Random cleavage of superhelical SV 40 DNA S1 nuclease.

SV40 DNA FO I is randomly cleaved by S1 nuclease both at moderate (50 mM) and higher salt concentrations (250 mM NaC1). Full length linear S1 cleavage products of SV40 DNA when digested with various restriction endonucleases revealed fragments that were electrophoretically indistinguishable from the products found after digestion of superhelical SV40 DNA FO I with the corresponding enzyme. Concordingly, when the linear S1 generated duplexes were melted and renatured, circular duplexes were formed in addition to complex larger structures. This indicated that cleavage must have occurred at different sites. The double-strand-cleaving activity present in S1 nuclease preparations requires circular DNA as a substrate, as linear SV40 DNA is not cleaved. With regard to these properties S1 nuclease resembles some of the complex type I restriction nucleases from Escherichia coli which also cleave SV40 DNA only once, and, completely at random.     

Double-strand cleavage of DNA by a polyamide-phenazine-di-N-oxide conjugate

Phenazine and its derivatives have been widely applied as nucleic acid cleavage agents due to active oxygen activating the C–H bond of the substrate. However, diffusion of oxygen radicals limits their potential applications in the DNA-targeted metal-free drug. Introduction of groove binder moiety such as polyamide enhanced the regional stability of radical molecules and reduced cytotoxicity of the drugs. In this work, we described the design and synthesis of a polyamide-modified phenazine-di-N-oxide as a DNA double-strand cleavage agent. The gel assays showed the hybrid conjugates can effectively break DNA double strands in a non-random manner under physiological conditions. The probable binding mode to DNA was investigated by sufficient spectral experiments, revealing weak interaction between hybrid ligand and nucleic acid molecules. The results of our study have implications on the design of groove-binding hybrid molecules as new artificial nucleases and may provide a strategy for developing efficient and safe DNA cleavage reagents.     

Specific cleavage of tRNA by nuclease S1.

Nuclease S1 specifically hydrolizes tRNAs in their anticodon loops, forming new 5' phosphate and 3' OH ends. Some single-stranded regions are not cut by nuclease S1. The strong preference of nuclease S1 for the anticodon region can be used for rapid identification of an anticodon-containing oligonucleotide and subsequent identification of the probable amino acid specificity of tRNA.     

Chemical cleavage of mismatch (CCM) to locate base mismatches in heteroduplex DNA

This protocol describes the use of the chemical cleavage of mismatch (CCM) method to assess whether a region of DNA contains mutations and to localize them. Compared with other mutation-detection techniques (such as single strand-conformation polymorphism (SSCP) analysis, denaturing high-performance liquid chromatography (DHPLC) and denaturing gradient gel electrophoresis (DGGE)) that detect mutations in short DNA fragments and require highly specific melting temperatures, CCM has a higher diagnostic sensitivity suited to the detection of mutations in tumor genes, and can analyze amplicons < or = 2 kb in length. To detect mutations, PCR heteroduplexes are incubated with two mismatch-specific reagents. Hydroxylamine modifies unpaired cytosine and potassium permanganate modifies unpaired thymine. The samples are then incubated with piperidine, which cleaves the DNA backbone at the site of the modified mismatched base. Cleavage products are separated by electrophoresis, revealing the identity and location of the mutation. The CCM method can efficiently detect point mutations as well as insertions and deletions. This protocol can be completed in 10 h.     

S1 nuclease does not cleave DNA at single-base mis-matches

Three assays have been designed to detect the cleavage of duplex phi X174 DNA at single-base mis-matches. Studies with S1 nuclease failed to detect cleavage at mis-matches. S1 nuclease digestion at 37 and 55 degrees C failed to produce a preferential degradation of a multiply mis-matched heteroduplex when compared to a mis-match-free homo-duplex as analyzed by sedimentation on sucrose gradients. Other heteroduplex templates were not cleaved by S1 nuclease at a defined single-base mis-match when assayed by gel electrophoresis or by marker rescue. In all cases, the amount of S1 nuclease employed was at least 10-times more than that required to render a single-stranded phi X174 DNA molecule completely acid soluble. The rate of hydrolysis of single-base mis-matches by S1 nuclease was estimated to be less than 0.016% of the rate at a base in single-strand phi X174 DNA. In no instance did we detect activity by S1 nuclease directed at mis-matched sites in our template molecules. Similarly, the single-strand specific endonuclease from Neurospora crassa does not cleave heteroduplex templates at a defined single-base mis-match when assayed by marker rescue.     

Analogous cleavage of DNA by micrococcal nuclease and a 1-10-phenanthroline-cuprous complex

We have examined the DNA cleavage specificity of a 1,10-phenanthroline-cuprous complex and find this agent to recognize the same sites and cleave with the same relative preferences as micrococcal nuclease. In contrast, DNase I and bleomycin-ferrous complex cleave the same 5000 bp D. melanogaster histone gene DNA with different specificities, although some of the sites appear to be recognized and cleaved by all four reagents. Our results suggest that the reagents used probably detect discrete conformational perturbations along the DNA double helix.     

Stereochemical course of DNA hydrolysis by nuclease S1

Nuclease S1 hydrolyzes the Sp-diastereomer of 5'-O-(2'-deoxyadenosyl)-3'-O-thymidyl phosphorothioate in H2(18)O to [18O]deoxyadenosine 5'-O-phosphorothioate which can be phosphorylated enzymatically to the Sp-diastereomer of [alpha-18O]deoxyadenosine 5'-O-(1-thiotriphosphate). 31P nmr spectroscopy shows the oxygen-18 in this compound to be in a nonbridging position at the alpha-phosphorus, indicating that the hydrolysis reaction catalyzed by nuclease S1 proceeds with inversion of configuration at phosphorus. This result is compatible with a direct nucleophilic attack of H2O at phosphorus without the involvement of a covalent enzyme intermediate.     

Sequence specific cleavage of DNA by micrococcal nuclease.

Micrococcal nuclease is shown to cleave DNA under conditions of partial digestion in a specific manner. Sequences of the type 5'CATA and 5'CTA are attacked preferentially, followed by exonucleolytic degradation at the newly generated DNA termini. GC-rich flanking sequences further increase the probability of initial attack. Unexpectedly, long stretches containing only A and T are spared by the nuclease. These results, which were obtained with spared by the nuclease. These results, which were obtained with mouse satellite DNA and two fragments from the plasmid pBR22, do not support the previous contention that it is the regions of high At-content which are initially cleaved by micrococcal nuclease. This specificity of micrococcal nuclease complicates its use in experiments intended to monitor the nucleoprotein structure of a DNA sequence in chromatin.     

Sequence-dependent S1 nuclease hypersensitivity of a heteronomous DNA duplex

Using cloned (dG-dA)n X (dC-dT)n DNA duplexes [GA)n) as models of homopurine-homopyrimidine S1-hypersensitive sites, we show that cleavage of the alternate (non-B, non-Z) DNA structure by S1 nuclease is length-dependent, in both supercoiled and linear forms, which are similar because of the identity of their nicking profiles. However, the length of flanking sequences, the presence of borders, and the DNA topology affect the equilibrium between the alternate structure and B-DNA. The B form of (GA)38 has a 10.4-base pair helical repeat, but the two phosphodiester backbones have different conformations (heteronomous DNA with a dinucleotide repeat unit). Extension experiments reveal that the alternate structure is also heteronomous, in agreement with the nicking patterns generated by S1 and mung bean nucleases and by venom phosphodiesterase. Sensitivity to the latter enzyme at pH 9.0 indicates that the alternate DNA does not appear only in the low pH of the S1 nuclease reaction. Moreover, Hoogsteen G-CH+ base-pairing does not seem to be a prerequisite for the appearance of sensitivity because S1 still recognizes the structure even when all Gs are methylated at N-7. This is consistent with the results of chemical probing of the structure using dimethyl sulfate and diethyl pyrocarbonate at various pH values, which show absence of protection at guanine N-7. However, diethyl pyrocarbonate treatment at low pH results in hyper-reactivity of A residues.     

Sequence selective double strand DNA cleavage by peptide nucleic acid (PNA) targeting using nuclease S1.

A novel method for sequence specific double strand DNA cleavage using PNA (peptide nucleic acid) targeting is described. Nuclease S1 digestion of double stranded DNA gives rise to double strand cleavage at an occupied PNA strand displacement binding site, and under optimized conditions complete cleavage can be obtained. The efficiency of this cleavage is more than 10 fold enhanced when a tandem PNA site is targeted, and additionally enhanced if this site is in trans rather than in cis orientation. Thus in effect, the PNA targeting makes the single strand specific nuclease S1 behave like a pseudo restriction endonuclease.     

Caffeine enhancement of digestion of DNA by nuclease S1.

The activity of Aspergillus orzae nuclease S1 on DNA has been investigated under varying pH and metal ion conditions. Nuclease S1 was found to preferentially digest denatured DNA. With native DNA as substrate the enzyme could only digest the DNA when caffeine was added to the reaction mixture. The enzyme was more active in sodium acetate buffer (pH 4.5), than in either standard saline citrate (PH 7.0) or sodium phosphate buffer (pH 6.8). Caffeine was also found to affect the thermal stability of DNA, resulting in a melting profile characterized by two transitions. The first transition (poorly defined) was below the normal melting temperature of the DNA, while the next transition was at the normal melting temperature of the DNA, while the next transition was at the normal melting temperature of the DNA. The susceptibility of caffeine-treated DNA to nuclease digestion seems to be a result of the local unwinding that caffeine causes in the regions of DNA that melt in the first transition. This selective destabilization presumably sensitizes the unwound regions to nuclease hydrolysis. The hydrolysates of the DNA digested by nuclease S1 were subjected first to ion exchange chromatography followed by paper chromatography. The results from this partial characterization of the digestion products showed that they contain mononucleotides as well as oligonucleotides of varying lengths. The base composition of the mononucleotide digests suggests that caffeine has greater preference for interacting with A-T base-pairs in DNA.     

Correlation of enzyme-induced cleavage sites on negatively superhelical DNA between prokaryotic topoisomerase I and S1 nuclease

Negatively superhelical pNS1 DNA with a molecular weight of 2.55 MDa (4 kbp) was found to contain 13 specific, unbasepaired sites that are sensitive to a single-strand-specific S1 nuclease cleavage. The S1-cleavage occurred once at these sites. In the absence of added Mg2+, the topoisomerase I purified from Haemophilus gallinarum formed a complex with the superhelical pNS1 DNA which has a hidden strand cleavage. Extensive proteinase K digestion of the complex led to cleavage of the DNA chain. Then the proteinase K-cleaved product was digested with S1, which can cut the opposite strand at the preexisting strand cleavage to generate unit-length linear DNA. Restriction endonuclease analysis of the linear DNA shows that the topoisomerase-induced cleavage occurred once at ten specific sites on the DNA. The topoisomerase caused mainly single-strand cleavage at these sites, but infrequently also caused double-strand cleavage at the same sites. Of interest is the fact that these sites considerably coincide with the S1-cleavable, unbasepaired sites.     

S1 nuclease sensitivity of a double-stranded telomeric DNA sequence.

We examined structural properties of poly d(C4A2).d(T2G4), the telomeric DNA sequence of the ciliated protozoan Tetrahymena. Under conditions of high negative supercoiling, poly d(C4A2).d(T2G4) inserted in a circular plasmid vector was preferentially sensitive to digestion with S1 nuclease. Only the C4A2 strand was sensitive to first-strand S1 cutting, with a markedly skewed pattern of hypersensitive sites in tracts of either 46 or 7 tandem repeats. Linear poly d(C4A2).(T2G4) showed no preferential S1 sensitivity, no circular dichroism spectra indicative of a Z-DNA conformation, no unusual Tm, and no unusual migration in polyacrylamide gel electrophoresis. The S1 nuclease sensitivity properties are consistent with a model proposed previously for supercoiled poly d(CT).d(AG) (Pulleyblank et al., Cell 42:271-280, 1985), consisting of a double-stranded, protonated, right-handed underwound helix. We propose that this structure is shared by related telomeric sequences and may play a role in their biological recognition.     

Induction of double-strand breaks by S1 nuclease, mung bean nuclease and nuclease P1 in DNA containing abasic sites and nicks.

Defined DNA substrates containing discrete abasic sites or paired abasic sites set 1, 3, 5 and 7 bases apart on opposite strands were constructed to examine the reactivity of S1, mung bean and P1 nucleases towards abasic sites. None of the enzymes acted on the substrate containing discrete abasic sites. Under conditions where little or no non-specific DNA degradation was observed, all three nucleases were able to generate double-strand breaks when the bistranded abasic sites were 1 and 3 base pairs apart. However, when the abasic sites were further apart, the enzymes again failed to cleave the DNA. These results indicate that single abasic sites do not cause sufficient denaturation of the DNA to allow incision by these single-strand specific endonucleases. The reactivity of these enzymes was also investigated on DNA substrates that were nicked by DNasel or more site-specifically by endonuclease III incision at the discrete abasic sites. The three nucleases readily induced a strand break opposite such nicks.     

P1 nuclease cleavage is dependent on length of the mismatches in DNA

P1 nuclease is one of the most extensively used single-strand DNA specific nucleases in molecular biology. In modern biology, it is used as an enzymatic probe to detect altered DNA conformations. It is well documented that P1 cleaves single-stranded nucleic acids and single-stranded DNA regions. The fact that P1 can act under a wide range of conditions, including physiological pH and temperature make it the most commonly used enzymatic probe in DNA structural studies. Surprisingly, to this date, there is no study to characterize the influence of length of mismatches on P1 sensitivity. Using a series of radioactively labeled oligomeric DNA substrates-containing mismatches, we find that P1 nuclease cleavage is dependent on the length of mismatches. P1 does not cleave DNA when there is a single-base mismatch. P1 cleavage efficiency is optimum when mismatch length is 3 nt or more. Changing the position of the mismatches also does not make any difference in cleavage efficacy. These novel findings on P1 properties have implications for its use in DNA structure and DNA repair studies.