Structure of (dG)n.(dC)n under superhelical stress and acid pH

We have recently shown that a (GA)n.(TC)n tract undergoes a sharp structural transition under superhelical stress (V.I. Lyamichev, S.M. Mirkin and M.D. Frank-Kamenetskii, J. Biomol. Struct. Dyn. 2,327 (1985]. Unlike the well studied transitions to the cruciform and to the Z form, this novel transition was strongly pH-dependent. We have found the (dG)n.(dC)n insert to undergo a pH-dependent structural transition similar to that of the (GA)n.(TC)n tract. These new data meet our earlier expectations and disagree with the data of D.E. Pulleyblank, D.B. Haniford and A.R. Morgan, Cell 42, 271 (1985). We conclude that a novel DNA structure (the H-form) is typical of homopurine-homopyrimidine mirror repeats (the H palindromes) under superhelical stress and/or acid pH. In the H-form the homopyrimidine strand forms a hairpin while half of the homopurine strand interacts with the hairpin forming a triplex, the other half of the homopurine strand being unstructured (V.I. Lyamichev, S.M. Mirkin and M.D. Frank-Kamenetskii, J. Biomol. Struct. Dyn. 2, 3, 667 (1986].     

A structural transition in d(AT)n.d(AT)n inserts within superhelical DNA

We have constructed plasmids carrying d(AT)n.d(AT)n inserts of different lengths. Two-dimensional gel electrophoresis patterns show that an increase in the negative superhelicity of these DNAs brings about a structural transition within the inserts, resulting in a reduction of the superhelical stress. However, this reduction corresponds to the expected values neither for cruciform nor the Z form. Those DNA topoisomers in which the structural transition had occurred proved to be specifically recognizable by single-strand-specific endonuclease S1, with the cleavage site situated at the centre of the insert. These data, as well as kinetic studies, suggest that the cloned d(AT)n.d(AT)n sequences adopt a cruciform rather than the Z-form structure. We discuss plausible reasons of the discrepancy between the observed superhelical stress release and that expected for the transition of the insert to the cruciform state.     

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).     

Localization of low-melting regions in phage T7 DNA

Specific fragmentation of T7 DNA at glyoxal-fixed denatured regions by the S1 endonuclease followed by restriction analysis made it possible to localize four low-melting regions in phage T7 DNA. These regions have the following coordinates:0.5-1.2;14.8+/-0.3;46.3+/-0.5; 98.4+/-0.3 (in T7 DNA length units). The location of the low-melting regions was refined by means of electron-microscopic denaturation mapping and gel electrophoresis of partially denatured DNA. The obtained localization of the low-melting regions is consistent with the available data on the sequence of T7 DNA. The map of low-melting regions was compared with the genetic map of T7 DNA.Images     

Native supercoiling of DNA: The effects of DNA gyrase and ω protein in E. coli

Molecular Genetics and Genomics - This study deals with the effects of a temperature-sensitive (ts) mutation at the gene encoding the DNA gyrase B subunit (gyrB ts) and a deletion of the top gene...     

Improved sensitivity for solid-support invasive cleavage reactions with flow cytometry analysis

A new configuration of the solid-support invasive cleavage reaction provides a small reaction-volume format for high-sensitivity discrimination of nucleic acid targets with single nucleotide differences. With target concentrations as low as 2 amol/assay, the solid-support invasive cleavage reaction clearly distinguishes single base mutations. Two oligonucleotides tethered to the solid support hybridize to the target nucleic acid, forming a tripartite substrate that can be recognized and cleaved by Cleavase, a structure-specific 5'-nuclease. Each cleavage event yields fluorescence signal on the surface. When microspheres serve as the solid-support surface, analysis by fluorometer imparts real-time information about change in the reaction signal over time. Flow cytometry provides an alternative detection technology that collects endpoint information about the reaction signal on individual microspheres. A reaction volume of 10 microL with as few as 3000 microspheres is sufficient to distinguish single nucleotide differences at target concentrations less than 200 fM. This sensitivity level is within the range required for analysis of SNPs in genomic DNA. In addition, the flow cytometry format has multiplexing potential, making the microsphere-based invasive cleavage assay attractive for high-throughput genomic applications.     

An invasive cleavage assay for direct quantitation of specific RNAs.

RNA quantitation is becoming increasingly important in basic, pharmaceutical, and clinical research. For example, quantitation of viral RNAs can predict disease progression and therapeutic efficacy. Likewise, gene expression analysis of diseased versus normal, or untreated versus treated, tissue can identify relevant biological responses or assess the effects of pharmacological agents. As the focus of the Human Genome Project moves toward gene expression analysis, the field will require a flexible RNA analysis technology that can quantitatively monitor multiple forms of alternatively transcribed and/or processed RNAs (refs 3,4). We have applied the principles of invasive cleavage and engineered an improved 5'-nuclease to develop an isothermal, fluorescence resonance energy transfer (FRET)-based signal amplification method for detecting RNA in both total RNA and cell lysate samples. This detection format, termed the RNA invasive cleavage assay, obviates the need for target amplification or additional enzymatic signal enhancement. In this report, we describe the assay and present data demonstrating its capabilities for sensitive (<100 copies per reaction), specific (discrimination of 95% homologous sequences, 1 in > or =20,000), and quantitative (1.2-fold changes in RNA levels) detection of unamplified RNA in both single- and biplex-reaction formats.     

Photofootprinting of DNA triplexes.

We have used a photofootprinting assay to study intermolecular and intramolecular DNA triplexes. The assay is based on the fact that the DNA duplex is protected against photodamage (specifically, against the formation of the (6-4) pyrimidine photoproducts) within a triplex structure. We have shown that this is the case for PyPuPu (YRR) as well as PyPuPy (YRY) triplexes. Using the photofootprinting assay, we have studied the triplex formation under a variety of experimentally defined conditions. At acid pH, d(C)n.d(G)n.d(C)n and d(CT)n.d(GA)n.d(CT)n triplexes are detected by this method. The d(CT)n.d(GA)n.d(CT)n triplexes are additionally stabilized by divalent cations and spermidine. PyPuPu triplexes are pH-independent and are stabilized by divalent cations, such as Mg++ and Zn++. The effect depends on the type of cation and on the DNA sequence. The d(CT)n.d(GA)n.d(GA)n triplex is stabilized by Zn++, but not by Mg++, whereas the d(C)n.d(G)n.d(G)n triplex is stabilized by Mg++. In H-DNA, virtually the entire pyrimidine chain is protected against photodimerization, whereas only half of the pyrimidine chain participating in a triplex is protected in the CGG intramolecular triplex.     

A stable complex between homopyrimidine oligomers and the homologous regions of duplex DNAs

When plasmid DNA duplexes carrying the regular homopurine-homopyrimidine inserts (dGA)n, (dTC)n and (dG)n, (dC)n are preincubated with homologous labeled oligo(dPy) ((dTC)n and (dC)n respectively) at acid pH, the label co-electrophoreses with the duplex DNA. Thus, a very strong complex is formed. Complementary oligo(dPu) does not form a complex under these conditions. No binding is observed for oligo(dPy) with non-homologous inserts as well as with vector plasmids without inserts. The complex is formed equally well with linear, nicked or superhelical DNA. The complex is not detected at pH greater than 6. Complex formation leads to very little, if any, unwinding of the duplex. The observed complex appears to be the Py.Pu.Py triplex consisting of TAT and CGC base-triads with protonated cytosines. Two-dimensional gel electrophoresis patterns show that the presence of homologous oligo(dPy) destabilizes the formation of the H form.     

A comparison of eubacterial and archaeal structure-specific 5'-exonucleases.

The 5'-exonuclease domains of the DNA polymerase I proteins of Eubacteria and the FEN1 proteins of Eukarya and Archaea are members of a family of structure-specific 5'-exonucleases with similar function but limited sequence similarity. Their physiological role is to remove the displaced 5' strands created by DNA polymerase during displacement synthesis, thereby creating a substrate for DNA ligase. In this paper, we define the substrate requirements for the 5'-exonuclease enzymes from Thermus aquaticus, Thermus thermophilus, Archaeoglobus fulgidus, Pyrococcus furiosus, Methanococcus jannaschii, and Methanobacterium thermoautotrophicum. The optimal substrate of these enzymes resembles DNA undergoing strand displacement synthesis and consists of a bifurcated downstream duplex with a directly abutted upstream duplex that overlaps the downstream duplex by one base pair. That single base of overlap causes the enzymes to leave a nick after cleavage and to cleave several orders of magnitude faster than a substrate that lacks overlap. The downstream duplex needs to be 10 base pairs long or greater for most of the enzymes to cut efficiently. The upstream duplex needs to be only 2 or 3 base pairs long for most enzymes, and there appears to be interaction with the last base of the primer strand. Overall, the enzymes display very similar substrate specificities, despite their limited level of sequence similarity.