The cleavage of Drosophila melanogaster DNA by restriction endonucleases

Drosophila melanogaster DNA, together with λ and E. coli DNAs as controls, was digested with three different restriction endonucleases: EcoR_I, Hind, and Hae. The size distributions of the segments were characterized by gel electrophoresis. More than 85% of the D. melanogaster DNA was found in a broad distribution of segment lengths consistent with random location of restriction sites. However, some DNA was spared and recovered in very long (≥20500bp) segments. These segments proved to be mostly simple sequence DNA. No complex spared segments could be found in Hind and Hae digests, while 50% of the spared EcoR_I segments had a complexity exceeding that of the E. coli DNA spared by this enzyme. These data do not support the hypothesis that chromomeres contain long regions of purely tandemly repeating sequences.     

Effect of polyamines and basic proteins on cleavage of DNA by restriction endonucleases

We have investigated the effect of the polyamines spermine, spermidine, and putrescine and the prokaryotic histone-like proteins NS1 and NS2 on the restriction endonuclease EcoRI catalyzed cleavage of plasmid and bacteriophage DNAs. At low concentrations of spermine and spermidine, the rate of DNA cleavage by EcoRI is increased, while high concentrations of spermine as well as of spermidine are inhibitory. These phenomena are also observed with other restriction endonucleases. They are, therefore, probably due to the interaction of the polyamines with the DNA. Putrescine does not have such an effect within the concentration range investigated. Remarkably, low concentrations of spermine and spermidine very efficiently suppress EcoRI activity. An inhibition of the EcoRI-catalyzed cleavage of DNA is also observed with NS1 and NS2, an effect that can be mimicked with other basic proteins that interact with DNA. The results are discussed in terms of the mechanism of restriction in vivo.     

A fluorimetric assay for on-line detection of DNA cleavage by restriction endonucleases

We have developed an assay for online detection of DNA cleavage by restriction endonucleases, suitable for the high throughput screening of the activity and flanking sequence preference of restriction endonuclease variants. For this purpose oligodeoxynucleotides were used, labeled with either 6-FAM or TAMRA whose fluorescence is quenched by a neighboring DABCYL group. After endonucleolytic cleavage the products are too short to remain double-stranded and the fluorophor labeled strand is released with concomitant increase in fluorescence which can be easily quantified. Employing this method, cleavage reactions can be monitored continuously, allowing for fast detection of specific activity as well as determination of kinetic parameters. To demonstrate the reliability of our assay we measured K(M) and k(cat) values for the restriction endonuclease EcoRV and obtained results similar to those obtained with established assays. Moreover, our method makes it possible to observe the cleavage of two different substrates differing in the sequences flanking the EcoRV site and labeled with different fluorophors in competition in a single experiment. This assay can be carried out in a microplate format, which allows for the analysis of many restriction endonuclease variants in parallel.     

Cleavage of phosphorothioate-substituted DNA by restriction endonucleases

M13 RF DNA was synthesized in vitro in the presence of various single deoxynucleoside 5'-O-(1-thiotriphosphate) phosphorothioate analogues, and the three other appropriate deoxynucleoside triphosphates using a M13 (+)-single-stranded template, Escherichia coli DNA polymerase I and T4 DNA ligase. The resulting DNAs contained various restriction endonuclease recognition sequences which had been modified at their cleavage points in the (-)-strand by phosphorothioate substitution. The behavior of the restriction enzymes AvaI, BamHI, EcoRI, HindIII, and SalI towards these substituted DNAs was investigated. EcoRI, BamHI, and HindIII were found to cleave appropriate phosphorothioate-substituted DNA at a reduced rate compared to normal M13 RF DNA, and by a two-step process in which all of the DNA is converted to an isolable intermediate nicked molecule containing a specific discontinuity at the respective recognition site presumably in the (+)-strand. By contrast, SalI cleaved substituted DNA effectively without the intermediacy of a nicked form. AvaI, however, is only capable of cleaving the unsubstituted (+)-strand in appropriately modified DNA.     

Infrequent cleavage of cloned Anabaena variabilis DNA by restriction endonucleases from A. variabilis.

A cosmid vector has been constructed, using a lambda replicon. A library of cosmids from Anabaena variabilis ATCC 29413 based on use of this vector is shown to be highly deficient in sites for the two type II restriction endonucleases found in that organism.     

Kinetic models of translocation, head-on collision, and DNA cleavage by type I restriction endonucleases

Digestion of linear DNA by type I restriction endonucleases is generally activated following the head-on collision of two translocating enzymes. However, the resulting distributions of cleavage loci along the DNA vary with different enzymes; in some cases, cleavage is located in a discrete region midway between a pair of recognition sites while in other cases cleavage is broadly distributed and occurs at nearly every intervening locus. Statistical models for DNA translocation, collision, and cleavage are described that can account for these observations and that are generally applicable to other DNA-based motor proteins. If translocation is processive (stepping forward is significantly more likely than DNA dissociation), then the linear distribution of an ensemble of proteins can be described simply using a Poisson relationship. The pattern of cleavage sites resulting from collision between two processive type I enzymes over a distance d can then be described by a binomial distribution with a standard deviation 0.5 x d1/2. Alternatively, if translocation is nonprocessive (stepping forward or dissociating become equally likely events), the linear distribution is described by a continuum of populated states and is thus extended. Comparisons of model data to the kinetics of DNA translocation and cleavage discount the nonprocessive model. Instead, the observed differences between enzymes are due to asynchronous events that occur upon collision. Therefore, type I restriction enzymes can be described as having processive DNA translocation but, in some cases, nonprocessive DNA cleavage.     

A comparative analysis of the cleavage by restriction endonucleases of the kinetoplast DNA of fish trypanosomes

A comparative study of the kinetoplast DNA (kpDNA) from 8 isolates of fish trypanosomes has been performed. The data of restriction analysis have revealed that these trypanosomes differ from species studied before. The size of minicircles is about 1600 bp and that of maxicircles--30-36 Kbp. The restriction analysis of the maxicircles allows us to divide studied isolates in at least two groups. Another evidence has been obtained that it is impossible to identify species of fish trypanosomes using host species.     

Relaxed circular SV40 DNA as cleavage intermediate of two restriction endonucleases.

We have determined the mode of cleavage of superhelical SV40 DNA (Form I) by restriction endonucleases EcoRI and HpaII at 37 degrees C. By analysis with agarose gel electrophoresis and direct examination with dark field electron microscopy, we found that a large amount of the single-nicked circular DNA (Form II) was produced before the linear SV40 DNA (Form III) appeared. Thus, both restriction enzymes cleave only one strand of the superhelical DNA first. The second cleavage on the complementary strand occurred after a lag period. The first order rate constant for the second cleavage by EcoRI endonuclease was determined and a kinetic reaction scheme for both enzymes is proposed.     

'Red pigment' from ADE-2 mutants of S. cerevisiae prevents DNA cleavage by restriction endonucleases

Protection of DNA from cleavage by restriction endonucleases EcoRI, HindIII, BamHI, and Bg/II with red pigment, produced by ADE-2 mutants of Saccharomyces cerevisiae is demonstrated. Purification of yeast DNA from pigment can be achieved by chromatography on hydroxyapatite columns.     

Spectrophotometric method of studying the cleavage of DNA-duplexes by restriction endonucleases]

A spectrophotometric method for continuous monitoring the cleavage of DNA duplexes by type II restriction endonucleases was proposed. The time course of cleavage of a 14-membered DNA duplex by MvaI endonuclease was obtained. The spectrophotometric method is characterized by rapidity and high precision in determining the kinetic parameters of the reaction. It can be recommended for testing the preparations for the presence of restriction endonucleases, rapid determination of the activity of any restriction endonucleases, highly precise quantitative analysis of the restriction enzyme catalysed reactions.     

Structural basis for sequence-dependent DNA cleavage by nonspecific endonucleases

Nonspecific endonucleases hydrolyze DNA without sequence specificity but with sequence preference, however the structural basis for cleavage preference remains elusive. We show here that the nonspecific endonuclease ColE7 cleaves DNA with a preference for making nicks after (at 3′O-side) thymine bases but the periplasmic nuclease Vvn cleaves DNA more evenly with little sequence preference. The crystal structure of the ‘preferred complex’ of the nuclease domain of ColE7 bound to an 18 bp DNA with a thymine before the scissile phosphate had a more distorted DNA phosphate backbone than the backbones in the non-preferred complexes, so that the scissile phosphate was compositionally closer to the endonuclease active site resulting in more efficient DNA cleavage. On the other hand, in the crystal structure of Vvn in complex with a 16 bp DNA, the DNA phosphate backbone was similar and not distorted in comparison with that of a previously reported complex of Vvn with a different DNA sequence. Taken together these results suggest a general structural basis for the sequence-dependent DNA cleavage catalyzed by nonspecific endonucleases, indicating that nonspecific nucleases could induce DNA to deform to distinctive levels depending on the local sequence leading to different cleavage rates along the DNA chain.     

Strand specific cleavage of phosphorothioate-containing DNA by reaction with restriction endonucleases in the presence of ethidium bromide.

A method for achieving strand specific nicking of DNA has been developed. Phosphorothioate groups were incorporated enzymatically into the (-)strand of M13 RF IV DNA. When such DNA is reacted with restriction endonucleases in the presence of ethidium bromide nicked DNA (RF II) is produced. All of the restriction enzymes tested linearised phosphorothioate-containing DNA in the absence of this dye. The strand specificity of the reaction was investigated by employing the ethidium bromide mediated nicking reaction in the phosphorothioate-based oligonucleotide-directed mutagenesis method. The mutational efficiencies obtained were in the region of 64-89%, indicating that these restriction enzymes hydrolyse the phosphodiester bond at the cleavage site of the unsubstituted (+)strand.     

Cleavage and protection of locked nucleic acid-modified DNA by restriction endonucleases

Locked nucleic acid (LNA) is one of the most prominent nucleic acid analogues reported so far. We herein for the first time report cleavage by restriction endonuclease of LNA-modified DNA oligonucleotides. The experiments revealed that RsaI is an efficient enzyme capable of recognizing and cleaving LNA-modified DNA oligonucleotides. Furthermore, introduction of LNA nucleotides protects against cleavage by the restriction endonucleases PvuII, PstI, SacI, KpnI and EcoRI.     

Digestion of highly modified bacteriophage DNA by restriction endonucleases.

The ability of thirty Type II restriction endonucleases to cleave five different types of highly modified DNA has been examined. The DNA substrates were derived from relatively large bacteriophage genomes which contain all or most of the cytosine or thymine residues substituted at the 5-position. These substituents were a proton (PBS1 DNA), a hydroxymethyl group (SP01 DNA), a methyl group (XP12 DNA), a glucosylated hydroxymethyl group (T4 DNA), or a phosphoglucuronated, glucosylated 4,5-dihydroxypentyl group (SP15 DNA). Although PBS1 DNA and SP01 DNA were digested by most of the enzymes, they were cleaved much more slowly than was normal DNA by many of them. 5-Methylcytosine-rich XP12 DNA and the multiply modified T4 and SP15 DNAs were resistant to most of these endonucleases. The only enzyme that cleaved all five of these DNAs was TaqI, which fragmented them extensively.     

Linear diffusion of restriction endonucleases on DNA

We have investigated the dependence of the rate of cleavage of DNA by EcoRI, HindIII, and BamHI on the chain length of the substrate. In order to keep the influence of flanking sequences and of nonspecific binding identical for all substrates we have carried out all experiments with the same plasmid DNA which had been digested previously with a variety of different restriction enzymes to give a set of substrates of different lengths. Our results show that depending on the buffer conditions long substrates are cleaved faster than small ones. We interpret these findings to mean that under certain conditions a linear diffusion of the enzymes on the DNA is involved in localizing the recognition sites. For EcoRI the mean diffusion length is approximately 1000 base pairs at 1 mM MgC12 which can be shown by diffusion theory to correspond to a linear diffusion coefficient of 5 X 10(-10) cm2 s-1. At 10 mM MgCl2 the linear diffusion of EcoRI is negligible and does not lead to a significant enhancement of the rate of site localization. In the presence of nonsaturating amounts of one of the prokaryotic histone-like protein Hu (NS 2) small and large DNA substrate are cleaved with identical rate by EcoRI indicating that other proteins bound to the DNA constitute a barrier across which linear diffusion cannot take place. We conclude that linear diffusion, albeit detectable under certain conditions in vitro, probably is of little importance for the process of site localization in vivo.     

Factors affecting the digestion of moss DNA by restriction endonucleases

Abstract

The age of gametophytic tissues, de-starching, inclusion of PVP in the extraction medium, and column purification of isolated DNA have little or no effect upon the restriction of total DNA of Physcomitrella patens (Hedw.) Bruch, Schimp. & W.Gümbel. The relative longevity of restriction enzymes also appears unimportant. However, the extent of digestion of moss DNA by a given restriction endonuclease appears to correlate inversely with the number of cytosine residues in its recognition sequence that are susceptible to methylation in plant cells. Inclusion of spermidine in the restriction buffer slightly enhances restriction by a few specific endonucleases. This knowledge has practical significance when designing experiments in which it is desirable that restriction of isolated DNA samples is taken to completion.