Cleavage of Circular, Superhelical Simian Virus 40 DNA to a Linear Duplex by S1 Nuclease

S(1) nuclease, the single-strand specific nuclease from Aspergillus oryzae can cleave both strands of circular covalently closed, superhelical simian virus 40 (SV40) DNA to generate unit length linear duplex molecules with intact single strands. But circular, covalently closed, nonsuperhelical DNA, as well as linear duplex molecules, are relatively resistant to attack by the enzyme. These findings indicate that unpaired or weakly hydrogen-bonded regions, sensitive to the single strand-specific nuclease, occur or can be induced in superhelical DNA. Nicked, circular SV40 DNA can be cleaved on the opposite strand at or near the nick to yield linear molecules. S(1) nuclease may be a useful reagent for cleaving DNAs at regions containing single-strand nicks. Unlike the restriction endonucleases, S(1) nuclease probably does not cleave SV40 DNA at a specific nucleotide sequence. Rather, the sites of cleavage occur within regions that are readily denaturable in a topologically constrained superhelical molecule. At moderate salt concentrations (75 mM) SV40 DNA is cleaved once, most often within either one of the two following regions: the segments defined as 0.15 to 0.25 and 0.45 to 0.55 SV40 fractional length, clockwise, from the EcoR(I) restriction endonuclease cleavage site (defined as the zero position on the SV40 DNA map). In higher salt (250 mM) cleavage occurs preferentially within the 0.45 to 0.55 segment of the map.     

Selection of cleavage site by mammalian tRNA 3' processing endoribonuclease

Mammalian tRNA 3' processing endoribonuclease (3' tRNase) removes 3' trailers from pre-tRNAs by cleaving the RNA immediately downstream of the discriminator nucleotide. Although 3' tRNase can recognize and cleave any target RNA that forms a pre-tRNA-like complex with another RNA, in some cases cleavage occurs at multiple sites near the discriminator. We investigated what features of pre-tRNA determine the cleavage site using various pre-tRNAArg variants and purified pig enzyme. Because the T stem-loop and the acceptor stem plus a 3' trailer are sufficient for recognition by 3' tRNase, we constructed variants that had additions and/or deletions of base-pairs in the T stem and/or the acceptor stem. Pre-tRNAs lacking one and two acceptor stem base-pairs were cleaved one and two nucleotides and two and three nucleotides, respectively, downstream of the discriminator. On the other hand, pre-tRNA variants containing extra acceptor stem base-pairs were cleaved only after the discriminator. The cleavage site was shifted to one and two nucleotides downstream of the discriminator by deleting one base-pair from the T stem, but was not changed by additional base-pairs in the T stem. Pre-tRNA variants that contained an eight base-pair acceptor stem plus a six base-pair T stem, an eight base-pair acceptor stem plus a four base-pair T stem, or a six base-pair acceptor stem plus a six base-pair T stem were all cleaved after the original nucleotide. In general, pre-tRNA variants containing a total of more than 11 bp in the acceptor stem and the T stem were cleaved only after the discriminator, and pre-tRNA variants with a total of N bp (N is less than 12) were cleaved 12-N and 13-N nt downstream of the discriminator. Cleavage efficiency of the variants decreased depending on the degree of structural changes from the authentic pre-tRNA. This suggests that the numbers of base-pairs of both the acceptor stem and the T stem are important for recognition and cleavage by 3' tRNase.     

Survey and mapping of restriction endonuclease cleavage sites in bacteriophage T7 DNA.

A survey of restriction endonucleases having different cleavage specificities has identified 10 that do not cut wild-type bacteriophage T7 DNA, 11 that cut at six or fewer sites, four that cut at 18 to 45 sites, and 12 that cut at more than 50 sites. All the cleavage sites for the 13 enzymes that cut at 26 or fewer sites have been mapped. Cleavage sites for each of the 10 enzymes that do not cut T7 DNA would be expected to occur an average of 9 to 10 times in a random nucleotide sequence the length of T7 DNA. A possible explanation for the lack of any cleavage sites for these enzymes might be that T7 encounters enzymes having these specificities in natural hosts, and that the sites have been eliminated from T7 DNA by natural selection. Five restriction endonucleases were found to cut within the terminal repetition of T7 DNA; one of these, KpnI, cuts at only three additional sites in the T7 DNA molecule. The length of the terminal repetition was estimated by two independent means to be approximately 155 to 160 base-pairs.     

Micrococcal nuclease as a DNA structural probe: its recognition sequences, their genomic distribution and correlation with DNA structure determinants

We have analyzed micrococcal nuclease (MNase) DNA cleavage patterns at the sequence level by examining 2.3 X 10(3) base-pairs of data derived from the Drosophila melanogaster 44D larval cuticle locus. Within this region, MNase preferentially cleaved 140 sites. Clusters of these sites appear to generate the preferential MNase eukaryotic DNA cleavage sites seen on agarose gels at roughly 100 to 300 base-pair intervals. These clusters of preferential cleavage sites rarely occur within gene coding regions. The analysis revealed that duplex DNA sequences preferentially cleaved by MNase are generally determined by a single strand sequence: d(A-T)n, where n greater than or equal to 1, flanked by a 5' dC or dG. Cleavage of the other strand is generally staggered 5' by several nucleotides and occurs even if such sequences are absent on that strand. An empirical predictive DNA cleavage model derived from a statistical analysis of the sequence level data was applied to seven eukaryotic gene loci of known sequence. The predicted patterns were in good general agreement with the previously observed eukaryotic gene/spacer cleavage pattern. Statistical analysis also revealed that sites of predicted preferential DNA cleavage occur less frequently in protein coding regions than for randomized sequences of the same length and nucleotide content. Comparison of the MNase cleavage patterns to the sequence-dependent pattern of binding energies between duplex DNA strands indicates that MNase preferentially cleaves sequences with low helix stability.     

Changes in site specificity of single-strand-specific endonucleases on supercoiled PM2 DNA with temperature and ionic environment.

Mung bean nuclease sites in supercoiled PM2 DNA at neutral pH were located by linearizing the singly-nicked circular DNA product with venom phosphodiesterase followed by restriction endonuclease mapping. The locations of the sites varied with small changes in temperature and in concentration of NaC1 or magnesium ion. Different environmental changes which affect duplex stability in the same direction showed similar effects on the number of sites and in some cases resulted in identical cleavage patterns. Venom phosphodiesterase and P1 nuclease showed cleavage patterns similar to mung bean nuclease under the same environmental conditions and showed similar variations in cleavage patterns when environmental conditions were changed. Relaxed, closed-circular DNA was slowly cleaved at numerous sites whose locations did not vary with environment. Changes in site specificity are likely the result of environmental effects on the conformation of supercoiled DNA as opposed to effects on the single-strand-specific endonucleases themselves.     

An endonuclease activity of venom phosphodiesterase specific for single-stranded and superhelical DNA.

A homogeneous preparation of venom phosphodiesterase from Crotalus adamanteus possesses an intrinsic endonuclease activity, specific for superhelical (form I) and single-stranded DNA. The phosphodiesterase degrades single-stranded T7 DNA by endonucleolytic cleavages. Duplex T7 DNA is hydrolyzed by the liberation of acid-soluble products simultaneously from the 3' and 5' termini but without demonstrable internal scissions in duplex regions. Since venom phosphodiesterase is known to hydrolyze oligonucleotides stepwise from the 3' termini, the cleavage at the 5' end of duplex T7 DNA is ascribed to an endonuclease activity. Form I PM2 DNA is nicked to yield first relaxed circles and then linear DNA which is subsequently hydrolyzed only from the chain termini. The linear duplex DNA intermediates consist of a discrete series of fragments (11 are usually resolved on agarose gels) with initial molecular weights ranging from 6.3 x 10(6) (the intact PM2 DNA size) to approximately 1 x 10(6). The cleavage of the form I molecule must, therefore, occur at a limited number of unique sites. The enzyme also cleaves nonsuperhelical, covalently closed circular PM2 DNA but at a 10(4) times slower rate. Both the endonuclease activity on form I DNA and the known exonuclease activity co-migrate on polyacrtkanude gels, are optimally active at pH 9, are stimulated by small concentrations of Mg2+, and are similarly inactivated by heat, reducing agents, and EDTA.     

Locating Interrupted Hydrogen Bonding in the Secondary Structure of PM2 Circular DNA by Comparative Denaturation Mapping

Previous studies with HCHO have revealed a reaction with superhelical DNA that strongly suggests that this DNA consists of small regions of interrupted secondary structure. To map these sites in PM2 DNA, the following set of experiments was performed using electron microscopy. (i) A denaturation map of nicked form II was obtained using Inman's alkaline-HCHO conditions. (ii) The superhelical form I was reacted with HCHO at 30 C until equilibrium was achieved at the interrupted sites (3.6% reactivity). The excess HCHO was removed rapidly and X-ray treatment was employed to nick these prereacted molecules. These form II molecules containing HCHO (form II HCHO) were also subjected to denaturation mapping. It would be expected that the HCHO-unpaired regions would serve as induction sites for the propagation of melting. Hence, depending on the location of the induction sites; we would anticipate either the creation of new regions of melting or a normal denaturation map shifted to lower pH values. Comparison of the development of progressive denaturation of form II and form II HCHO reveals that the latter is the case. The denaturation maps of form II are highly organized patterns of adenine-thymine (AT)-rich regions, with a total of five regions at extreme pH conditions. There are six highly organized regions for form II HCHO, i.e., smaller adjacent loops, at low denaturation conditions where no denaturation is seen for form II. These coalesce into the pattern for form II containing four of five A-T-rich regions observed for form II. Hence we conclude that the regions of altered hydrogen bonding in superhelical PM2 DNA are four to six in number and they map in the A-T-rich regions of the DNA.     

Factors influencing resistance of UV-irradiated DNA to the restriction endonuclease cleavage

DNA molecules of pUC19, pBR322 and PhiX174 were irradiated by various doses of UV light and the irradiated molecules were cleaved by about two dozen type II restrictases. The irradiation generally blocked the cleavage in a dose-dependent way. In accordance with previous studies, the (A + T)-richness and the (PyPy) dimer content of the restriction site belongs among the factors that on average, cause an increase in the resistance of UV damaged DNA to the restrictase cleavage. However, we observed strong effects of UV irradiation even with (G + C)-rich and (PyPy)-poor sites. In addition, sequences flanking the restriction site influenced the protection in some cases (e.g. HindIII), but not in others (e.g. SalI), whereas neoschizomer couples SmaI and AvaI, or SacI and Ecl136II, cleaved the UV-irradiated DNA similarly. Hence the intrastrand thymine dimers located in the recognition site are not the only photoproduct blocking the restrictases. UV irradiation of the A-form generally made the irradiated DNA less resistant to restrictase cleavage than irradiation in the B-form and in some cases, the A-form completely protected the UV-irradiated DNA against the damage recognized by the restrictases. The present results also demonstrate that the UV irradiation approach used to generate partial digests in genomic DNA studies, can be extended to the (G + C)-rich and (PyPy)-poor restriction sites. The present extensive and quantitative data can be used in genomic applications of UV damage probing by restrictases.     

Sequence specificity of 125I-labelled Hoechst 33258 damage in six closely related DNA sequences

The sequence selectivity of [125I]Hoechst 33258 in six 340 base-pair DNA sequences has been investigated. [125I]Hoechst 33258, which is a bis-benzimidazole and binds to the minor groove of B-DNA, preferentially binds to A + T-rich regions of DNA. Six out of nine strong binding sites contained four or more consecutive A.T base-pairs, while the other three strong binding sites were AAGGATT, TATAGAAA (the peak of damage was in the run of 3 A residues) and AAA. One of the six weak binding sites had five consecutive A.T base-pairs, two of the weak binding sites had three, and three did not have any. In addition to genomic 340 base-pair alpha RI-DNA (which is a tandem repeat in human cells), five 340 base-pair alpha RI-DNA clones were generated that differed from the genomic "consensus" sequence by a number of random base alterations. The effect of these base changes on the sequence specificity of [125I]Hoechst 33258 damage indicated that of the base changes that interrupted 14 binding sites, six decreased and eight did not change the extent of damage, while two sites changed position. Of the base alterations that augmented 17 binding sites, five increased, two decreased and ten did not alter the degree of cleavage, while ten sites changed position. It was concluded from the data that, while runs of consecutive A.T base-pairs was the most important parameter that determines [125I]Hoechst 33258 binding, other factors including position in the DNA sequence, nearest neighbour and long-range interactions were also important.     

A comparison of the phage T4 gene 32 protein and Escherichia coli RNA polymerase binding sites on hamster papovavirus DNA

Phage T4 gene 32 protein and Escherichia coli RNA polymerase were bound to hamster papovavirus DNA. The binding regions were identified by electron microscopy employing a protein-free spreading technique. After gene 32 protein treatment four denaturation regions could be mapped, at 0.04–0.12, 0.30–0.36, 0.50–0.60 and 0.75–0.90 DNA map units, respectively, using the unique BamHI cleavage site as zero point. Eight RNA polymerase binding sites can be found which are localized at positions 0.05; 0.11; 0.18; 0.31; 0.57; 0.66; 0.76 and 0.82. A comparison of the RNA polymerase binding sites with the gene 32 protein denaturation pattern reveals a correspondence of six of eight polymerase binding sites with (A + T)-rich regions within the hamster papovavirus genome.     

Enhancement of S1 nuclease-susceptibility of negatively superhelical DNA by netropsin

It was evidenced that the antibiotic netropsin enhances the single-strand-specific nuclease S1-susceptibility of negatively superhelical DNA. In contrast, an intercalating drug inhibited S1 action on the superhelical DNA. Negatively superhelical DNA is known to possess several (or a number of) unbasepaired sites sensitive to S1 cleavage. S1 cleaves generally the DNA once at these sites to result in production of the full-length linear form. However netropsin-bound DNA had a tendency to be cleaved by S1 simultaneously at plural sites producing several species of linear DNAs smaller than full-length size.     

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.     

Formation of MboII vectors and cassettes using asymmetric MboII linkers

Class-IIS restriction endonucleases such as MboII cleave DNA at a specified distance away from their recognition sequences. This feature was exploited to cleave DNA at previously inaccessible locations by preparing special asymmetric linker/adapters containing the MboII recognition sequence. These could be joined to DNA fragments and subsequently cleaved by MboII. Attachment of a 3' phosphate to one of the two different oligodeoxynucleotides comprising the asymmetric duplex prevented ligation at the improper end of the linker. Plasmids were constructed containing a unique BamHI or BclI site between the recognition and cleavage site of MboII. These sites were used to introduce a foreign fragment into the plasmid at a position permitting MboII to cleave within the newly inserted fragment. Once cleaved at the unique MboII site, another DNA fragment was inserted. DNA was thus inserted at a sequence not previously accessible to specific cleavage by a restriction enzyme. A cassette containing an identifiable marker, the lac operator, between two oppositely oriented MboII/BamHI linkers was made and tested in a random insertion linker mutagenesis experiment.     

Altering the specificity of restriction endonuclease: effect of replacing Mg2+ with Mn2+.

In the presence of 100 mM Tris buffer (pH 7.5) and 1-10 mM Mg2+ EcoRI endonuclease cleaves DNA at a specific nucleotide sequence and in a characteristic way: -GAATTC-. But if Mg2+ is replaced by Mn2+, the specificity of the cleavage is relaxed and cleavages occur at many other sites; moreover, there appears to be a hierarchy of cleavage rates at the pseudo-EcoRI restriction sites. For example, SV40 DNA is cleaved only once in the usual digestion conditions, but with Mn2+ more than ten cleavages are made; the five most rapidly cleaved SV40 DNA map locations are 0/1.0 larger than 0.93 larger than 0.33 approximately equal to 0.42 larger than 0.29 approximately equal to 0.40 larger than 0.25. Mn2+ also alters the restriction specificity of HindIII but not HpaII endonuclease.     

Location of the T4 gene 32 protein-binding site on polyoma virus DNA

Three easily denatured regions can be demonstrated in polyoma virus DNA. T4 gene 32 protein which binds to single stranded DNA, but not to duplex DNA, will specifically bind to any of these sites when viral DNA is in its superhelical configuration. These sites were mapped relative to a unique E. coli R_I endonuclease cleavage site by electron microscopy.     

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.     

Long (dA)n.(dT)n tracts can form intramolecular triplexes under superhelical stress.

Plasmids containing long tracts of (dA)n.(dT)n have been prepared and their conformations examined in linear and supercoiled DNA using a series of chemical and enzymic probes which are known to be sensitive to unusual DNA structures. Under superhelical stress and in the presence of magnesium the sequence T69.A69 adopts a conformation at pH 8.0 consistent with the formation of an intramolecular DNA triplex. Site specific cleavage of the supercoiled plasmid by single-strand specific nucleases occurs within the A.T insert; the 5'-end of the purine strand is sensitive to reaction with diethylpyrocarbonate while the central 5-6 bases of the pyrimidine strand are reactive to osmium tetroxide. By contrast shorter inserts of A33.T33 and A23.T23 do not appear to form unusual structures.