Osmium tetroxide probing of local DNA structure in linear and supercoiled plasmids containing curvature-inducing sequences

Recombinant plasmids pK1A108, pK3A108, pK4A108 and pK5/6T217 containing 80 +/- 1 base pair inserts with different curvature-inducing sequences were studied using the DNA structure probe osmium tetroxide in the presence of pyridine (Os, py). The insertion sequences of the plasmids pK1A108, pK3A108, and pK4A108 are strongly related while the degree of curvature increases from pK1A108 (no curvature) less than pK3A108 less than pK4A108 less than pK5/6T217. The Os, py probe reacts selectively with single-stranded and distorted double-stranded regions in the DNA double helix. Nuclease S1 was used to recognize and cleave regions made permanently single-stranded due to osmium recognize and cleave regions made permanently single-stranded due to osmium modification. In linearized plasmids treatment with Os, py produced no S1-detectable site-specific modification. This result is in agreement with models suggested for DNA curvature; in general, continuous base pairing and base stacking is considered through different sequence blocks as well as through structural junctions. Os, py-probing of the plasmids in the supercoiled state also resulted in no S1-detectable site-specific modification within the inserts of pK1A108, pK3A108, and pK4A108 plasmids (while the regions containing inverted repeat nucleotide sequences in these plasmids were site-specifically modified). In contrast, supercoiled pK5/6T217 DNA was site-specifically modified within the curvature-inducing insert sequence. The nucleotide sequence of the insert of this plasmid strongly differs from the insertion sequences of the other three plasmids; it is extremely AT-rich and contains regularly arranged dAGAGA and dATATA sequences. The structural distortion observed in supercoiled pK5/6T217 is most probably due to the presence of these sequences in a particular arrangement in the insertion sequence.     

Intrinsic curvature of DNA influences LacR-mediated looping

Protein-mediated DNA looping is a common mechanism for regulating gene expression. Loops occur when a protein binds to two operators on the same DNA molecule. The probability of looping is controlled, in part, by the basepair sequence of inter-operator DNA, which influences its structural properties. One structural property is the intrinsic or stress-free curvature. In this article, we explore the influence of sequence-dependent intrinsic curvature by exercising a computational rod model for the inter-operator DNA as applied to looping of the LacR-DNA complex. Starting with known sequences for the inter-operator DNA, we first compute the intrinsic curvature of the helical axis as input to the rod model. The crystal structure of the LacR (with bound operators) then defines the requisite boundary conditions needed for the dynamic rod model that predicts the energetics and topology of the intervening DNA loop. A major contribution of this model is its ability to predict a broad range of published experimental data for highly bent (designed) sequences. The model successfully predicts the loop topologies known from fluorescence resonance energy transfer measurements, the linking number distribution known from cyclization assays with the LacR-DNA complex, the relative loop stability known from competition assays, and the relative loop size known from gel mobility assays. In addition, the computations reveal that highly curved sequences tend to lower the energetic cost of loop formation, widen the energy distribution among stable and meta-stable looped states, and substantially alter loop topology. The inclusion of sequence-dependent intrinsic curvature also leads to nonuniform twist and necessitates consideration of eight distinct binding topologies from the known crystal structure of the LacR-DNA complex.     

Centromere parC of plasmid R1 is curved

The centromere sequence parC of Escherichia coli low-copy-number plasmid R1 consists of two sets of 11 bp iterated sequences. Here we analysed the intrinsic sequence-directed curvature of parC by its migration anomaly in polyacrylamide gels. The 159 bp long parC is strongly curved with anomaly values (k-factors) close to 2. The properties of the parC curvature agree with those of other curved DNA sequences. parC contains two regions of 5-fold repeated iterons separated by 39 bp. We modified 4 bp within this intermediate sequence so that we could analyse the two 5-fold repeated regions independently. The analysis shows that the two repeat regions are not independently curved parts of parC but that the overall curvature is a property of the whole fragment. Since the centromere sequence of an E.coli plasmid as well as eukaryotic centromere sequences show DNA curvature, we speculate that curvature might be a general property of centromeres.     

Scaffold/matrix attachment regions and intrinsic DNA curvature

Recent approaches have failed to detect nucleotide sequence motifs in Scaffold/Matrix Attachment Regions (S/MARs). The lack of any known motifs, together with the confirmation that some S/MARs are not associated to any peculiar sequence, indicates that some structural elements, such as DNA curvature, have a role in chromatin organization and on their efficiency in protein binding. Similar to DNA curvature, S/MARs are located close to promoters, replication origins, and multiple nuclear processes like recombination and breakpoint sites. The chromatin structure in these regulatory regions is important to chromosome organization for accurate regulation of nuclear processes. In this article we review the biological importance of the co-localization between bent DNA sites and S/MARs. Published in Russian in Biokhimiya, 2006, Vol. 71, No. 5, pp. 598–606.     

Sequence definition and organization of a human repeated DNA

DNA sequence data for a DNA repeated sequence, found largely in centromeres of specific human chromosomes is presented. The sequence consists of two tandem 169 and 171 base-pair units that show 27% base variation with each other. In contrast the dimer is more faithfully copied in longer tandem repeats, such as the sequenced 680 base-pair tetramer. In the major sequence of the tetramer, base variation of the order of only 1%, in comparison to the complete dimer is seen. A minimum of two steps in the formation of this sequence is proposed, consisting of evolution of a tandem dimer of two 170 base-pair variant units of a related family within the human genome, and later saltation or amplification of this dimer. No evidence that these sequences contained or evolved from a simpler 6 to 20 base-pair repeat was found, and no homology with known simpler human satellites could be discerned. In reviewing and comparing the literature on repeated DNAs it appears that overall length and tandem repetition are the critical features, rather than individual unit repeat length or secondary structural potential, in defining these sequences as a class and their special centromeric functions and higher chromosome order. The possibility that such sequences arise from a reservoir of interspersed sequences that are common to at least several species is discussed.     

A-tract clusters may facilitate DNA packaging in bacterial nucleoid

Molecular mechanisms of bacterial chromosome packaging are still unclear, as bacteria lack nucleosomes or other apparent basic elements of DNA compaction. Among the factors facilitating DNA condensation may be a propensity of the DNA molecule for folding due to its intrinsic curvature. As suggested previously, the sequence correlations in genome reflect such a propensity [Trifonov and Sussman (1980) Proc. Natl Acad. Sci. USA, 77, 3816–3820]. To further elaborate this concept, we analyzed positioning of A-tracts (the sequence motifs introducing the most pronounced DNA curvature) in the Escherichia coli genome. First, we observed that the A-tracts are over-represented and distributed ‘quasi-regularly’ throughout the genome, including both the coding and intergenic sequences. Second, there is a 10–12 bp periodicity in the A-tract positioning indicating that the A-tracts are phased with respect to the DNA helical repeat. Third, the phased A-tracts are organized in ~100 bp long clusters. The latter feature was revealed with the help of a novel approach based on the Fourier series expansion of the A-tract distance autocorrelation function. Since the A-tracts introduce local bends of the DNA duplex and these bends accumulate when properly phased, the observed clusters would facilitate DNA looping. Also, such clusters may serve as binding sites for the nucleoid-associated proteins that have affinities for curved DNA (such as HU, H-NS, Hfq and CbpA). Therefore, we suggest that the ~100 bp long clusters of the phased A-tracts constitute the ‘structural code’ for DNA compaction by providing the long-range intrinsic curvature and increasing stability of the DNA complexes with architectural proteins.     

Point mutation in the polyomavirus enhancer alters local DNA conformation

A point mutation in the enhancer of polyomavirus host range mutant, PyEC F441, permits productive infection of the murine embryonal carcinoma cell line, F9. This mutation at nucleotide position 5258 introduces a local conformational change in naked viral DNA. The effect of all four possible nucleotide sequences at position 5258 on local DNA conformation was analyzed by gel electrophoresis of fragments produced by ligation of synthetic oligonucleotides having these sequences. The results indicated that both the wild-type and the F441 sequences introduced local structural polymorphism that can lead to DNA bending. The wild-type sequence had a greater effect on DNA curvature than the F441 sequence. The two other sequences at nucleotide 5258 did not appear to introduce detectable amounts of DNA curvature.     

An Escherichia coli protein that preferentially binds to sharply curved DNA

We attempted to find Escherichia coli proteins which preferentially bind to a curved DNA sequence even in the presence of an excess amount of a non-curved DNA sequence as a competitor, mainly by means of a DNA-binding gel retardation assay. Since the two sequences used had nearly the same nucleotide compositions, including consecutive dA5 stretches, we reasoned that this strategy would allow us to identify proteins which preferentially recognize an overall DNA curvature. We purified such a protein from E. coli. Its preferential binding to the curved DNA was found to be inhibited by distamycin, which removes the curvature from appropriate DNA sequences. The purified protein was identified as the E. coli nucleoid protein, H-NS.     

Tandem-repetitive noncoding DNA: forms and forces

A model of sequence-dependent, unequal crossing-over and gene amplification (slippage replication) has been stimulated in order to account for various structural features of tandemly repeated DNA sequences. It is shown that DNA whose sequence is not maintained by natural selection will exhibit repetitive patterns over a wide range of recombination rates as a result of the interaction of unequal crossing-over and slippage replication, processes that depend on sequence similarity. At high crossing-over frequencies, the nucleotide patterns generated in the simulations are simple and highly regular, with short, nearly identical sequences repeated in tandem. Decreasing recombination rates increase the tendency to longer and more-complex repeat units. Periodicities have been observed down to very low recombination rates (one or more orders of magnitude lower than mutation rate). At such low rates, most of the sequences contain repeats which have an extensive substructure and a high degree of heterogeneity among each other; often higher-order structures are superimposed on a tandem array. These results are compared with various structural properties of tandemly repeated DNAs known from eukaryotes, the spectrum ranging from simple-sequence DNAs, particularly the hypervariable mini-satellites, to the classical satellite DNAs, located in chromosomal regions of low recombination, e.g., heterochromatin.     

Evolutionary force of AT‐rich repeats to trap genomic and episomal DNAs into the rice genome: lessons from endogenous pararetrovirus

In plant genomes, the incorporation of DNA segments is not a common method of artificial gene transfer. Nevertheless, various segments of pararetroviruses have been found in plant genomes in recent decades. The rice genome contains a number of segments of endogenous rice tungro bacilliform virus‐like sequences (ERTBVs), many of which are present between AT dinucleotide repeats (ATrs). Comparison of genomic sequences between two closely related rice subspecies, japonica and indica, allowed us to verify the preferential insertion of ERTBVs into ATrs. In addition to ERTBVs, the comparative analyses showed that ATrs occasionally incorporate repeat sequences including transposable elements, and a wide range of other sequences. Besides the known genomic sequences, the insertion sequences also represented DNAs of unclear origins together with ERTBVs, suggesting that ATrs have integrated episomal DNAs that would have been suspended in the nucleus. Such insertion DNAs might be trapped by ATrs in the genome in a host‐dependent manner. Conversely, other simple mono‐ and dinucleotide sequence repeats (SSR) were less frequently involved in insertion events relative to ATrs. Therefore, ATrs could be regarded as hot spots of double‐strand breaks that induce non‐homologous end joining. The insertions within ATrs occasionally generated new gene‐related sequences or involved structural modifications of existing genes. Likewise, in a comparison between Arabidopsis thaliana and Arabidopsis lyrata, the insertions preferred ATrs to other SSRs. Therefore ATrs in plant genomes could be considered as genomic dumping sites that have trapped various DNA molecules and may have exerted a powerful evolutionary force.     

DNA methylation can enhance or induce DNA curvature.

Oligomers of different palindromic sequences (EcoRI, BamHI, and ClaI linkers) were ligated to form distributions of multimers. These long ligation ladders were methylated using corresponding methylases. The migration of the unmethylated as well as the methylated multimer distributions was analysed in 10% polyacrylamide gels. The migration anomaly of these sequences is interpreted in terms of the curvature of the DNA helix axis. The double-stranded oligomer dCGGAATTCCG is considerably curved in its unmodified form. Its curvature is strongly enhanced when the central dAs are methylated. This result is predicted by a model for DNA curvature. Multimers of the closely related sequence dCGGGATCCCG are straight. When methylated at the central dAs or at the most central dCs, a small curvature of the helix axis is induced. The double-stranded oligomer dCCATCGATGG is straight in its unmodified form as well as when it is methylated. Thus, DNA curvature can be induced or enhanced by methylation. However, DNA methylation at palindromic sequences seems not always to influence the linear path of the DNA helix axis.     

48 NMR study of the interaction between G-quadruplexes and small molecules

Guanine-rich oligonucleotides are able to adopt secondary DNA structures, known as G-quadruplexes. Such G-rich sequences are found in human telomeres, promoter regions of oncogenes, 5′ untranslated regions (UTRs) of mRNAs and human intronic sequences. Studies have shown that small molecules can induce anti-cancer effect through stabilizing or promoting G-quadruplex formation. In order to design and develop a potent drug, structural details on the interaction between small molecules and G-quadruplexes are invaluable. In this study, we seek to understand the structural determinants involved in the interaction between G-quadruplexes and small molecules. NMR spectroscopy is employed to resolve the structures of two intramolecular G-quadruplexes bound to small molecules. These resolved complexes allow us to structurally design new potent drugs for their application in anti-cancer therapy.     

Exposition of a family of RNA m(5)C methyltransferases from searching genomic and proteomic sequences.

The Escherichia coli fmu gene product has recently been determined to be the 16S rRNA m(5)C 967 methyltransferase. As such, Fmu represents the first protein identified as an S -adenosyl-L-methionine (AdoMet)- dependent RNA m(5)C methyltransferase whose amino acid sequence is known. Using the amino acid sequence of Fmu as an initial probe in an iterative search of completed DNA sequence databases, 27 homologous ORF products were identified as probable RNA m(5)C methyltransferases. Further analysis of sequences in undeposited genomic sequencing data and EST databases yielded more than 30 additional homologs. These putative RNA m(5)C methyltransferases are grouped into eight subfamilies, some of which are predicted to consist of direct genetic counterparts, or orthologs. The enzymes proposed to be RNA m(5)C methyltransferases have sequence motifs closely related to signature sequences found in the well-studied DNA m(5)C methyltransferases and other AdoMet-dependent methyltransferases. Structure-function correlates in the known AdoMet methyltransferases support the assignment of this family as RNA m(5)C methyltransferases.     

GenBank.

The GenBank sequence database continues to expand its data coverage, quality control, annotation content and retrieval services. GenBank is comprised of DNA sequences submitted directly by authors as well as sequences from the other major public databases. An integrated retrieval system, known as Entrez, contains data from GenBank and from the major protein sequence and structural databases, as well as related MEDLINE abstracts. Users may access GenBank over the Internet through the World Wide Web and through special client-server programs for text and sequence similarity searching. FTP, CD-ROM and e-mail servers are alternate means of access.