Bent DNA Structures Research Articles

Analysis of structural flexibility of damaged DNA using thiol-tethered oligonucleotide duplexes.

Bent structures are formed in DNA by the binding of small molecules or proteins. We developed a chemical method to detect bent DNA structures. Oligonucleotide duplexes in which two mercaptoalkyl groups were attached to the positions facing each other across the major groove were prepared. When the duplex contained the cisplatin adduct, which was proved to induce static helix bending, interstrand disulfide bond formation under an oxygen atmosphere was detected by HPLC analyses, but not in the non-adducted duplex, when the two thiol-tethered nucleosides were separated by six base pairs. When the insert was five and seven base pairs, the disulfide bond was formed and was not formed, respectively, regardless of the cisplatin adduct formation. The same reaction was observed in the duplexes containing an abasic site analog and the (6–4) photoproduct. Compared with the cisplatin case, the disulfide bond formation was slower in these duplexes, but the reaction rate was nearly independent of the linker length. These results indicate that dynamic structural changes of the abasic site- and (6–4) photoproduct-containing duplexes could be detected by our method. It is strongly suggested that the UV-damaged DNA-binding protein, which specifically binds these duplexes and functions at the first step of global-genome nucleotide excision repair, recognizes the easily bendable nature of damaged DNA.

Transcription Factor-dependent DNA Bending Governs Promoter Recognition by the Mitochondrial RNA Polymerase

Promoter recognition is the first and the most important step during gene expression. Our studies of the yeast (Saccharomyces cerevisiae) mitochondrial (mt) transcription machinery provide mechanistic understandings on the basic problem of how the mt RNA polymerase (RNAP) with the help of the initiation factor discriminates between promoter and non-promoter sequences. We have used fluorescence-based approaches to quantify DNA binding, bending, and opening steps by the core mtRNAP subunit (Rpo41) and the transcription factor (Mtf1). Our results show that promoter recognition is not achieved by tight and selective binding to the promoter sequence. Instead, promoter recognition is achieved by an induced-fit mechanism of transcription factor-dependent differential conformational changes in the promoter and non-promoter DNAs. While Rpo41 induces a slight bend upon binding both the DNAs, addition of the Mtf1 results in severe bending of the promoter and unbending of the non-promoter DNA. Only the sharply bent DNA results in the catalytically active open complex. Such an induced-fit mechanism serves three purposes: 1) assures catalysis at promoter sites, 2) prevents RNA synthesis at non-promoter sites, and 3) provides a conformational state at the non-promoter sites that would aid in facile translocation to scan for specific sites.

Wormlike Chain Theory and Bending of Short DNA

The probability distributions for bending angles in double helical DNA obtained in all-atom molecular dynamics simulations are compared with theoretical predictions. The computed distributions remarkably agree with the wormlike chain theory and qualitatively differ from predictions of the subelastic chain model. The computed data exhibit only small anomalies in the apparent flexibility of short DNA and cannot account for the recently reported AFM data. It is possible that the current atomistic DNA models miss some essential mechanisms of DNA bending on intermediate length scales. Analysis of bent DNA structures reveal, however, that the bending motion is structurally heterogeneous and directionally anisotropic on the length scales where the experimental anomalies were detected. These effects are essential for interpretation of the experimental data and they also can be responsible for the apparent discrepancy.

DNA Bending versus DNA End Joining Activity of HMGB1 Protein Is Modulated in Vitro by Acetylation

The ability of HMGB1 protein to recognize bent DNA and to induce bending in linear duplex DNA defines HMGB1 as an architectural factor. It has already been demonstrated that the binding affinity of the protein for various bent DNA structures is enhanced upon in vivo acetylation at Lys2. Here we investigate how this modification of HMGB1 affects its ability to bend DNA. We report that the modified protein cannot bend short DNA fragments but, instead, stimulates joining of the same fragments via their ends. The same properties are exhibited in vivo by acetylated HMGB1 lacking its acidic tail. Further, in vitro acetylation of the truncated protein at Lys81 (possible upon tail removal only) restores the protein's bending ability, while the level of stimulation of DNA end joining is strongly reduced. We conclude, therefore, that the ability of HMGB1 to bend DNA or to stimulate end joining is modulated in vitro by acetylation. In an attempt to explain the properties of in vivo-acetylated HMGB1, its complexes with DNA have been analyzed by both protein-DNA cross-linking and atomic force microscopy. Unlike the parental protein, bound mainly within the internal sequences, acetylated HMGB1 binds preferentially to DNA ends. We propose that the loading of acetylated protein on DNA ends accounts for both the failure to bend DNA and the stimulation of DNA end joining.

Spontaneous sharp bending of DNA: role of melting bubbles

The role of centrally located and distributed base pair mismatches (‘melting bubbles’) on localized bending and stiffness of short dsDNA fragments is evaluated using time-dependent fluorescence lifetime measurements. Distributed melting bubbles are found to induce larger bending angles and decreased levels of stiffness in DNA than centrally located ones of comparable overall size. Our results indicate that spontaneous local opening-up of the DNA duplex could facilitate sharp bending of short DNA strands even in the absence of DNA binding proteins. We also find that the occurrence of two closely spaced melting bubbles will generally be favored when a large energetic barrier must be overcome in forming the desired bent DNA structure.

Linearization and integration of DNA into cells preferentially occurs at intrinsically curved regions from human LINE-1 repetitive element

A bent DNA library was constructed from human genomic DNA, from which a new clone belonging to the human LINE-1 sequence family was isolated and characterized. This clone, with a length of 378 base pairs and termed HBC-1 (human bent clone-1), contained an intrinsically occurring curved DNA structure. By permutation analysis, the center of curvature of this fragment was mapped onto the nucleotide position 886 from the 5′ terminus of the complete LINE-1 sequence. Reporter plasmids, which contain HBC-1, were effectively integrated into human chromosome, indicating that the bent DNA structure provides a preferential donor site for the integration of human LINE-1 sequences. The present finding may provide an explanation as to why some inactivated LINE-1 sequences on human chromosomes carry the deletion at their 5′ termini.

Intrinsic DNA bends: an organizer of local chromatin structure for transcription.

DNA with a curved trajectory of its helix axis is called bent DNA, or curved DNA. Interestingly, biologically important DNA regions often contain this structure, irrespective of the origin of DNA. In the last decade, considerable progress has been made in clarifying one role of bent DNA in prokaryotic transcription and its mechanism of action. However, the role of bent DNA in eukaryotic transcription remains unclear. Our recent study raises the possibility that bent DNA is implicated in the "functional packaging" of transcriptional regulatory regions into chromatin. In this article, I review recent progress in bent DNA research in eukaryotic transcription, and summarize the history of bent DNA research and several subjects relevant to this theme. Finally, I propose a hypothesis that bent DNA structures that mimic a negative supercoil, or have a right-handed superhelical writhe, organize local chromatin infrastructure to help the very first interaction between cis-acting DNA elements and activators that trigger transcription.

A common feature shared by bent DNA structures locating in the eukaryotic promoter region.

Eukaryotic promoters often contain a bent DNA structure, suggesting that this structure plays some role in transcription. To reveal the role, we need more information on the promoters that contain or flank a bent DNA structure. In this study, we collected such promoters by the following approach: we first isolated human genomic DNA fragments that contained at least one bent DNA structure, then shotgun cloned them into a promoter trap vector, screened DNA fragments that functioned as a promoter, and finally found the promoters of interest by determining the bent DNA locus and the region expressing promoter activity. From 1,187 recombinant plasmids, we isolated 51 that showed promoter activity. Structural and functional analyses of randomly selected 10 clones with inserts of 548-913 bp demonstrated 11 sequences that could drive transcription. Unexpectedly, all of these clones met our purpose: i.e., each segment that showed a promoter activity (67-179 bp) was very close to the bent DNA structure (spanning about 150 bp in all clones), and in some cases overlapped it. More interestingly, these bent DNA structures all had a superhelical writhe. We propose a hypothesis that in the bent-DNA-containing eukaryotic promoters. bent DNA organizes local chromatin infrastructure appropriately for transcription initiation.

Effect of upstream DNA architecture on transcription of a human LINE-1 retrotransposon sequence.

We have cloned a human genomic DNA fragment that contains a complex bent DNA structure and a LINE-1 (L1) sequence. The bent DNA structure spans from 70 to -440 relative to the transcription start site of L1, and contains a left-handed sup erhelical structure from about 70 to -200. When we changed the rotational orientation of the bent DNA relative to the L1 promoter by inserting double-stranded oligonucleotides between positions -3 and -4, L1 promoter activity was profoundly affected Inserts of 5 and 16 by altered the rotational position by about 180°, and stimulated transcription 2- to 3-fold Insertions of 11 and 21 base pairs, which altered the rotational orientati on minimally, had much less effect. We present here the first experimental evidence that upstream DNA architecture influences the machinery employed in L1 transcription.

HMG-Domain Protein Recognition of Cisplatin 1,2-Intrastrand d(GpG) Cross-Links in Purine-Rich Sequence Contexts

HMG-domain proteins bind strongly to bent DNA structures, including cruciform and cisplatin-modified duplexes. Such protein-platinated DNA complexes, formed where the DNA is modified by the active cis but not the inactive trans isomer of diamminedichloroplatinum(II), are implicated in the cytotoxic mechanism of the drug. A series of oligonucleotide duplexes with deoxyguanosine nucleosides flanking a cis-[Pt(NH(3))(2)¿d(GpG)-N7(1),-N7(2)¿] cross-link have been synthesized. These probes were used to determine the flanking sequence dependence of the affinity of the individual HMG domains of HMG1 toward cisplatin-modified DNA. Nine related sequences, where N(1) and N(2) are not dG and GG is the 1,2-intrastrand cisplatin adduct in N(1)GGN(2), were previously investigated [Dunham, S. U., and Lippard, S. J. (1997) Biochemistry 36, 11428-11436]. Three of the seven remaining possible sequences for which N(1) and/or N(2) was dG were prepared here by using normal deoxyguanosine, but the rest, where N(1) is dG and N(2) is dA, dC, T, or dG, could not be isolated in pure form. These sequences were accessed by using the synthetic bases 7-deazaadenine and 7-deazaguanine, which lack the nucleophilic N7 atom in the purine ring. Deaza nucleotides accurately mimic the properties of the natural bases, allowing the interaction of the HMG-domain proteins with cisplatin-modified DNA to be examined. These experiments reveal that the flexibility of A.T versus G.C flanking base pairs, rather than base-specific contacts, determines HMG1domA protein selectivity. This conclusion was supported by use of mutant HMG1domA and HMG1domB proteins, which exhibit identical flanking sequence selectivity. The methods and results obtained here not only improve our understanding of how proteins might mediate cisplatin genotoxicity but also should apply more generally in the investigation of how other proteins interact with damaged DNA.

The unusual structures of the hot-regions flanking large-scale deletions in human mitochondrial DNA.

Large-scale deletions of mitochondrial DNA (mtDNA) are common events that have been found to occur in human ageing and in patients with mitochondrial myopathies. The mechanisms by which these deletions occur remain unclear, but several mechanisms have been proposed, such as slipped-mispairing, illegitimate recombination, and oxidative reactions elicited by free radicals. In addition, the DNA topological stress and local DNA structures have been suggested as the important factors in eliciting the recombinational events. Upon examination of 128 breakpoints of human mtDNA deletions that have been published in the past 8 years, we found that these large-scale deletions often occur at some 'hot-regions'. We thus hypothesized that there exist unusual structures in these regions of human mtDNA that are important for eliciting the deletions. To test this hypothesis, we used PCR techniques to amplify the sequences of the so-called hot-regions and analysed the PCR products by two-dimensional gel electrophoresis. We found that the sequences of nucleotide position (np) 5221-5988, np 6928-7493, np 7901-8732 and np 15327-16228 exhibited retarded mobilities like bent DNA structures; np 5989-6750, np 13282-13653 and np 13282-14850 showed increased mobilities like anti-bent DNA structures. Moreover, except for the sequences of np 1175-1766 found in 12 S and 16 S rRNA genes exhibiting abnormal mobility like bent DNA structures, we did not observe significant mobility abnormalities in the np 499-5545 region where deletions rarely occurred. We thus conclude that these hot-regions assume some kinds of unusual DNA structures, which may render these regions more sensitive to the attack of free radicals or serve as recognition motifs for certain recombination machinery that is involved in the large-scale deletions of human mtDNA.

Solution structure of a two-base DNA bulge complexed with an enediyne cleaving analog.

Nucleic acid bulges have been implicated in a number of biological processes and are specific cleavage targets for the enediyne antitumor antibiotic neocarzinostatin chromophore in a base-catalyzed, radical-mediated reaction. The solution structure of the complex between an analog of the bulge-specific cleaving species and an oligodeoxynucleotide containing a two-base bulge was elucidated by nuclear magnetic resonance. An unusual binding mode involves major groove recognition by the drug carbohydrate unit and tight fitting of the wedge-shaped drug in the triangular prism pocket formed by the two looped-out bulge bases and the neighboring base pairs. The two drug rings mimic helical DNA bases, complementing the bent DNA structure. The putative abstracting drug radical is 2.2 +/- 0.1 angstroms from the pro-S H5' of the target bulge nucleotide. This structure clarifies the mechanism of bulge recognition and cleavage by a drug and provides insight into the design of bulge-specific nucleic acid binding molecules.

A Role for a Bent DNA Structure in E2F-Mediated Transcription Activation

We examined the role of promoter architecture, as well as that of the DNA-bending capacity of the E2F transcription factor family, in the activation of transcription. DNA phasing analysis revealed that a consensus E2F site in the E2F1 promoter possesses an inherent bend with a net magnitude of 40 +/-2 degrees and with an orientation toward the major groove relative to the center of the E2F site. The inherent DNA bend is reversed upon binding of E2F, generating a net bend with a magnitude of 25 +/- 3 degrees oriented toward the minor groove relative to the center of the E2F site. We also found that three members of the E2F family, in conjunction with the DP1 protein, bend the DNA toward the minor groove, suggesting that DNA bending is a characteristic of the entire E2F family. The Rb-E2F complex, on the other hand, does not reverse the intrinsic DNA bend. Analysis of a series of E2F1 deletion mutants defined E2F1 sequences which are not required for DNA binding but are necessary for the DNA-bending capacity of E2F. An internal region of E2F1, previously termed the marked box, which is highly homologous among E2F family members, was particularly important in DNA bending. We also found that a bent DNA structure can be a contributory component in the activation of the E2F1 promoter but is not critical in the repression of that promoter in quiescent cells. This finding suggests that E2F exhibits characteristics typical of modular transcription factors, with independent DNA-binding and transcriptional activation functions, but also has features of architectural factors that alter DNA structure.

Regulatory noise in prokaryotic promoters: how bacteria learn to respond to novel environmental signals.

Various features of the regulation of pathways for biodegradation of recalcitrant compounds by Pseudomonas provide insights into the mechanisms by which operons evolve to acquire conditionally active promoters that permit the corresponding genes to be transcribed only when required. The "regulatory noise hypothesis' proposes that transcriptional control systems develop responsiveness to new signals due to the leakiness and lack of specificity of preexisting promoters and regulators. When needed, these may become more specific through suppression of undesirable signals and further fine-tuning of the recruited proteins to interact with distinct chemicals. This hypothesis is supported by the sophisticated regulation of sigma 54-dependent promoters of the TOL (toluene biodegradation) operons, which can be activated to various degrees by heterologous proteins. Such "illegitimate' activation is suppressed by bent DNA structures, either static or protein induced, between promoter core elements. Therefore, not only the regulators but also the DNA sequences participate in the process that gives rise to novel specificities.

Integration host factor suppresses promiscuous activation of the sigma 54-dependent promoter Pu of Pseudomonas putida.

In the presence of m-xylene, the Pu promoter of the TOL plasmid of Pseudomonas putida is activated by the prokaryotic enhancer-binding protein XylR. The intervening DNA segment between the upstream activating sequences (UASs) and those for RNA polymerase binding contains an integration host factor (IHF) attachment site that is required for full transcriptional activity. In the absence of IHF, the Pu promoter can be cross-activated by other members of the sigma 54-dependent family of regulatory proteins. Such illegitimate activation does not require the binding of the heterologous regulators to DNA and it is suppressed by bent DNA structures, either static or protein induced, between the promoter core elements (UAS and RNA polymerase recognition sequence). The role of IHF in some sigma 54 promoters is, therefore, not only a structural aid for assembling a correct promoter geometry but also that of an active suppressor (restrictor) of promiscuous activation by heterologous regulators for increased promoter specificity.

An intrinsically bent region upstream of the transcription start site of the rRNA genes of Arabidopsis thaliana interacts with an HMG-related protein.

Intrinsically bent DNA structures are thought to play crucial roles as functionally important modules in promoter/enhancer elements. Here we show the existence of a DNA bending centre within the intergenic region of the tandemly arranged rRNA genes of Arabidopsis thaliana. By use of a circular permutation assay, this bending centre was localized to position -284 to -256 upstream of the transcription start site. A 143 bp fragment containing the bent region was shown to interact with several nuclear proteins. Evidence is presented that one of these proteins is related to the high-mobility-group (HMG) proteins, a group thought to be involved in transcription and replication processes.

The maize chromosomal HMGa protein recognizes structural features of DNA and increases DNA flexibility.

The abundant maize high-mobility group protein HMGa belongs to the chromosomal, non-histone proteins and consists of a basic region containing the HMG-box DNA-binding domain and a highly acidic carboxy-terminal tail. The full-length HMGa protein and a truncated version lacking the acidic tail were synthesized in Escherichia coli and tested for their ability to induce DNA-bending in a ligase mediated circularization assay with short DNA fragments. It is shown that the recombinant HMGa protein as well as its truncated form efficiently cause circularization of the tested DNA fragments without an obvious requirement for stable DNA-binding. They bind furthermore preferentially to A/T-rich linear DNA or bent DNA structures such as four-way junctions and DNA minicircles. The DNA-binding properties and the ability to increase DNA flexibility suggest a general role of the HMGa protein in assisting the formation of nucleoprotein complexes, possibly by facilitating interactions of proteins bound to adjacent DNA sites.

Distribution of bent DNA structures in the fission yeast centromere

To gain a clue as to the functional significance of DNA curvature, we experimentally characterized the distribution of bent DNA structures throughout the 35-kb cen1 sequence, one of the isolated functional centromeric DNA of the fission yeast, Schizosaccharomyces pombe. It was revealed that a relatively large central portion of cen1, covering a 2.2kb DNA sequence, displays a remarkable DNA curvature.

A highly conserved repetitive sequence from Physarum polycephalum contains nucleotide arrangements similar to replicator sequences

An interspersed repetitive sequence from Physarum polycephalum has been cloned and analysed. The 394 bp sequence is highly conserved and contains several homopolymeric (dA)-(dT) tracts capable of forming bent DNA structures and a 10/11 match to the yeast-ARS-consensus sequence. The repetition frequency of the described sequence is about 3000 to 7000, a number that would fit with the distribution of replicator segments in Physarum.

Abolition of intrinsically bent DNA structure components in AT clusters by netropsin interaction; titration viscometric investigations.

It is argued that the enhancement of the apparent DNA contour length by the specifically binding non-intercalating drug netropsin (Nt) (Reinert et al., NAR 9, 2335, 1981) at very low Nt/DNA-phosphate ratios essentially is the result of an abolition of periodically arranged intrinsic helix bends in A.T rich tracts of base pairs. In the preceding paper the existence of pronounced DNA tertiary structure components has been postulated for (two species of) natural eukaryotic DNA. The resulting model suggests local apparent solenoid-related DNA tertiary structure components at high sodium ion concentration cs, partly/totally molten out at 45/60 C. With decreasing cs the tertiary structure components have been found to be gradually reduced, at least below cs = 0.010 M, as titration viscometrically revealed by a gradual rise of the apparent DNA contour length (Reinert et al., JBSD 9, 537, 1991). Hence, we performed titration viscometric analyses about Nt interaction with calf thymus DNA (ctDNA) at cs = 0.075 M, 0.010 M and 0.004 M Na+. The concomitant DNA conformational changes are quantitatively described in terms of the relative changes of both DNA persistence length and hydrodynamically operative apparent DNA contour length for the three first resolved interaction modes below a Nt/DNA-P ratio of 0.03. These experiments, together with previous respective analyses at cs = 0.20 M Na+ and different temperatures (l.c.), suggest that those DNA sites binding Nt most strongly predominantly are responsible for the formation of solenoid-related DNA tertiary structure components. Most probably these are A tract-containing sequences. As the essential factor for their apparent elongation effect at low Na+ concentrations, a gradual alteration of the number of base pairs per helix turn seems to occur below cs = 0.010 M Na+ and, concomitantly, a change in phasing between intrinsic helix bends and helix screw.