S1 Cleavage Research Articles

Tertiary base pair interactions in slipped loop-DNA: an NMR and model building study.

Short direct repeat sequences are often found in regulatory regions of various genes; in some cases they display hypersensitivity to S1 nuclease cleavage in supercoiled plasmids. A non-standard DNA structure (Slipped Loop Structure, or SLS) has been proposed for these regions in order to explain the S1 cleavage data; the formation of this structure may be involved in the regulation of transcription. The structure can be generally classified as a particular type of pseudoknot. To date, no detailed stereochemical model has been developed. We have applied one-dimensional 1H NMR spectroscopy to study a synthetic DNA, 55 nucleotides in length, which cannot fold as a standard hairpin but which may favor the SLS formation. AT base pairs were identified, consistent only with the formation of an additional, tertiary miniduplex in the SLS. An all-atom stereochemically sound model has been developed for the SLS with the use of conformational calculations. The model building studies have demonstrated that the tertiary miniduplex can be formed for one of the plausible SLS isomers, but not for the other.

EVOLUÇÃO TECTÔNICA BRASILIANA DA FAIXA PARAGUAI: A ESTRUTURAÇÃO DA REGIÃO DE CUIABÁ

Late Proterozoic sediments and metasediments are present along the transition zone between the Amazonian Craton to the northwest and the Paraguay Belt to the southeast in the Cuiaba region, Mato Grosso. In the Paraguay Belt, these rocks have been affected by the BraS1liano Orogeny, characterized by increaS1ng deformation and metamorphism from the craton towards the fold belt The metamorphic evolution is determined by the illite crystallinity index. Four closely related phases of deformation (D1 to D4) are distinguished in the Paraguay Belt. The trend directions of Dl, D2 and D3 are almost identical (NE-SW) whereas D4 is transverse (NW-SE). The first phase Dl is the most preeminent, and is contemporaneous with the regional metamorphism. It produced tight and isoclinal Fl folds to the east and open folds F1 to the west, and associated S1 cleavage. The D2 and D3 phase conS1st mainly of local crenulation cleavages (S2 and S3). D4 phase is characterized by regional slight transverse folds.

A single-stranded gap in human immunodeficiency virus unintegrated linear DNA defined by a central copy of the polypurine tract

The structure of unintegrated human immunodeficiency virus type 1 (HIV-1) DNA from acutely infected human lymphoid cells was analyzed by nuclease S1 cleavage. We observed a unique, discrete single-stranded gap in unintegrated linear DNA molecules, located near the center of the genome. Oligonucleotide primer extension experiments determined that the downstream limit of this gap coincides with the last nucleotide of a central copy of the polypurine tract found in all sequenced lentivirus genomes. Other retroviruses have only one copy of the polypurine tract at the 5' boundary of the 3' long terminal repeat, which has been shown to determine initiation of retroviral DNA plus-strand synthesis. We conclude from our observations that the central repeat of the polypurine tract can create an additional site for plus-strand synthesis initiation in lentiviruses. The central single-stranded gap was not found in circular DNA molecules, the vast majority of them carrying only one long terminal repeat. This finding suggests that the generation of such circular molecules is associated with early DNA ligation events.

The age of cleavage development in the Ross orogen, northern Victoria Land, Antarctica: evidence from 40Ar/39Ar whole-rock slate ages

Metamorphic conditions accompanying regional S1 cleavage development and whole-rock slate 40Ar/39Ar age spectra indicate that the Ross orogeny occurred at ca 500 Ma. Subsequent deformation associated with development of a major thrust overprinted the regional cleavage. Existing cleavage micas were not themally reset and syntectonic mica did not crystallize during thrusting. A regional, low-grade thermal event of Middle Jurassic age may be suggested by the variably discordant age spectra.

Localized melting and structural changes in the SV40 origin of replication induced by T-antigen.

Replication of simian virus 40 (SV40) DNA is dependent upon the binding of the viral T-antigen to the SV40 origin of replication. Structural changes in the origin of replication induced by binding of T-antigen were probed by chemical modifications of the DNA. In the presence of ATP, T-antigen rendered two of three domains in the SV40 core origin hypersensitive to attack by either dimethyl sulfate or potassium permanganate (KMnO4). One of these domains, the early palindrome, was shown to contain an 8-bp region of melted DNA as determined from methylation of cytosine residues and by nuclease S1 cleavage of methylated DNA. DNA melting was not dependent upon either the hydrolysis of ATP or the binding of T-antigen to an adjacent site (site I). A second domain, the A/T element, was extensively modified by KMnO4 but no significant melting was detected. Rather, the pattern of modification indicates that T-antigen caused a conformational change of the double-stranded DNA in this region. These results suggest that T-antigen, in the presence of ATP, destabilizes the SV40 origin by melting and structurally deforming two flanking regions within the core origin sequence. These DNA structural changes may provide access to other replication factors, allowing complete denaturation of the SV40 origin and the initiation of SV40 DNA replication.

Site-specific chemical modification of B-Z junctions in supercoiled DNA as detected by nuclease S1 digestion, inhibition of restriction cleavage and nucleotide sequencing.

Structural distortions on the boundary between right-handed and left-handed segments in the superhelical plasmid pPK2 (a derivative of pUC19 containing (dC-dG)n segments cloned into polylinker) were studied by means of chemical probes. Strong osmium tetroxide, pyridine (Os,py) modification of DNA at native superhelical density (sigma) was found in four thymines surrounding the (dC-dG)13 segment. These results correlated with restriction cleavage inhibition (due to modification): BamHI cleavage was strongly inhibited, unlike the neighbouring XbaI and SalI (weak or no inhibition). In the (dC-dG)8 segment considerably weaker modification of the B-Z junctions was observed, accompanied by weak inhibition of BamHI cleavage, while the neighbouring SmaI and KpnI were not affected. Os,py modification of DNA at native sigma was not detected by nuclease S1 cleavage at and (dC-dG)n segment. However, this enzyme recognized and cleaved at the B-Z junction, osmium modified at more negative sigma. The results obtained with the glyoxal and diethyl pyrocarbonate modification support the idea of very narrow B-Z junctions at native sigma.

Structure and deformation history of the Central Wales Synclinorium, northeast Dyfed: Evidence for a long-lived basement structure

The Central Wales Synclinorium in northeast Dyfed is markedly asymmetrical, dominated by strongly southeast verging NE–SW trending F1 folds with an axial planar to mildly clockwise-transecting cleavage. Within the Pysgotwr Fault Belt, a narrow braided array of steep faults developed in the core of the structure, the F1 folds and S1 cleavage are locally refolded into en echelon ENE–WSW F2 folds with an associated S2 cleavage. F2 and S2 geometry and orientation strongly suggest development in response to an important element of oblique simple shear involving components of sinistral strikeslip and NW-down dip-slip. The Pysgotwr Fault Belt is anomalous in the context of fold-dominated mid-Wales and appears to be associated with a long-lived basement structure which significantly influenced Welsh Basin topography in early Silurian time.

A new method for constructing linker scanning mutants

A new procedure for the construction of linker scanning mutants is described. A plasmid containing the target DNA is randomly linearized and slightly shortened by a novel combination of established methods. After partial apurination with formic acid a specific nick or small gap is introduced at the apurinic site by exonuclease III, followed by nuclease S1 cleavage of the strand opposite the nick/gap. Synthetic linkers are ligated to the ends and plasmids having the linker inserted in the target DNA are enriched. Putative linker scanning mutants are identified by their topoisomer patterns after relaxation with topoisomerase I. This technique allows the distinction of plasmids differing in length by a single basepair. We have used this rapid and efficient strategy to generate a set of 32 linker scanning mutants covering the chicken lysozyme promoter from -208 to +15.

DNA structure equilibria in the human c-myc gene.

We have employed analytical S1 nuclease analysis to identify sites with altered DNA secondary structure in the human c-myc gene. We have mapped several sites of that kind in vitro at one-base resolution but have focused our attention on one particularly stable conformational isomer which occurs approximately 270 base pairs upstream from the preferred transcription origin. We have analyzed the kinetics of that conformational equilibrium as a function of supercoil density and enzyme concentration and find that DNA structure in this region is adequately modeled as a two-state equilibrium between an undistorted (S1 nuclease insensitive) and a distorted (S1-sensitive) state. We find that at fixed supercoil density, S1 nuclease cleavage at this DNA segment can be altered in vitro by a DNA sequence change as far away as 1500 bases. We also find that the S1 nuclease cleavage at this site can be dramatically enhanced by the binding of small RNA molecules. On the basis of an analysis of S1 cutting kinetics and an analysis of DNA sequence at the S1 cleavage site, we conclude that RNA may bind directly to DNA, thereby shifting the underlying conformational equilibrium. Together, these data suggest that as a class, short RNA molecules could serve as site-specific regulatory elements in the myc gene and elsewhere.

Supercoil-dependent recognition of specific DNA sites by chromosomal protein HMG 2

The ability of the chromosomal high mobility group protein HMG 2 to recognize supercoil-dependent structures within the chicken adult beta-globin gene was investigated by examining its ability to protect such sites from digestion by S1 nuclease. Low molar ratios of HMG 2 were found to be sufficient for complete inhibition of S1 cleavage of a supercoiled plasmid containing the globin gene. Furthermore, HMG 2 protected an S1 cleavage site within the 5'-flanking region of the globin gene, in preference to a palindromic S1 site within the plasmid vector.

Structures of homopurine-homopyrimidine tract in superhelical DNA.

For homopurine-homopyrimidine tracts in superhelical DNA, we propose a structure involving Watson-Crick and Hoogsteen paired triple helixes, hairpin loops and unstructured domains. Topologically, the whole structure is equivalent to an open region. The proposed structure is consistent with available S1 cleavage, pH and alkylation data and energetics under superhelical stress; this new structure is a much more probable candidate than the one proposed by us recently (V.I. Lyamichev, S.M. Mirkin & M.D. Frank-Kamenetskii, J. Biomole. Str. Dyns 3, 327-338, 1985).

The Orlock Bridge Fault: a major Late Caledonian sinistral fault in the Southern Uplands terrane, British Isles

ABSTRACTA detailed examination of the boundary between Peach and Home's (1899) Northern and Central Belts, in the Lower Palaeozoic rocks of the Southern Uplands of Scotland and their Irish continuation in Longford–Down, demonstrates it to be a major Caledonian sinistral wrench fault. Maps and descriptions of outcrops of the fault at Slieve Glah in County Cavan, at Orlock Bridge on the NE coast of Down, at Cairngarroch on the Rhinns of Galloway and at Garvald, 11 km SW of Dunbar, are presented. A distinctive fault fabric, clearly the result of repeated sinistral slip at the first three localities, characterises these outcrops along the 400 km trace. The fabric elements include: (1) lenticular shearing of arenites in the fault protolith, (2) a fault-associated phyllonitic fabric, with abundant pressure-solution seams, overprinting the regional S1, (3) numerous foliation-parallel quartz segregations, (4) refolding of the regional S1 cleavage, segregation veins and the phyllonitic fabric in at least two generations of steeply plunging, sinistrally-verging folds, (5) a non-penetrative crenulation cleavage, (6) a locally developed sinistral S–C fabric, and (7) typical protomylonite, mylonite and ultramylonite textures in thin sections of the fault rocks. The zone of fault-associated deformation varies from a few metres across at Garvald to over 1 km at Slieve Glah. The fault thus differs from other tract-defining faults in the Southern Uplands, particularly in its clear evidence of ductile, quasi-plastic, deformation at depth, in its lack of associated imbrication, in its significant refolding and overprinting of the regional S1 cleavage and in its uniquely large stratigraphic effects.The main fabric-generating movement postdates the regional, accretion-related, S1 cleavage but predates minor Caledonian (c. 400 Ma) intrusions. For its entire length the fault separates the Northern Belt, with proof only of Upper Ordovician turbidites, and the Central Belt, where turbidite deposition began in the Silurian. The age of the base of the turbidites decreases southward by some six graptolite zones at the fault trace. The fault thus either (a) excises the equivalent of four tracts from a single accretionary-prism terrane, or (b) juxtaposes two distinct terranes, one of late Ordovician and the other of Silurian age. In either event the sinistral slip is probably in excess of 400 km.

Characterization of a supercoil-dependent S1 sensitive site 5' to the Drosophila melanogaster hsp 26 gene.

We have analyzed the prominent supercoil-dependent S1 nuclease cleavage site 5' to hsp 26 in the plasmid 88B13, which contains 11.7 kilobases from the Drosophila locus 67B1. The double stranded cleavage product is generated by initial nicking on the purine strand, six preferred sites occurring between positions -96 and -90 (relative to the start of transcription) with weaker ones extending to position -84, followed by cleavage on the pyrimidine strand at positions -86 and -84. A derivative of 88B13, 88B13-X, was generated by insertion of an Xho I linker at position -84; this does not affect the positions or strand specificity of the S1 cleavage in that region. A small deletion, delta 41.1, removes the homopurine/homopyrimidine stretch from positions -86 to -132 and is no longer sensitive to cleavage by S1 nuclease 5' to hsp 26. Mung bean and P1 nucleases recognize the same site 5' to hsp 26 and give the same general pattern of cleavage. All three nucleases show an initial cleavage of 88B13 DNA at this site at pH 5.5 but not at pH 6.5, indicating that the DNA structure there may be pH dependent in vitro.

Recognition of the Structural Distortions at the Junctions Between B and Z Segments in Negatively Supercoiled DNA by Osmium Tetroxide

It has been shown for the first time that conformational junction between contiguous right-handed B and left-handed Z segments can be recognized by a chemical probe. Plasmid pRW751 containing (dC-dG)13 and (dC-dG)16 blocks was treated with osmium tetroxide, pyridine (a reagent known to be single-strand selective) at physiological ionic conditions (0.1 and 0.2 M NaCl) and neutral pH. Mapping of the osmium binding sites by restriction enzyme digestion followed by nuclease S1 cleavage has revealed selective binding of osmium at, or near to, the end of the (dC-dG)n segments proximal to the 95 bp lac sequence. The junction of the shorter (dC-dG)13 segment was modified to a substantially greater extent than that of the longer segment. Partial inhibition of DNA cleavage by BamHI was observed at the restriction sites neighbouring to the both (dC-dG)n segments as a result of DNA modification by osmium tetroxide. The site-selective modification occurred only in supercoiled and not in relaxed molecules. Differences in the sensitivity of the B/Z junctions in pRW751 to the osmium tetroxide were explained by different structural features of these junctions.

Nuclease S1-sensitive sites in multigene families: human U2 small nuclear RNA genes.

We show here that human U2 small nuclear RNA genes contain a 'strong nuclease S1 cleavage site' (SNS1 site), a sequence that is very sensitive to digestion by nuclease S1. This site is located 0.50-0.65 kb downstream of the U2 RNA coding region. It comprises a 0.15-kb region in which (dC-dT)n:(dA-dG)n co-polymeric stretches represent greater than 90% of the sequence. Nuclease S1 is able to excise unit length repeats of the human U2 RNA genes both from cloned fragments and total human genomic DNA. The precise locations of the cleavage sites are dependent on the superhelicity of the substrate DNA. In negatively supercoiled substrates, cleavages are distributed over the entire 0.15-kb region, but in linearized substrates, they occur within a more limited region, mainly at the boundary of the SNS1 site closest to the human U2 RNA coding region. Nuclease S1 cleavage of negatively supercoiled substrates occurs at pHs as high as 7.0; in contrast, cleavage of linearized substrates requires a pH less than 5.0, indicating that supercoiling contributes to the sensitivity of this site. Mung bean nuclease gives results similar to that observed with nuclease S1.

Low-grade metamorphism and accretion tectonics: Southern Uplands terrain, Scotland

AbstractPrevious studies of low-grade metamorphism in the Southern Uplands accretionary terrain indicated prehnite-pumpellyite facies/anchizone conditions developed throughout the area, except for local preservation of trench-slope sediments and an accreted seamount at zeolite facies/advanced diagenetic grade. New graptolite reflectance data are presented that show a general northward increase in temperature in the Southern Uplands. The results from two cross-strike traverses in the southern and central belts in contemporaneous sequences, using illite crystallinity, illite lateral spacing (bo) , and graptolite reflectance, indicate the development of systematic accretion-related low-grade metamorphism. Well-developed and constant anchizone conditions occur throughout the NE (Langholm) traverse, associated with common, F1 accretion-related folding and a regionally penetrative S1 cleavage. In the SW (Kirkcudbright) traverse, however, the youngest, last accreted packets are preserved at a transitional diagenetic stage and lack a penetrative S1 cleavage. Illite crystallinity, graptolite reflectance, and bo increase systematically northward through earlier accreted packets, reaching values of the NE traverse only at the northern end. The concomitant increase of bo with illite crystallinity suggests the relatively high P-low T trajectory characteristic of subduction zones. Integration of metamorphic and structural data relates increasing intensity of aceretion-related F1 folding, developmertt of S1 fabric, and onset of later fold phases to grade of metamorphism and structural level within the accretionary pile.

S1-sensitive sites in the supercoiled double-stranded form of tomato golden mosaic virus DNA component B: identification of regions of potential alternative secondary structure and regulatory function.

The sensitivity of the supercoiled double-stranded form of the DNA of tomato golden mosaic virus (TGMV), a geminivirus, to the single-strand specific enzyme S1 nuclease has been demonstrated. Specific S1 cleavage sites were identified in TGMV DNA component B by cloning into the single-strand bacteriophage vector M13 mp8 and sequencing of the inserted DNA. Analysis of the DNA sequence at the sites of S1 sensitivity in TGMV DNA component B revealed several possible regions of alternative secondary structure which were clustered in an intergenic region upstream of the starts of the two major open reading frames which are in opposite orientations. This region contains putative transcriptional promoter and modulatory sequences and a possible replication origin. The extreme S1 sensitivity of the supercoiled form of TGMV DNA component A precluded its cloning under the conditions employed for selective cleavage of DNA component B.

Human U1 RNA genes contain an unusually sensitive nuclease S1 cleavage site within the conserved 3' flanking region.

We find that the cloned DNAs of human U1 small nuclear RNA genes contain two nuclease S1-sensitive sites, one about 1.8 kilobases downstream of the U1 RNA coding region and the other around 0.3 kilobase upstream. The downstream site is unusually sensitive to the nuclease, being cleaved in both linear and negatively supercoiled DNAs. The extent of cleavage at this site is enhanced at lower pH and reduced concentrations of NaCl; the effects of salt are more apparent on linear than supercoiled DNAs. The nuclease S1 sensitivity of this downstream site is dependent on the presence of the sequence (dC-dT)n X (dA-dG)n, where n = 15-25. (One gene with n = 5 is resistant to nuclease S1 cleavage in this region.) In contrast, the nuclease S1 site upstream of the coding region is cleaved only when the DNA is supercoiled. This site also has a homopyrimidine X homopurine bias in the DNA strands, but the sequence is less regular. In the course of these studies, we detected several discrepancies between our restriction maps of some U1 RNA genes and those published by others. Our maps demonstrate that all seven cloned human U1 RNA genes are very similar in sequence for as much as 2.3 kilobases downstream of the U1 RNA coding region.

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.

A sequence conserved in both the chicken and mouse alpha 2(I) collagen promoter contains sites sensitive to S1 nuclease.

We have examined the S1 nuclease sensitivity in the promoter of both the chicken and mouse alpha 2(I) collagen genes. When these DNAs are introduced into supercoiled plasmids and digested with S1 nuclease, a discrete region containing one or more cleavages is found in each promoter. These S1 cleavage sites were mapped by the distance of the S1 site from known restriction enzyme cleavage sites. In the chicken gene, the S1-sensitive segment is located 180 to 200 base pairs preceding the start site of transcription, whereas in the mouse promoter it is between -145 to -165 base pairs. This site in the chicken promoter maps to the segment that has previously been shown to be S1 and DNase I hypersensitive in chromatin. Although these S1 sites are found at different distances from the start site of transcription in the two promoters, the sequences at these sites are strongly conserved between the two species. Each sequence consists of an identical tandem repeat containing a short palindrome within each repeat. Since the DNA sequence does not exhibit the features that would favor either a left-handed Z-DNA configuration or a cruciform structure, an alternative model is discussed that could account for the S1 sensitivity of these sequences. The conservation of these sequences and their S1 sensitivity suggests they play a role in the activation or regulation of the alpha 2(I) collagen gene promoters.