Unusual DNA Structure Research Articles

Chromatin Pull-Down Methodology Based on DNA Triple Helix Formation.

Identification of the protein complexes associated with defined DNA sequence elements is essential to understand the numerous transactions in which DNA is involved, such as replication, repair, transcription, and chromatin dynamics. Here we describe two protocols, IDAP (Isolation of DNA Associated Proteins) and CoIFI (Chromatin-of-Interest Fragment Isolation), that allow for isolating DNA/protein complexes (i.e., nucleoprotein elements) by means of a DNA capture tool based on DNA triple helix (triplex) formation. Typically, IDAP is used to capture proteins that bind to a given DNA element of interest (e.g., a specific DNA sequence, an unusual DNA structure, a DNA lesion) that can be introduced at will into plasmids. The plasmids are immobilized by means of a triplex-forming probe on magnetic beads and incubated in nuclear extracts; by using in parallel a control plasmid (that lacks the DNA element of interest), proteins that preferentially bind to the DNA element of interest are captured and identified by mass spectrometry. Similarly, CoIFI also uses a triplex-forming probe to capture a specific chromatin fragment from a cultured cell line that has been engineered to contain multiple copies of the DNA element of interest.

5-Formylcytosine does not change the global structure of DNA.

The mechanism by which the recently identified DNA modification 5-formylcytosine (fC) is recognized by epigenetic writer and reader proteins is not known. Recently, an unusual DNA structure, F-DNA, has been proposed as the basis for enzyme recognition of clusters of fC. We used NMR and X-ray crystallography to compare several modified DNA duplexes with unmodified analogs and found that in the crystal state the duplexes all belong to the A family, whereas in solution they are all members of the B family. We found that, contrary to previous findings, fC does not significantly affect the structure of DNA, although there are modest local differences at the modification sites. Hence, global conformation changes are unlikely to account for the recognition of this modified base, and our structural data favor a mechanism that operates at base-pair resolution for the recognition of fC by epigenome-modifying enzymes.

Twisting right to left: A…A mismatch in a CAG trinucleotide repeat overexpansion provokes left-handed Z-DNA conformation.

Conformational polymorphism of DNA is a major causative factor behind several incurable trinucleotide repeat expansion disorders that arise from overexpansion of trinucleotide repeats located in coding/non-coding regions of specific genes. Hairpin DNA structures that are formed due to overexpansion of CAG repeat lead to Huntington’s disorder and spinocerebellar ataxias. Nonetheless, DNA hairpin stem structure that generally embraces B-form with canonical base pairs is poorly understood in the context of periodic noncanonical A…A mismatch as found in CAG repeat overexpansion. Molecular dynamics simulations on DNA hairpin stems containing A…A mismatches in a CAG repeat overexpansion show that A…A dictates local Z-form irrespective of starting glycosyl conformation, in sharp contrast to canonical DNA duplex. Transition from B-to-Z is due to the mechanistic effect that originates from its pronounced nonisostericity with flanking canonical base pairs facilitated by base extrusion, backbone and/or base flipping. Based on these structural insights we envisage that such an unusual DNA structure of the CAG hairpin stem may have a role in disease pathogenesis. As this is the first study that delineates the influence of a single A…A mismatch in reversing DNA helicity, it would further have an impact on understanding DNA mismatch repair.

APTE: identification of indirect read-out A-DNA promoter elements in genomes.

BackgroundTranscriptional regulation is normally based on the recognition by a transcription factor of a defined base sequence in a process of direct read-out. However, the nucleic acid secondary and tertiary structure can also act as a recognition site for the transcription factor in a process known as indirect read-out, although this is much less understood. We have previously identified such a transcriptional control mechanism in early Xenopus development where the interaction of the transcription factor ilf3 and the gata2 promoter requires the presence of both an unusual A-form DNA structure and a CCAAT sequence. Rapid identification of such promoters elsewhere in the Xenopus and other genomes would provide insight into a less studied area of gene regulation, although currently there are few tools to analyse genomes in such ways.ResultsIn this paper we report the implementation of a novel bioinformatics approach that has identified 86 such putative promoters in the Xenopus genome. We have shown that five of these sites are A-form in solution, bind to transcription factors and fully validated one of these newly identified promoters as interacting with the ilf3 containing complex CBTF. This interaction regulates the transcription of a previously uncharacterised downstream gene that is active in early development.ConclusionsA Perl program (APTE) has located a number of potential A-form DNA promotor elements in the Xenopus genome, five of these putative targets have been experimentally validated as A-form and as targets for specific DNA binding proteins; one has also been shown to interact with the A-form binding transcription factor ilf3. APTE is available from http://www.port.ac.uk/research/cmd/software/ under the terms of the GNU General Public License.

G4 motifs in human genes

The G4 motif, G(≥3) N(x) G(≥3) N(x) G(≥3) N(x) G(≥3) , is enriched in some genomic regions and depleted in others. This motif confers the ability to form an unusual four-stranded DNA structure, G4 DNA. G4 DNA is associated with genomic instability, which may explain depletion of G4 motifs from some genes and genomic regions. Conversely, G4 motifs are enriched downstream of transcription start sites, where they correlate with pausing. The uneven distribution of G4 motifs in the genome strongly suggests that mechanisms of selection act not only on one-dimensional genomic sequence, but also on structures formed by genomic DNA. The biological roles of G4 structures illustrate that, to understand genome function, it is important to consider the dynamic structural potential implicit in the G4 motif.

Chemical Shifts for the Unusual DNA Structure in Pf1 Bacteriophage from Dynamic-Nuclear-Polarization-Enhanced Solid-State NMR Spectroscopy

Solid-state NMR spectra, including dynamic nuclear polarization enhanced 400 MHz spectra acquired at 100 K, as well as non-DNP spectra at a variety of field strengths and at temperatures in the range 213-243 K, have allowed the assignment of the (13)C and (15)N resonances of the unusual DNA structure in the Pf1 virion. The (13)C chemical shifts of C3' and C5', considered to be key reporters of deoxyribose conformation, fall near or beyond the edges of their respective ranges in available databases. The (13)C and (15)N chemical shifts of the DNA bases have above-average values for AC4, AC5, CC5, TC2, and TC5, and below average values for AC8, GC8, and GN2, pointing to an absence of Watson-Crick hydrogen bonding, yet the presence of some type of aromatic ring interaction. Crosspeaks between Tyr40 of the coat protein and several DNA atoms suggest that Tyr40 is involved in this ring interaction. In addition, these crosspeak resonances and several deoxyribose resonances are multiply split, presumably through the effects of ordered but differing interactions between capsid protein subunits and each type of nucleotide in each of the two DNA strands. Overall, these observations characterize and support the DNA model proposed by Liu and Day and refined by Tsuboi et al., which calls for the most highly stretched and twisted naturally occurring DNA yet encountered.

The CTCF insulator protein forms an unusual DNA structure

BackgroundThe CTCF insulator protein is a highly conserved zinc finger protein that has been implicated in many aspects of gene regulation and nuclear organization. The protein has been hypothesized to organize the human genome by forming DNA loops.ResultsIn this paper, we report biochemical evidence to support the role for CTCF in forming DNA loops. We have measured DNA bending by CTCF at the chicken HS4 β-globin FII insulator element in vitro and have observed a unique DNA structure with aberrant electrophoretic mobility which we believe to be a DNA loop. CTCF is able to form this unusual DNA structure at two other binding sites: the c-myc P2 promoter and the chicken F1 lysozyme gene silencer. We also demonstrate that the length though not the sequence of the DNA downstream of the binding site is important for the ability of CTCF to form this unusual DNA structure. We hypothesize that a single CTCF protein molecule is able to act as a "looper" possibly through the use of several of its zinc fingers.ConclusionsCTCF is able to form an unusual DNA structure through the zinc finger domain of the protein. This unusual DNA structure is formed in a directional manner by the CTCF protein. The findings described in this paper suggest mechanisms by which CTCF is able to form DNA loops, organize the mammalian genome and function as an insulator protein.

Unusual DNA Structure and DNA Damage Recognition: Structure and Dynamic Markers

Nucleic acids play a central role in many biological processes, including information storage, gene expression, serving as messengers or structural components and even catalysis. Their diverse roles have made them targets of interest to diagnose and treat an array of human disorders such as infections, degenerative diseases and cancer. Nature has evolved proteins and ligands that recognize specific nucleic acid sequences or structures and control their function, demonstrating that this can be efficiently accomplished. This has led to the development of wide variety of synthetic molecules that selectively bind to nucleic acids. In turn, this has precipitated numerous studies which showed that nucleic acid structures and their dynamic properties must be understood in order to efficiently target specific sequences or structures.

An Alternative DNA Structure Is Necessary for Pilin Antigenic Variation in Neisseria gonorrhoeae

Pathogens can use DNA recombination to promote antigenic variation (Av) of surface structures to avoid immune detection. We identified a cis-acting DNA sequence near the antigenically variable pilin locus of the human pathogen, Neisseria gonorrhoeae. This 16-base pair guanine (G)-rich sequence was required for pilin Av and formed a guanine quartet (G4) structure in vitro. Individual mutations that disrupted the structure also blocked pilin Av and prevented nicks required for recombination from occurring within the G4 region. A compound that binds and stabilizes G4 structures also inhibited pilin Av and prevented nicks from occurring on the G-rich strand. This site constitutes a recombination initiation sequence/structure that directs gene conversion to a specific chromosomal locus.

Nature's pH meter

A new pH nanosensor changes color in acidic cell compartments by forming an unusual four-stranded DNA structure.

Molecular models for intrastrand DNA G-quadruplexes

BackgroundIndependent surveys of human gene promoter regions have demonstrated an overrepresentation of G3Xn1G3Xn2G3Xn3G3 motifs which are known to be capable of forming intrastrand quadruple helix structures. In spite of the widely recognized importance of G-quadruplex structures in gene regulation and growing interest around this unusual DNA structure, there are at present only few such structures available in the Nucleic Acid Database. In the present work we generate by molecular modeling feasible G-quadruplex structures which may be useful for interpretation of experimental data.ResultsWe have used all quadruplex DNA structures deposited in the Nucleic Acid Database in order to select a list of fragments entailing a strand of three adjacent G's paired with another strand of three adjacent G's separated by a loop of one to four residues. These fragments were further clustered and representative fragments were finally selected. Further fragments were generated by assemblying the two strands of each fragment with loops from different fragments whenever the anchor G's were superimposable. The fragments were used to assemble G quadruplex based on a superimposability criterion.ConclusionMolecular models have been generated for a large number of G3Xn1G3Xn2G3Xn3G3 sequences. For a given sequence not all topologies are possible with the available repertoire of fragments due to steric hindrance and low superimposability. Since all molecular models are generated by fragments coming from observed quadruplex structures, molecular models are in principle reliable and may be used for interpretation of experimental data. Some examples of applications are given.

G4-forming Sequences in the Non-transcribed DNA Strand Pose Blocks to T7 RNA Polymerase and Mammalian RNA Polymerase II

DNA sequences rich in runs of guanine have the potential to form G4 DNA, a four-stranded non-canonical DNA structure stabilized by formation and stacking of G quartets, planar arrays of four hydrogen-bonded guanines. It was reported recently that G4 DNA can be generated in Escherichia coli during transcription of plasmids containing G-rich sequences in the non-transcribed strand. In addition, a stable RNA/DNA hybrid is formed with the transcribed strand. These novel structures, termed G loops, are suppressed in recQ(+) strains, suggesting that their persistence may generate genomic instability and that the RecQ helicase may be involved in their dissolution. However, little is known about how such non-canonical DNA structures are processed when encountered by an elongating polymerase. To assess whether G4-forming sequences interfere with transcription, we studied their effect on transcription elongation by T7 RNA polymerase and mammalian RNA polymerase II. We used a reconstituted transcription system in vitro with purified polymerase and initiation factors and with substrates containing G-rich sequences in either the transcribed or non-transcribed strand downstream of the T7 promoter or the adenovirus major late promoter. We report that G-rich sequences located in the transcribed strand do not affect transcription by either polymerase, but when the sequences are located in the non-transcribed strand, they partially arrest both polymerases. The efficiency of arrest increases with negative supercoiling and also with multiple rounds of transcription compared with single events.

Quartets in G‐major

The First International Meeting on Quadruplex DNA took place between 21 and 24 April 2007, in Louisville, Kentucky, USA. The meeting followed two regional workshops (pan‐Pacific and European) in Hawai (2005) and Slovenia (2006), and was organized by J. Chaires and L. Hurley. ![][1] The conference literally started with a bang! The meeting brought together around 110 scientists from all over the world to present their most recent data on quadruplex DNA, and the opening reception coincided with Thunder Over Louisville , a massive fireworks display marking the start of the Kentucky Derby Festival. The focus of the conference was the family of nucleic acid secondary structures known as guanine (G)‐quadruplexes. These can be formed by certain guanine‐rich sequences in the presence of monovalent cations and are stabilized by G‐quartets (Fig 1). Interest in this area has intensified in recent years because G‐quadruplex structures have roles in crucial biological processes and can be targeted for therapeutic intervention (Maizels, 2006; Neidle & Balasubramanian, 2006; Riou, 2004). G‐quadruplexes might also have applications in areas ranging from supramolecular chemistry and nanotechnology to medicinal chemistry (Alberti et al , 2006; Davis, 2004; Petraccone et al , 2005). The aim of this meeting was to assess the current status of this unusual DNA structure, which was discovered more than 40 years ago by our plenary lecturer, D. Davies (Bethesda, MD, USA), and his colleagues (Gellert et al , 1962). The meeting also coincided with the tenth anniversary of a seminal paper that catalysed much interest in the field and laid the foundations for therapeutic possibilities (Sun et al , 1997). Here, we summarize a few of the highlights from the broad range of topics discussed. Figure 1. The quadruplex. (A) A tetrameric, dimeric and monomeric G‐quadruplex composed of three G‐quartets (top). (B) A schematic model of DNA secondary … [1]: /embed/graphic-1.gif

Performing DNA computation with RecA-mediated triple-stranded DNA structure

A new method of DNA computing using an unusual triple-stranded DNA structure mediated by RecA protein is introduced and a minor 3-variable instance of the satisfiability (SAT) problem is solved. Separations were performed by using biotinylated oligodeoxynucleotide probes coated with RecA protein. During the computation, sequence-specific recognition of double-stranded DNA by homologous oligodeoxynucleotides was fulfilled in the presence of ATPγS. Captured by the magnetic particles coated with streptavidin, the desirable double-stranded DNA strands were retained and extracted. It is suggested that the methods employed here may help decrease the errors in DNA computation and solve some larger size NP- complete problems.

Relationship between two tandemly arranged and light-induced glutathione S-transferase genes from the ciliated protozoa Blepharisma japonicum

Recently we reported a light-induced cDNA encoding glutathione S-transferase (GST) from the ciliated protozoa Blepharisma japonicum, which possessed photosensitive pigments. In this study, a novel cDNA encoding GST was further isolated, and the two GSTs (BjGST1 and BjGST2) showed high sequence identity of 86%. Phylogenetic trees indicated that the BjGSTs were distantly related to known classes of GSTs, and they could form a protozoa-specific class. The recombinant proteins also existed as homo- or heterodimers that exhibited different enzyme activities, appreciating the functional differentiation. Furthermore, the transcription levels of BjGST genes were coordinately regulated in response to light stimulation. In addition, the genomic structure analysis revealed that the two genes were tandemly arranged through an approximately 500-bp spacer region of unusual DNA structure containing cis-acting elements related to oxidative stress response. These results demonstrate that the two BjGSTs are expressed simultaneously and act cooperatively against photooxidative stress.

G-quadruplex formation within the promoter of the KRAS proto-oncogene and its effect on transcription

In human and mouse, the promoter of the KRAS gene contains a nuclease hypersensitive polypurine–polypyrimidine element (NHPPE) that is essential for transcription. An interesting feature of the polypurine G-rich strand of NHPPE is its ability to assume an unusual DNA structure that, according to circular dichroism (CD) and DMS footprinting experiments, is attributed to an intramolecular parallel G-quadruplex, consisting of three G-tetrads and three loops. The human and mouse KRAS NHPPE G-rich strands display melting temperature of 64 and 73°C, respectively, as well as a K+-dependent capacity to arrest DNA polymerase. Photocleavage and CD experiments showed that the cationic porphyrin TMPyP4 stacks to the external G-tetrads of the KRAS quadruplexes, increasing the Tm by ∼20°C. These findings raise the intriguing question that the G-quadruplex formed within the NHPPE of KRAS may be involved in the regulation of transcription. Indeed, transfection experiments showed that the activity of the mouse KRAS promoter is reduced to 20% of control, in the presence of the quadruplex-stabilizing TMPyP4. In addition, we found that G-rich oligonucleotides mimicking the KRAS quadruplex, but not the corresponding 4-base mutant sequences or oligonucleotides forming quadruplexes with different structures, competed with the NHPPE duplex for binding to nuclear proteins. When vector pKRS-413, containing CAT driven by the mouse KRAS promoter, and KRAS quadruplex oligonucleotides were co-transfected in 293 cells, the expression of CAT was found to be downregulated to 40% of the control. On the basis of these data, we propose that the NHPPE of KRAS exists in equilibrium between a double-stranded form favouring transcription and a folded quadruplex form, which instead inhibits transcription. Such a mechanism, which is probably adopted by other growth-related genes, provides useful hints for the rational design of anticancer drugs against the KRAS oncogene.

Recombination, rearrangement, reshuffling, and divergence in a centromeric region of rice.

Centromeres have many unusual biological properties, including kinetochore attachment and severe repression of local meiotic recombination. These properties are partly an outcome, partly a cause, of unusual DNA structure in the centromeric region. Although several plant and animal genomes have been sequenced, most centromere sequences have not been completed or analyzed in depth. To shed light on the unique organization, variability, and evolution of centromeric DNA, detailed analysis of a 1.97-Mb sequence that includes centromere 8 (CEN8) of japonica rice was undertaken. Thirty-three long-terminal repeat (LTR)-retrotransposon families (including 11 previously unknown) were identified in the CEN8 region, totaling 245 elements and fragments that account for 67% of the region. The ratio of solo LTRs to intact elements in the CEN8 region is approximately 0.9:1, compared with approximately 2.2:1 in noncentromeric regions of rice. However, the ratio of solo LTRs to intact elements in the core of the CEN8 region ( approximately 2.5:1) is higher than in any other region investigated in rice, suggesting a hotspot for unequal recombination. Comparison of the CEN8 region of japonica and its orthologous segments from indica rice indicated that approximately 15% of the intact retrotransposons and solo LTRs were inserted into CEN8 after the divergence of japonica and indica from a common ancestor, compared with approximately 50% for previously studied euchromatic regions. Frequent DNA rearrangements were observed in the CEN8 region, including a 212-kb subregion that was found to be composed of three rearranged tandem repeats. Phylogenetic analysis also revealed recent segmental duplication and extensive rearrangement and reshuffling of the CentO satellite repeats.

Mechanism of Gene Repair Open for Discussion

During the last decade, chimeric RNA-DNA oligonucleotides (RDOs) and single-stranded oligodeoxynucleotides have been used to make permanent and specific sequence changes in the genome, with the ultimate goal of curing human genetic disorders caused by mutations. There have been large variations observed in the rate of gene repair in these studies. This has been due, at least in part, to the lack of standardized assay conditions and the paucity of mechanistic studies in the early developmental stages. Previously, it was proposed that strand pairing is the rate-limiting step and mismatch DNA repair is involved in the gene repair process. We propose an alternative model, in which an oligonucleotide is assimilated to the target DNA during active transcription, leading to formation of a transient D-loop. The trafficking of RNA polymerase is interrupted by the D-loop, and the stalled RNA polymerase complex may signal for recruitment of DNA repair proteins, including transcription-coupled DNA repair and nucleotide-excision repair. Thus, oligonucleotides can be considered as a class of DNA-damaging agents that cause a transient but major structural change in DNA. Understanding of the recognition and repair pathways to process this unusual DNA structure may have relevance in physiologic processes, transcription, and DNA replication.

Highly polymorphic repeat region in the CETP promoter induces unusual DNA structure

Genetic variation in the human cholesteryl ester transfer protein (CETP) promoter is associated with HDL cholesterol levels and cardiovascular disease with much of the genetic variation in CETP attributed to the promoter region. In this region, there are several single nucleotide polymorphisms as well as a variable length tandem repeat located 1946 base pairs upstream of the CETP transcription start that is highly polymorphic with respect to both length and sequence. There are more than 10 different long alleles and these vary in their repeat structure. We find that the short allele of this repeat is associated with high HDL cholesterol levels in vivo ( P<0.0001). In males, this association is independent of the nearby −629 polymorphism. In addition, the variable length GAAA repeat can stimulate an adjacent GGGGA repeat to form a structure that hinders DNA amplification and sequencing. This structure also has an effect in vivo as shown by orientation effects and cloning efficiency in Escherichia coli.

Replication of a cis-syn thymine dimer at atomic resolution.

Ultraviolet light damages DNA by catalysing covalent bond formation between adjacent pyrimidines, generating cis-syn cyclobutane pyrimidine dimers (CPDs) as the most common lesion. CPDs block DNA replication by high-fidelity DNA polymerases, but they can be efficiently bypassed by the Y-family DNA polymerase pol eta. Mutations in POLH encoding pol eta are implicated in nearly 20% of xeroderma pigmentosum, a human disease characterized by extreme sensitivity to sunlight and predisposition to skin cancer. Here we have determined two crystal structures of Dpo4, an archaeal pol eta homologue, complexed with CPD-containing DNA, where the 3' and 5' thymine of the CPD separately serves as a templating base. The 3' thymine of the CPD forms a Watson-Crick base pair with the incoming dideoxyATP, but the 5' thymine forms a Hoogsteen base pair with the dideoxyATP in syn conformation. Dpo4 retains a similar tertiary structure, but each unusual DNA structure is individually fitted into the active site for catalysis. A model of the pol eta-CPD complex built from the crystal structures of Saccharomyces cerevisiae apo-pol eta and the Dpo4-CPD complex suggests unique features that allow pol eta to efficiently bypass CPDs.