaction of endonuclease V with [14C]formaldehyde (14CH2O) in the presence of the reducing agent sodium cyanoborohydride (Na-CNBH3), it was discovered that 0.8 methylation/endonuclease V molecule was required to reduce both the glycosylase and the phosphodiester lyase activities by 70-80%. Pyrimidine dimer-specific binding was not eradicated at a level of methylation equivalent to 0.8 CH3/endonuclease V molecule but was eradicated at higher levels of methylation. Endonuclease V that had been modified with an average of 1.6 CH3/molecule was digested with Staphylococcus aureus strain V8 protease and the peptides subsequently separated by reverse-phase high performance liquid chromatography. Radiolabel was found exclusively on the peptide including the amino terminus, as determined by the percent amino acid composition. Neither intact CH3-endonuclease V nor radiolabeled peptides were able to be sequenced by Edman degradation indicating blockage of the amino terminus by methylation. This study shows strong evidence for the unusual involvement of the alpha NH2 moiety in the chemical mechanisms of endonuclease V. A reaction mechanism that incorporates these findings is presented.     

Molecular analysis of plasmid DNA repair within ultraviolet-irradiated Escherichia coli. I. T4 endonuclease V-initiated excision repair

The process by which DNA-interactive proteins locate specific sequences or target sites on cellular DNA within Escherichia coli is a poorly understood phenomenon. In this study, we present the first direct in vivo analysis of the interaction of a DNA repair enzyme, T4 endonuclease V, and its substrate, pyrimidine dimer-containing plasmid DNA, within UV-irradiated E. coli. A pyrimidine dimer represents a small target site within large domains of DNA. There are two possible paradigms by which endonuclease V could locate these small target sites: a processive mechanism in which the enzyme "scans" DNA for dimer sites or a distributive process in which dimers are located by random three-dimensional diffusion. In order to discriminate between these two possibilities in E. coli, an in vivo DNA repair assay was developed to study the kinetics of plasmid DNA repair and the dimer frequency (i.e. the number of dimer sites on a given plasmid molecule) in plasmid DNA as a function of time during repair. Our results demonstrate that the overall process of plasmid DNA repair initiated by T4 endonuclease V (expressed from a recombinant plasmid within repair-deficient E. coli) occurs by a processive mechanism. Furthermore, by reducing the temperature of the repair incubation, the endonuclease V-catalyzed incision step has been effectively decoupled from the subsequent steps including repair patch synthesis, ligation, and supercoiling. By this manipulation, it was determined that the overall processive mechanism is composed of two phases: a rapid processive endonuclease V-catalyzed incision reaction, followed by a slower processive mechanism, the ultimate product of which is the dimer-free supercoiled plasmid molecule.     

An electron microscopic method for studying and mapping the region of weak sequence homology between simian virus 40 and polyoma DNAs.

A procedure for investigating the possibility of small amounts of partial DNA sequence homology between two defined DNA molecules has been developed and used to test for sequence homology between simian virus 40 and polyoma DNAs. This procedure, which does not necessitate the use of separated viral DNA strands, involves the construction of hybrid DNA molecules containing a simian virus 40 DNA molecule covalently joined to a polyoma DNA molecule, using the sequential action of EcoRI restriction endonuclease and Escherichia coli DNA ligase. Denaturation of such hybrid DNA molecules then makes it possible to examine intramolecularly rather than intermolecularly renatured molecules. Visualization of these intramolecularly renatured “snapback” molecules with duplex regions of homology by electron microscopy reveals a 15% region of weak sequence homology. This region is denatured at about 35 °C below the melting temperature of simian virus 40 DNA and therefore corresponds to about 75% homology. This region was mapped on both the simian virus 40 and polyoma genomes by the use of Hemophilus parainfluenzae II restriction endonuclease cleavage of the simian virus 40 DNA prior to EcoRI cleavage and construction of the hybrid molecule. The 15% region of weak homology maps immediately to the left of the EcoRI restriction endonuclease cleavage site in the simian virus 40 genome and halfway around from the EcoRI restriction endonuclease cleavage site in the polyoma genome.     

Mechanism of action of Escherichia coli endonuclease III

Endonuclease III isolated from Escherichia coli has been shown to have both N-glycosylase and apurinic/apyrimidinic (AP) endonuclease activities. A nicking assay was used to show that the enzyme exhibited a preference for form I DNA when DNA containing thymine glycol was used as a substrate. This preference was reduced or eliminated either when the DNA was relaxed or when the type of damage was altered to urea residues or AP sites. The combined N-glycosylase/AP endonuclease activity was at least 10-fold higher than the AP endonuclease activity alone when urea-containing DNA was used as a substrate as compared to AP DNA. When DNA containing thymine glycol was used as a substrate, the combined N-glycosylase/AP endonuclease activity was about 2-fold higher than the AP endonuclease activity. Yet, when DNA containing thymine glycol or urea was used as substrate, no apurinic sites remained. Furthermore, magnesium selectively inhibited endonuclease III activity when AP DNA was used as a substrate but had no effect when DNA containing either urea or thymine glycol was used as substrate. These data suggest that both the N-glycosylase and AP endonuclease activities of endonuclease III reside on the same molecule or are in very tight association and that these activities act in concert, with the N-glycosylase reaction preceding the AP endonuclease reaction.     

Single molecule detection of DNA looping by NgoMIV restriction endonuclease

Single molecule fluorescence resonance energy transfer (FRET) and fluorescence correlation spectroscopy were used to investigate DNA looping by NgoMIV restriction endonuclease. Using a linear double-stranded DNA (dsDNA) molecule labeled with a fluorescence donor molecule, Cy3, and fluorescence acceptor molecule, Cy5, and by varying the concentration of NgoMIV endonuclease from 0 to 3 x 10(-6) M, it was possible to detect and determine diffusion properties of looped DNA/protein complexes. FRET efficiency distributions revealed a subpopulation of complexes with an energy transfer efficiency of 30%, which appeared upon addition of enzyme in the picomolar to nanomolar concentration range (using 10(-11) M dsDNA). The concentration dependence, fluorescence burst size analysis, and fluorescence correlation analysis were all consistent with this subpopulation arising from a sequence specific interaction between an individual enzyme and a DNA molecule. A 30% FRET efficiency corresponds to a distance of approximately 65 A, which correlates well with the distance between the ends of the dsDNA molecule when bound to NgoMIV according to the crystal structure of this complex. Formation of the looped complexes was also evident in measurements of the diffusion times of freely diffusing DNA molecules with and without NgoMIV. At very high protein concentrations compared to the DNA concentration, FRET and fluorescence correlation spectroscopy results revealed the formation of larger DNA/protein complexes.     

Properties of the main endonuclease specific for apurinic sites of Escherichia coli (endonuclease VI). Mechanism of apurinic site excision from DNA.

The main endonuclease for apurinic sites of Escherichia coli (endonuclease VI) has no action on normal strands, either in double-stranded or single-stranded DNA, or on alkylated sites. The enzyme has an optimum pH at 8.5, is inhibited by EDTA and needs Mg2+ for its activity; it has a half-life of 7 min at 40 degrees C. A purified preparation of endonuclease VI, free of endonuclease II activity, contained exonuclease III; the two activities (endonuclease VI and exonuclease III) copurified and were inactivated with the same half-lives at 40 degrees C. Endonuclease VI cuts the DNA strands on the 5' side of the apurinic sites giving a 3'-OH and a 5'-phosphate, and exonuclease III, working afterwards, leaves the apurinic site in the DNA molecule; this apurinic site can subsequently be removed by DNA polymerase I. The details of the excision of apurinic sites in vitro from DNA by endonuclease VI/exonuclease III, DNA polymerase I and ligase, are described; it is suggested that exonuclease III works as an antiligase to facilitate the DNA repair.     

Mitochondrial DNA in Fusarium oxysporum is a 46.5 kilobase pair circular molecule

Purified mitochondria were obtained from the phytopathogenic fungus Fusarium oxysporum f. sp. lycopersici by mechanical disruption of protoplasts, followed by differential and density gradient centrifugation. DNA, extracted from the mitochondria, was shown by electron microscopy and restriction endonuclease analysis to be a 46.5 kilobase pair circular molecule.     

Physical map of the DNA of bacteriophage phiB

A physical map of DNA phi B bacteriophage was constructed. Using the data of denaturation maps and heteroduplex analysis the positions of 10 (from 11) peaks were estimated. These peaks belong to DNA regions obtained after hydrolysis of phi B DNA by EcoRI endonuclease. This map is orientated to the end of the DNA molecule, which first entered the head, during phage maturation. The disposition of AT-enriched regions of the DNA molecule were shown.     

Crystal structure of NaeI-an evolutionary bridge between DNA endonuclease and topoisomerase

NAE:I is transformed from DNA endonuclease to DNA topoisomerase and recombinase by a single amino acid substitution. The crystal structure of NAE:I was solved at 2.3 A resolution and shows that NAE:I is a dimeric molecule with two domains per monomer. Each domain contains one potential DNA recognition motif corresponding to either endonuclease or topoisomerase activity. The N-terminal domain core folds like the other type II restriction endonucleases as well as lambda-exonuclease and the DNA repair enzymes MutH and Vsr, implying a common evolutionary origin and catalytic mechanism. The C-terminal domain contains a catabolite activator protein (CAP) motif present in many DNA-binding proteins, including the type IA and type II topoisomerases. Thus, the NAE:I structure implies that DNA processing enzymes evolved from a few common ancestors. NAE:I may be an evolutionary bridge between endonuclease and DNA processing enzymes.     

Netropsin, distamycin A, bis-netropsins as selective inhibitors of restrictases and DNAse I

The simultaneous analysis of DNAase I "footprinting" data and restriction endonucleases inhibition data was performed on the same DNA end-labelled fragment. The inhibition induced by netropsin, a number of bis-netropsins and distamycin A was investigated. These experiments led us to the following conclusions. The restriction endonucleases inhibition by the ligands is caused by the ligand molecules binding in the close vicinity to the restriction endonuclease recognition sequence. The zone of +/- 4 bp from the center of the restriction endonuclease recognition sequence can be defined as the zone of the influence of the bounded ligand on the restriction endonuclease. But in this case the intersection of recognition sequence and the binding site occupied by a single ligand molecule is not sufficient for the inhibition to occur. Restriction endonuclease cutting sites protected by netropsin can be predicted basing upon known nucleotide sequence specificity of netropsin. Netropsin and bis-netropsins show different nucleotide sequence specificity. This fact can be used for selective inhibition of restriction endonucleases.     

Enhancement of two apurinic/apyrimidinic endonuclease activities from normal but not xeroderma pigmentosum lymphoblastoid cells by nucleosome structure

The influence of nucleosomes on the activity of two chromatin-associated apurinic/apyrimidinic (AP) DNA endonuclease activities, pIs 9.2 and 9.8, from normal and xeroderma pigmentosum, complementation group A (XPA), lymphoblastoid cells was examined. These AP endonuclease activities were studied on non-nucleosomal and nucleosomal plasmid pWT830/pBR322 DNA which had been reconstituted with core (H2A, H2B, H3, H4) or total (core plus H1) histones from normal or XPA cells. Both nucleosomal and non-nucleosomal DNA was rendered partially AP by alkylation with 12.5 mM methyl methanesulfonate, followed by heating it at 70 degrees C, to produce approximately three AP sites per DNA molecule. The activities of both normal lymphoblastoid AP endonuclease activities on nucleosomal AP DNA, reconstituted with core histones, was approximately 2.5 times greater than that on non-nucleosomal AP DNA. When histone H1 was added to the system, this increase was reduced. XPA AP endonuclease activities, on the other hand, did not show any increase in activity on nucleosomal AP DNA reconstituted with core histones. These differences between normal and XPA endonuclease activities on AP nucleosomal DNA were the same regardless of whether histones from normal or XPA cells were used in the reconstituted system.     

Isolation of deletion and substitution mutants of adenovirus type 5

The infectivity of adenovirus type 5 DNA can be increased to about 5 x 10³ plaque-forming units per μg DNA if the DNA is isolated as a DNA-protein complex. Utilizing this improved infectivity, a method was developed for the selection of mutants lacking restriction endonuclease cleavage sites. The procedure involves three steps. First, the DNA-protein complex is cleaved with a restriction endonuclease. The Eco RI restriction endonuclease was used here. It cleaves adenovirus type 5 DNA to produce three fragments: fragment A (1–76 map units), fragment C (76–83 map units) and fragment B (10–83 map units). Second, the mixture of fragments is rejoined by incubating with DNA ligase, and, third, the modified DNA is used to infect cells in a DNA plaque assay. Mutants were obtained which lacked the endonuclease cleavage site at 0.83 map units. Such mutant DNAs were selected by this procedure because they were cleaved by the Eco RI endonuclease to produce only two fragments: a normal A fragment and a fused B/C fragment. These two fragments could be rejoined to produce a viable DNA molecule as a result of a bimolecular reaction with one ligation event; this exerted a strong selection for such molecules since a trimolecular reaction (keeping the C fragment in its proper orientation) and two ligation events were required to regenerate a wild-type molecule. The alterations resulting in the loss of the Eco RI endonuclease cleavage site at 0.83 map units include both deletion and substitution mutations. The inserted sequences in the substitution mutations are cellular in origin.     

The bacteriophage P1 restriction endonuclease

The bacteriophage P1 restriction endonuclease has been purified from Escherichia coli lysogenic for P1. This restriction endonuclease P has a sedimentation coefficient of 9.3 S. Unlike the E. coli K restriction endonuclease, endonuclease P does not require S-adenosylmethionine for breakage of DNA. S-adenosylmethionine does, however, stimulate the rate of double-strand breakage of DNA by endonuclease P. Hydrolysis of ATP by endonuclease P could not be detected under conditions in which the K restriction endonuclease massively degrades ATP.The enzyme makes a limited number of double-strand breaks in unmodified or heterologously modified λ DNA. In the presence of S-adenosylmethionine, it does not cut every DNA molecule to the same extent. Incubation of λ DNA with excess amounts of enzyme in the presence of S-adenosylmethionine results in less breakage of the DNA than with smaller amounts of enzyme. This effect is not seen in the absence of S-adenosylmethionine. The maximum amount of cutting in the absence of S-adenosylmethionine appears to be greater than the maximum amount of cutting in its presence. This is most likely due to the modification methylase activity of P1 restriction endonuclease.