The use of phosphorothioate-modified DNA in restriction enzyme reactions to prepare nicked DNA

The RF IV form of M13 DNA was synthesized enzymatically in vitro, using the viral (+)strand as template, to contain phosphorothioate-modified internucleotidic linkages of the Rp configuration on the 5' side of every base of a particular type in the newly-synthesized (-)strand. Twenty nine restriction enzymes were then tested for their reactions with the appropriate modified DNA types having a phosphorothioate linkage placed exactly at the cleavage site(s) of these enzymes in the (-)strand. Eleven of the seventeen restriction enzymes tested that had recognition sequences of five bases or more could be used to convert the phosphorothioate DNA entirely into the nicked form, either by simply allowing the reaction to go to completion with excess enzyme (Ava I, Ava II, Ban II, Hind II, Nci I, Pst I or Pvu I) or by stopping the reaction at the appropriate time before the nicked DNA is linearized (Bam HI, Bgl I, Eco RI or Hind III). Only modification of the exact cleavage site in the (-)strand could block linearization by the first class of enzymes. The results presented imply that the restriction enzyme-directed nicking of phosphorothioate M13 DNA occurs exclusively in the (+)strand.     

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.     

Stabilisation of DNA by Incorporation of Phosphothioate Groups

Abstract

The synthesis of Rp- and Sp-d (GGsAATTCC), an octanucleotide containing the recognition sequence for the restriction endonuclease Eco R1 as well as a phosphorothioate group at the cleavage site is described. Only the Rp-diastereomer is a substrate for Eco R1. Viral DNA, containing exclusively phosphate groups in the (+)strand, and phosphorothioate groups 5′-to deoxyadenosine in the (-)strand yields nicked DNA on cleavage by Eco R1 as an isolatable intermediate. Other restriction enzymes show a similar pattern of hydrolysis. - The influence of phosphorothioate groups on the B Z conformational change is demonstrated on phosphorothioate analogues of d(C-G)₄ and d(G-C)₄.     

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.     

On the DNA cleavage mechanism of Type I restriction enzymes

Although the DNA cleavage mechanism of Type I restriction–modification enzymes has been extensively studied, the mode of cleavage remains elusive. In this work, DNA ends produced by EcoKI, EcoAI and EcoR124I, members of the Type IA, IB and IC families, respectively, have been characterized by cloning and sequencing restriction products from the reactions with a plasmid DNA substrate containing a single recognition site for each enzyme. Here, we show that all three enzymes cut this substrate randomly with no preference for a particular base composition surrounding the cleavage site, producing both 5′- and 3′-overhangs of varying lengths. EcoAI preferentially generated 3′-overhangs of 2–3 nt, whereas EcoKI and EcoR124I displayed some preference for the formation of 5′-overhangs of a length of ~6–7 and 3–5 nt, respectively. A mutant EcoAI endonuclease assembled from wild-type and nuclease-deficient restriction subunits generated a high proportion of nicked circular DNA, whereas the wild-type enzyme catalyzed efficient cleavage of both DNA strands. We conclude that Type I restriction enzymes require two restriction subunits to introduce DNA double-strand breaks, each providing one catalytic center for phosphodiester bond hydrolysis. Possible models for DNA cleavage are discussed.     

Molecular evidence for a fourth species within the Isotoma viridis group (Insecta, Collembola)

For identification of single species within the Isotoma viridis group, we present polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) as a fast and efficient DNA-based molecular method. We used five PCR primers amplifying the cytochrome oxidase II (COII) region (760 bp) of the mitochondrial DNA. The sequences clearly separated four species ( I. viridis , I. riparia , I. anglicana and I. caerulea ) out of samples from Norway, Sweden, Germany and Switzerland. Examination of genetic variation and phylogenetic relationship did not support the separation of two colour pattern forms of I. viridis into distinct species. For RFLP, several restriction enzymes were tested for their ability to produce not only species-specific restriction fragment patterns but to discriminate more than one species per enzyme used with as few cleavage sites as possible. Such a design should render a clear fragment pattern when performing a double digest. These demands appear to be fulfilled best by the combination of the restriction enzymes Mfe I, Nci I and one of Aci I, Bst EII, Nde I, or Sfc I. From the enzymes tested in a previous study, Ase I proved to be reliable, whereas Mbo I can no longer be recommended.     

The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA.

M13 RF IV DNA may be prepared in vitro to contain phosphorothioate-modified internucleotidic linkages in the (-)strand only. Certain restriction enzymes react with this modified DNA to hydrolyze the (+)strand exclusively when a phosphorothioate linkage occurs at the normal cleavage point in the (-)strand. The reaction of Pvu I with M13mp2 RF IV DNA containing dCMPS residues in the (-)strand is of this type, and is exploited to allow subsequent digestion with exonuclease III of a portion of the (+)strand opposite different mutagenic mismatched oligonucleotide primers. Two methods are described by which this approach has been used to produce mutations in M13mp2 phage DNA with high efficiency as a result of simple and rapid in vitro manipulations. Plaques containing mutant phage in a genetically-pure form are obtained at a frequency of 40-66%, allowing their characterisation directly by sequence analysis without prior screening and plaque purification. The wide applicability of this approach is discussed.     

Inhibition of the restriction endonuclease BanII using modified DNA substrates. Determination of phosphate residues critical for the formation of an active enzyme-DNA complex

The restriction endonuclease BanII catalyzes the cleavage of double-stranded DNA and recognizes the degenerate sequence 5'-GPuGCPyC-3'. The poly-linker of M13mp18 contains one such sequence, 5'-GAGCTC-3'. The three other possible sites recognized by the enzyme were prepared by site-directed muta-genesis. The substitution of phosphate groups by phosphorothioate residues at some positions within the various recognition sites had relatively little effect on the rate of cleavage of the DNA. However, when the DNA contained a phosphorothioate group at the site of cleavage the rate of linearization of the DNA was decreased by a factor of 9. Interestingly, DNA which contained an additional phosphorothioate internucleotidic linkage immediately 3'-outside the recognition site could not be linearized by the enzyme. The results indicate that an important contact between enzyme and substrate is perturbed by the presence of the sulfur atom at this position.     

S-Adenosyl-L-methionine-dependent restriction enzymes

Restriction-modification (R-M) enzymes are classified into type I, II, III, and IV, based on their recognition sequence, subunit composition, cleavage position, and cofactor requirements. While the role of S-Adenosyl-L-methionine (AdoMet) as the methyl group donor in the methylation reaction is undisputed, its requirement in DNA cleavage reaction has been subject to intense study. AdoMet is a prerequisite for the DNA cleavage by most type I enzymes known so far, with the exception of R.EcoR124I. A number of new type II restriction enzymes belonging to the type IIB and IIG family were found to show AdoMet dependence for their cleavage reaction. The type III enzymes have been found to require AdoMet for their restriction function. AdoMet functions as an allosteric effector of the DNA cleavage reaction and has been shown to bring about conformational changes in the protein upon binding.     

The time-resolved kinetics of superhelical DNA cleavage by BamHI restriction endonuclease

The kinetics of the cleavage of superhelical plasmid DNA (pBR322) by the restriction endonuclease, BamHI, have been analyzed in terms a compartmental model consistent with the chemistry first proposed by Rubin and Modrich (Rubin, R. A., and Modrich, P. (1978) Nucleic Acids Res. 5, 2991-2997) for analysis of the kinetics of the restriction endonuclease, EcoRI. The model was defined in terms of two compartments representing DNA substrate (bound and free), two compartments representing nicked intermediate (bound and free), one compartment representing linear product, and one compartment for free enzyme. A simultaneous analysis of concentration changes over time of the three DNA forms (superhelical, nicked, and linear) at six different enzyme concentrations was undertaken employing this compartmental model using SAAM (Simulation Analysis And Modeling) software. Results showed that rate constants characterizing the association of enzyme with superhelical DNA (6.0 x 10(5) M-1 s-1) and nicked DNA (2.8 x 10(5) M-1 s-1) were similar in magnitude and rate constants characterizing cleavage of the first (1.2 x 10(-2) s-1) and second phosphodiester bonds (3.1 x 10(-2) s-1) were also similar. The analysis yields a kinetically determined equilibrium constant of 12.9 nM for the dissociation of nicked intermediate from the enzyme. The rate constant describing the release of the nicked intermediate from the enzyme has a value of 3.7 x 10(-3) s-1. By comparing the value of this release rate constant to the value of the constant describing the second cleavage event, it can be determined that only 10% of the nicked intermediate bound to the enzyme is released as free nicked DNA and that 90% of the nicked intermediate is processed to the linear form without being released. Hence, most of the DNA is cleaved as the result of a single enzyme-DNA recognition event. No steady state assumptions were made in the analysis. The approach was to directly solve the differential equations which described the kinetic processes using an interactive method. This study demonstrates the usefulness of this approach for the analysis of kinetics of protein-DNA interactions for the restriction endonucleases.     

A novel suicide substrate for DNA topoisomerases and site-specific recombinases.

DNA topoisomerases and DNA site-specific recombinases are biologically important enzymes involved in a diverse set of cellular processes. We show that replacement of a phosphodiester linkage by a 5'-bridging phosphorothioate linkage creates an efficient suicide substrate for calf thymus topoisomerase I and lambda integrase protein (Int). Although the bridging phosphorothioate linkage is cleaved by these enzymes, the 5'-sulfhydryl which is generated is not competent for subsequent ligation reactions. We use the irreversibility of Int-promoted cleavage to explore conditions and factors that contribute to various steps of lambda integrative recombination. The phosphorothioate substrates offer advantages over conventional suicide substrates, may be potent tools for inhibition of the relevant cellular enzymes and represent a unique tool for the study of many other phosphoryl transfer reactions.     

A gel electrophoresis assay for the simultaneous determination of topoisomerase I inhibition and DNA intercalation

The DNA maintenance enzyme, topoisomerase I, is thought to play crucial roles in all living cells and for this reason inhibitors of this enzyme have been much studied. In this paper we describe a gel electrophoresis method capable of characterizing and quantifying inhibition of topoisomerase I by selected compounds. Inhibitors of topoisomerase I are often associated with intercalative binding to DNA and the method can simultaneously determine intercalative binding (as DNA unwinding) except in the cases where inhibition is prohibitively strong. The method uses closed circular (plasmid) DNA and can separate single-strand nicked, linearized (double-strand nicked), fully relaxed, partially relaxed (topoisomers), and supercoiled forms of the plasmid so that topoisomerase-dependent DNA cleavage (poisoning) can also be determined. By quantifying poisoning, inhibition, and intercalation simultaneously and separately in relation to reference compounds it is possible to make quantitative determinations of these phenomena for comparative purposes. Data for the topoisomerase I inhibitor, luteolin, are presented.     

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.     

Scanning force microscopy of DNA translocation by the Type III restriction enzyme EcoP15I

Type III restriction enzymes are multifunctional heterooligomeric enzymes that cleave DNA at a fixed position downstream of a non-symmetric recognition site. For effective DNA cleavage these restriction enzymes need the presence of two unmethylated, inversely oriented recognition sites in the DNA molecule. DNA cleavage was proposed to result from ATP-dependent DNA translocation, which is expected to induce DNA loop formation, and collision of two enzyme-DNA complexes. We used scanning force microscopy to visualise the protein interaction with linear DNA molecules containing two EcoP15I recognition sites in inverse orientation. In the presence of the cofactors ATP and Mg(2+), EcoP15I molecules were shown to bind specifically to the recognition sites and to form DNA loop structures. One of the origins of the protein-clipped DNA loops was shown to be located at an EcoP15I recognition site, the other origin had an unspecific position in between the two EcoP15I recognition sites. The data demonstrate for the first time DNA translocation by the Type III restriction enzyme EcoP15I using scanning force microscopy. Moreover, our study revealed differences in the DNA-translocation processes mediated by Type I and Type III restriction enzymes.     

5''-3'' exonucleases in phosphorothioate-based oligonucleotide-directed mutagenesis.

The application of T7 and lambda exonuclease to phosphorothioate-based oligonucleotide-directed mutagenesis was investigated. Oligonucleotide primers designed to introduce single or double base mismatches, an insertion or a deletion (each of 16 bases) were annealed to M13 phage derivatives. Double stranded closed circular DNA (RF IV) containing phosphorothioate internucleotidic linkages in the (-)strand was prepared enzymatically from these templates. A nick was introduced into the (+)strand of the hetroduplex DNA. This nicked DNA (RF II) was subjected to treatment with T7 or lambda exonuclease. Both of these enzymes were able to degrade almost all of the viral (+)strand when presented with DNA containing one or two base mismatches. Repolymerisation of the DNA after the gapping reaction, followed by transfection into E. coli cells gave mutational efficiencies of up to 95%. In the case of RF II DNA prepared with insertion or deletion primers these exonucleases could only partially degrade the viral (+)strand but were nevertheless highly efficient in such mutagenesis experiments.     

Investigation of the inhibitory role of phosphorothioate internucleotidic linkages on the catalytic activity of the restriction endonuclease EcoRV

The inhibitory effect of phosphorothioate residues, located within one strand of double-stranded DNA, on the hydrolytic activity of the restriction endonuclease EcoRV was investigated. Specific incorporation of a phosphorothioate group at the site of cleavage yielded the sequence 5'-GATsATC-3'. This modified sequence was cleaved at a relative rate of 0.1 compared to the unmodified substrate. Substrates 5'-GATsAsTC-3' and 5'-GsATsATC-3', both containing one additional phosphorothioate substitution, were linearized at a rate of 0.04 relative to unmodified DNA. However, under the same conditions, fully dAMPS-substituted DNA was found to be virtually resistant to the hydrolytic activity of EcoRV. Further experiments showed that double-stranded DNA fragments generated by PCR containing phosphorothioate groups within both strands are potent inhibitors of EcoRV catalysis. The inhibition was independent of whether the inhibitor fragment contained an EcoRV recognition site. We concluded that substitution of the phosphate group at the site of cleavage by a phosphorothioate residue decreases the rate of EcoRV-catalyzed hydrolysis most significantly. Substitution of other phosphate groups within the recognition sequence plays a limited role in enzyme inhibition. The presence of multiple dNMPS residues at regions of the DNA removed from the EcoRV recognition site may decrease the amount of enzyme available for catalysis by nonspecific binding to EcoRV.     

Nuclease-induced DNA structural changes assessed by flow cytometry with the intercalating dye propidium iodide

A flow cytometric analysis of DNA structural changes induced by cleavage with nucleases was performed on isolated HeLa nuclei by assessing changes in stainability with the DNA-specific fluorochrome propidium iodide (PI). After mild digestion with DNAse I, micrococcal nuclease, or with the single-strand-specific S1 and Neurospora crassa nucleases, fluorescence intensity of nuclei stained with PI increased by about 15-30% above the value of undigested control samples. No significant modifications were observed with the restriction enzymes Eco RI, Alu I, and Not I. The DNAse I-induced increase in fluorescence intensity was also observed with the non-intercalating dye Hoechst 33258, but not with mithramycin. Nuclease-induced fluorescence intensity changes as determined with PI were found to be dependent on the dye concentration. A constant increase (about 20%) was measured at dye/DNA-P ratios greater than 0.11. Below this value (2 micrograms/ml PI), the fluorescence intensity of digested samples was 15-30% lower than that of undigested controls. This behaviour towards intercalating dyes is similar to that of the relaxed (nicked) vs. the supercoiled (intact) form of circular DNA. These results suggest that conformation- but not sequence-specific nucleases induce a relaxation of DNA supercoils.     

Studies of the recognition sequence of phi X174 gene A protein. Cleavage site of phi X gene A protein in St-1 RFI DNA.

It is already known that phi X gene A protein converts besides phi X RFI DNA also the RFI DNAs of the single-stranded bacteriophages G4, St-1, alpha 3 and phi K into RFII DNA. We have extended this observations for bacteriophages G14 and U3. Restriction enzyme analysis placed the phi X gene A protein cleavage site in St-1 RF DNA in the HinfI restriction DNA fragment F10 and in the overlapping HaeIII restriction DNA fragment Z7. The exact position and the nucleotide sequence at the 3'-OH end of the nick were determined by DNA sequence analysis of the single-stranded DNA subfragment of the nicked DNA fragment F10 obtained by gelelectrophoresis in denaturing conditions. A stretch of 85 nucleotides of St-1 DNA around the position of the phi X gene A protein cleavage site was established by DNA sequence analysis of the restriction DNA fragment Z7F1. Comparison of this nucleotide sequence with the previously determined nucleotide sequence around the cleavage site of phi X gene A protein in phi X174 RF DNA and G4 RF DNA revealed an identical sequence of only 10 nucleotides. The results suggest that the recognition sequence of the phi X174 gene A protein lies within these 10 nucleotides.     

Single-strand and double-strand cleavage at half-modified and fully modified recognition sites for the restriction nucleases Sau3a and Taqi

The influence of cytosine methylation on the cleavage of DNA by the restriction nucleases Sau3A and TaqI has been investigated. Bovine satellite DNA fragments containing a GATCGA sequence, i.e. a Sau3A site overlapping with a TaqI site have been used in this study. The methylation of these fragments has been determined by sequence analysis. It has been found that a TaqI site (TCGA) methylated at cytosine in both DNA strands is still sensitive to double-strand cleavage. A Sau3A site (GATC), however, is rendered resistant to double-strand cleavage by methylation of a single cytosine. Fragments containing the "half-modified" Sau3A site are nicked in the unmethylated DNA strand. It has been shown by sequence analysis of nicked DNA that the single-strand break occurs at the same position which is cleaved in unmodified DNA.