Alternative DNA Secondary Structures Research Articles

DNA fragility at topologically associated domain boundaries is promoted by alternative DNA secondary structure and topoisomerase II activity.

CCCTC-binding factor (CTCF) binding sites are hotspots of genome instability. Although many factors have been associated with CTCF binding site fragility, no study has integrated all fragility-related factors to understand the mechanism(s) of how they work together. Using an unbiased, genome-wide approach, we found that DNA double-strand breaks (DSBs) are enriched at strong, but not weak, CTCF binding sites in five human cell types. Energetically favorable alternative DNA secondary structures underlie strong CTCF binding sites. These structures coincided with the location of topoisomerase II (TOP2) cleavage complex, suggesting that DNA secondary structure acts as a recognition sequence for TOP2 binding and cleavage at CTCF binding sites. Furthermore, CTCF knockdown significantly increased DSBs at strong CTCF binding sites and at CTCF sites that are located at topologically associated domain (TAD) boundaries. TAD boundary-associated CTCF sites that lost CTCF upon knockdown displayed increased DSBs when compared to the gained sites, and those lost sites are overrepresented with G-quadruplexes, suggesting that the structures act as boundary insulators in the absence of CTCF, and contribute to increased DSBs. These results model how alternative DNA secondary structures facilitate recruitment of TOP2 to CTCF binding sites, providing mechanistic insight into DNA fragility at CTCF binding sites.

Cytosine epigenetic modifications and conformational changes in G‐quadruplex DNA: An ultraviolet resonance Raman spectroscopy study

Epigenetic modifications of DNA are known to play important regulatory roles in biological systems, especially in regulation of gene expression, and are associated with many types of human diseases, including cancer. Alternative DNA secondary structures, such as G‐quadruplexes, can also influence gene transcription, thus suggesting that such structures may represent a distinctive layer of epigenetic information. G‐quadruplex structures and DNA epigenetic modifications often go side by side, and recent evidence reveals that cytosine modifications within loops of G‐quadruplexes can play a role in modulating their stability and structural polymorphism. Therefore, the development and validation of experimental techniques that can easily and reliably analyse G‐quadruplex structures are highly desirable. In the present study, we propose to exploit the advantages of UV resonance Raman (UVRR) spectroscopy to investigate cytosine epigenetic modifications along with conformational changes in G‐quadruplex-forming DNA. Our findings show that clear and specific spectral changes occur when there is a change in a G‐quadruplex structure. Moreover, UVRR spectral analysis can indirectly distinguish the spectral variations occurring because of modifications in the guanine glycosidic conformations, as well as detect changes in the loops induced by H-bond formation or hydration of nitrogenous bases. The results further underscore the utility of UVRR spectroscopy for G‐quadruplex structure elucidation under biologically relevant solution conditions.

Pif1 Activity is Modulated by DNA Sequence and Structure.

The gene encoding the Pif1 helicase was first discovered in a Saccharomyces cerevisiae genetic screen as a mutant that reduces recombination between mitochondrial respiratory mutants and was subsequently rediscovered in a screen for genes affecting the telomere length in the nucleus. It is now known that Pif1 is involved in numerous aspects of DNA metabolism. All known functions of Pif1 rely on binding to DNA substrates followed by ATP hydrolysis, coupling the energy released to translocation along DNA to unwind duplex DNA or alternative DNA secondary structures. The interaction of Pif1 with higher-order DNA structures, like G-quadruplex DNA, as well as the length of single-stranded (ss)DNA necessary for Pif1 loading have been widely studied. Here, to test the effects of ssDNA length, sequence, and structure on Pif1's biochemical activities in vitro, we used a suite of oligonucleotide-based substrates to perform a basic characterization of Pif1 ssDNA binding, ATPase activity, and helicase activity. Using recombinant, untagged S. cerevisiae Pif1, we found that Pif1 preferentially binds to structured G-rich ssDNA, but the preferred binding substrates failed to maximally stimulate ATPase activity. In helicase assays, significant DNA unwinding activity was detected at Pif1 concentrations as low as 250 pM. Helicase assays also demonstrated that Pif1 most efficiently unwinds DNA fork substrates with unstructured ssDNA tails. As the chemical step size of Pif1 has been determined to be 1 ATP per translocation or unwinding event, this implies that the highly structured DNA inhibits conformational changes in Pif1 that couple ATP hydrolysis to DNA translocation and unwinding.

A transgenic cell line with inducible transcription for studying (CGG)n repeat expansion mechanisms.

There are more than 30 inherited human disorders connected with repeat expansion (myotonic dystrophy type I, Huntington’s disease, Fragile X syndrome). Fragile X syndrome is the most common reason for inherited intellectual disability in the human population. The ways of the expansion development remain unclear. An important feature of expanded repeats is the ability to form stable alternative DNA secondary structures. There are hypotheses about the nature of repeat instability. It is proposed that these DNA secondary structures can block various stages of DNA metabolism processes, such as replication, repair and recombination and it is considered as the source of repeat instability. However, none of the hypotheses is fully confirmed or is the only valid one. Here, an experimental system for studying (CGG)n repeat expansion associated with transcription and TCR-NER is proposed. It is noteworthy that the aberrations of transcription are a poorly studied mechanism of (CGG)n instability. However, the proposed systems take into account the contribution of other processes of DNA metabolism and, therefore, the developed systems are universal and applicable for various studies. Transgenic cell lines carrying a repeat of normal or premutant length under the control of an inducible promoter were established and a method for repeat instability quantification was developed. One type of the cell lines contains an exogenous repeat integrated into the genome by the Sleeping Beauty transposon; in another cell line, the vector is maintained as an episome due to the SV40 origin of replication. These experimental systems can serve for finding the causes of instability and the development of therapeutic agents. In addition, a criterion was developed for the quantification of exogenous (CGG)n repeat instability in the transgenic cell lines’ genome.

Substitution of Cytosine with Guanylurea Decreases the Stability of i-Motif DNA.

Both 5-aza-2'-deoxycytidine (decitabine) and its primary breakdown product, 2'-deoxyriboguanylurea (GuaUre-dR), have been shown to act as mutagens and epimutagens that cause replication stress and alter both DNA methylation and gene expression patterns. As cytosine analogues, both are expected to be preferentially incorporated into regions of GC skew where runs of cytosine residues are sequestered on one strand and guanine residues on the other. Given that such regions have been identified as sites with the potential for effects on gene expression and replication stress linked to formation of alternative DNA secondary structures, it is of interest to determine the influence that these base analogues might have on the stability of structures of this kind. Here we report that incorporation of GuaUre-dR into an i-motif-forming sequence decreases both the thermal and pH stability of an i-motif despite the apparent ability of GuaUre-dR to base pair with cytosine.

Identification of multiple genomic DNA sequences which form i-motif structures at neutral pH.

i-Motifs are alternative DNA secondary structures formed in cytosine-rich sequences. Particular examples of these structures, traditionally assumed to be stable only at acidic pH, have been found to form under near-physiological conditions. To determine the potential impact of these structures on physiological processes, investigation of sequences with the capacity to fold under physiological conditions is required. Here we describe a systematic study of cytosine-rich DNA sequences, with varying numbers of consecutive cytosines, to gain insights into i-motif DNA sequence and structure stability. i-Motif formation was assessed using ultraviolet spectroscopy, circular dichroism and native gel electrophoresis. We found that increasing cytosine tract lengths resulted in increased thermal stability; sequences with at least five cytosines per tract folded into i-motif at room temperature and neutral pH. Using these results, we postulated a folding rule for i-motif formation, analogous to (but different from) that for G-quadruplexes. This indicated that thousands of cytosine-rich sequences in the human genome may fold into i-motif structures under physiological conditions. Many of these were found in locations where structure formation is likely to influence gene expression. Characterization of a selection of these identified i-motif forming sequences uncovered 17 genomic i-motif forming sequence examples which were stable at neutral pH.

Optimized Virtual Screening Workflow for the Identification of Novel G-Quadruplex Ligands

G-quadruplexes, alternative DNA secondary structures present in telomeres, emerge as promising targets for the treatment of cancer, because they prevent telomere elongation and accordingly cell proliferation. Within this study, theoretically validated pharmacophore- and shape-based models as well as a theoretically validated docking protocol were generated and applied in parallel for virtual screening and the identification of novel G-quadruplex ligands. Top-ranked hits retrieved with all methods independently and in addition in a consensus approach were selected for biological testing. Of the 32 tested virtual hits seven selectively stabilized G-quadruplexes over duplex DNA in the fluorescence melting assay. For the five most active compounds, chemically closely related analogues were collected and subjected to in vitro analysis. Thereby, seven further novel G-quadruplex ligands could be identified. These molecules do not only represent novel scaffolds, but some of them are in addition even more potent G-quadruplex stabilizers than the established reference compound berberine. This study proposes an optimized in silico workflow for the identification of novel G-quadruplex stabilizers, which can also be applied in future studies. In addition, structurally novel and promising lead candidates with strong and selective G-quadruplex stabilizing properties are reported.

G-quadruplex formation in double strand DNA probed by NMM and CV fluorescence.

G-quadruplexes (GQs) are alternative DNA secondary structures that can form throughout the human genome and control the replication and transcription of important regulatory genes. Here, we established an ensemble fluorescence assay by employing two GQ-interacting compounds, N-methyl mesoporphyrin IX (NMM) and Crystal Violet (CV). This enables quantitative measurement of the GQ folding propensity and conformation specificity in both single strand (ss) and double strand (ds) DNA. Our GQ mapping indicates that the likelihood of GQ formation is substantially diminished in dsDNA, likely due to the competition from the Watson–Crick base pairing. Unlike GQ folding sequence in ssDNA which forms both parallel and antiparallel GQs, dsDNA displays only parallel folding. Additionally, we employed single molecule FRET to obtain a direct quantitation of stably formed-, weakly folded and unfolded GQ conformations. The findings of this study and the method developed here will enable identifying and classifying potential GQ-forming sequences in human genome.

Role of DNA secondary structures in fragile site breakage along human chromosome 10

The formation of alternative DNA secondary structures can result in DNA breakage leading to cancer and other diseases. Chromosomal fragile sites, which are regions of the genome that exhibit chromosomal breakage under conditions of mild replication stress, are predicted to form stable DNA secondary structures. DNA breakage at fragile sites is associated with regions that are deleted, amplified or rearranged in cancer. Despite the correlation, unbiased examination of the ability to form secondary structures has not been evaluated in fragile sites. Here, using the Mfold program, we predict potential DNA secondary structure formation on the human chromosome 10 sequence, and utilize this analysis to compare fragile and non-fragile DNA. We found that aphidicolin (APH)-induced common fragile sites contain more sequence segments with potential high secondary structure-forming ability, and these segments clustered more densely than those in non-fragile DNA. Additionally, using a threshold of secondary structure-forming ability, we refined legitimate fragile sites within the cytogenetically defined boundaries, and identified potential fragile regions within non-fragile DNA. In vitro detection of alternative DNA structure formation and a DNA breakage cell assay were used to validate the computational predictions. Many of the regions identified by our analysis coincide with genes mutated in various diseases and regions of copy number alteration in cancer. This study supports the role of DNA secondary structures in common fragile site instability, provides a systematic method for their identification and suggests a mechanism by which DNA secondary structures can lead to human disease.

118 DNA structure-induced genetic instability in mammals

Naturally occurring repetitive DNA sequences can adopt alternative (i.e. non-B) DNA secondary structures, and often co-localize with chromosomal breakpoint “hotspots,” implicating non-B DNA in translocation-related cancer etiology. We have found that sequences capable of adopting H-DNA and Z-DNA structures are intrinsically mutagenic in mammals. For example, an endogenous H-DNA-forming sequence from the human c-MYC promoter and a model Z-DNA-forming CpG repeat induced genetic instability in mammalian cells, largely in the form of deletions resulting from DNA double-strand breaks (Wang & Vasquez, 2004; Wang et al., 2006). Characterization of the mutants revealed microhomologies at the breakpoints, consistent with a microhomology-mediated end-joining repair of the double-strand breaks (Kha et al., 2010). We have constructed transgenic mutation-reporter mice containing these human H-DNA- and Z-DNA-forming sequences to determine their effects on genomic instability in a chromosomal context in a living organism (Wang et al., 2008). Initial results suggest that both H-DNA- and Z-DNA-forming sequences induced genetic instability in mice, suggesting that these non-B DNA structures represent endogenous sources of genetic instability and may contribute to disease etiology and evolution. Our current studies are designed to determine the mechanisms of DNA structure-induced genetic instability in mammals; the roles of helicases, polymerases, and repair enzymes in H-DNA and Z-DNA-induced genetic instability will be discussed.

Targeting DNA G-Quadruplex Structures with Peptide Nucleic Acids

Regulation of genetic functions based on targeting DNA or RNA sequences with complementary oligonucleotides is especially attractive in the post-genome era. Oligonucleotides can be rationally designed to bind their targets based on simple nucleic acid base pairing rules. However, the use of natural DNA and RNA oligonucleotides as targeting probes can cause numerous off-target effects. In addition, natural nucleic acids are prone to degradation in vivo by various nucleases. To address these problems, nucleic acid mimics such as peptide nucleic acids (PNA) have been developed. They are more stable, show less off-target effects, and, in general, have better binding affinity to their targets. However, their high affinity to DNA can reduce their sequence-specificity. The formation of alternative DNA secondary structures, such as the G-quadruplex, provides an extra level of specificity as targets for PNA oligomers. PNA probes can target the loops of G-quadruplex, invade the core by forming PNA-DNA guanine-tetrads, or bind to the open bases on the complementary cytosine-rich strand. Not only could the development of such G-quadruplex-specific probes allow regulation of gene expression, but it will also provide a means to clarify the biological roles G-quadruplex structures may possess.

Non-B DNA-forming Sequences and WRN Deficiency Independently Increase the Frequency of Base Substitution in Human Cells

Although alternative DNA secondary structures (non-B DNA) can induce genomic rearrangements, their associated mutational spectra remain largely unknown. The helicase activity of WRN, which is absent in the human progeroid Werner syndrome, is thought to counteract this genomic instability. We determined non-B DNA-induced mutation frequencies and spectra in human U2OS osteosarcoma cells and assessed the role of WRN in isogenic knockdown (WRN-KD) cells using a supF gene mutation reporter system flanked by triplex- or Z-DNA-forming sequences. Although both non-B DNA and WRN-KD served to increase the mutation frequency, the increase afforded by WRN-KD was independent of DNA structure despite the fact that purified WRN helicase was found to resolve these structures in vitro. In U2OS cells, ∼70% of mutations comprised single-base substitutions, mostly at G·C base-pairs, with the remaining ∼30% being microdeletions. The number of mutations at G·C base-pairs in the context of NGNN/NNCN sequences correlated well with predicted free energies of base stacking and ionization potentials, suggesting a possible origin via oxidation reactions involving electron loss and subsequent electron transfer (hole migration) between neighboring bases. A set of ∼40,000 somatic mutations at G·C base pairs identified in a lung cancer genome exhibited similar correlations, implying that hole migration may also be involved. We conclude that alternative DNA conformations, WRN deficiency and lung tumorigenesis may all serve to increase the mutation rate by promoting, through diverse pathways, oxidation reactions that perturb the electron orbitals of neighboring bases. It follows that such "hole migration" is likely to play a much more widespread role in mutagenesis than previously anticipated.

Brownian dynamics of double-stranded DNA in periodic systems with discrete salt

Numerical models of mesoscale DNA dynamics relevant to in vivo scenarios require methods that incorporate important features of the intracellular environment, while maintaining computational tractability. Because the explicit inclusion of ions leads to electrostatic calculations that scale as the square of the number of charged particles, such models typically handle these calculations using low-potential, mean-field approaches, rather than by considering the discrete interactions of ions. This allows approximation of the long-range, screened self-repulsion of DNA, but is unable to capture detailed electrostatic phenomena, such as short-range attractions mediated by ion-ion correlations. Here, we develop a dynamical model of explicitly double-stranded, sequence-specific DNA in a bulk environment consisting of other polyions and explicitly represented counterions and coions. DNA is represented as two interwound chains of charged Stokes spheres, and ions as free, monovalently charged Stokes spheres. Brownian dynamics simulations performed at salt concentrations of 0.1, 1, 10, and 100 mM demonstrate this model captures anticipated behaviors of the system, including increasing compaction of the polyion by the ionic atmosphere with increasing ionic strength. The decay of the distance dependence of the ion concentrations as one moves away from the polyion approaches their equilibrium values in quantitative agreement with predictions of Poisson-Boltzmann theory. The simulation results also demonstrate quantitative agreement with experimental measurements of the persistence length of B-DNA, which increases significantly at low ionic strengths. The model also captures behaviors intimating the importance of explicitly representing ionic and polyionic structure. These include penetration of the polyion interior by both coions and counterions, and counterion-mediated accumulation of coions near the surface of the polyion. Such phenomena are likely to play an important role in the formation of alternative DNA secondary structures, suggesting the present methods will prove valuable to dynamic models of superhelical stress-induced DNA structural transitions.

Structural features of trinucleotide repeats associated with DNA expansion

The mechanism of DNA expansion is not well understood. Recent evidence from genetic, in vivo, and in vitro studies has suggested a link between the formation of alternative DNA secondary structures by trinucleotide repeat tracts and their propensity to undergo expansion. This review will focus on structural features and the mechanism of expansion relevant to human disease.

Structural analysis of slipped-strand DNA (S-DNA) formed in (CTG)n. (CAG)n repeats from the myotonic dystrophy locus.

The mechanism of disease-associated trinucleotide repeat length variation may involve slippage of the triplet-containing strand at the replication fork, generating a slipped-strand DNA structure. We recently reported formation in vitro of slipped-strand DNA (S-DNA) structures when DNAs containing triplet repeat blocks of myotonic dystrophy or fragile X diseases were melted and allowed to reanneal to form duplexes. Here additional evidence is presented that is consistent with the existence of S-DNA structures. We demonstrate that S-DNA structures can form between two complementary strands containing equal numbers of repeats. In addition, we show that both the propensity for S-DNA formation and the structural complexity of S-DNAs formed increase with increasing repeat length. S-DNA structures were also analyzed by electron microscopy, confirming that the two strands are slipped out of register with respect to each other and confirming the structural polymorphism expected within long tracts of trinucleotide repeats. For (CTG)50.(CAG)50 two distinct populations of slipped structures have been identified: those involving </=10 repeats per slippage, which appear as bent/kinked DNA molecules, and those involving >10 repeats, which have multiple loops or hairpins indicative of complex alternative DNA secondary structures.

CTG repeats associated with human genetic disease are inherently flexible

The lengthening of tracts of CTG, CGG and GAA triplet repeats during progression of a pedigree has been associated with more than 12 human genetic diseases, including fragile X syndrome, myotonic dystrophy and Friedreich’s ataxia. These repetitive sequence elements have the potential to form alternative DNA secondary structures that may contribute to their instability. The alternative DNA secondary structures may mediate errors during DNA replication, repair or recombination of the triplet repeat, leading to expansion. Here we show that DNA composed of pure CTG or CGG repeats exhibits anomalously fast mobility on polyacrylamide gels, confirming a previous observation for DNA containing CTG and CGG triplet repeats flanked by mixed sequence DNA. Moreover, we show that even short tracts of duplex CTG repeats have an unusual helix structure. CTG repeats reduce overall curvature associated with phased A-tract or GGCC curves, but alone they do not introduce curvature into DNA. The reduction in curvature of phased A-tracts by CTG repeats is similar to that afforded by an interspersed flexible region associated with a (TT)·(TT) mispair. CTG-containing DNAs exhibit a rapid rate of cyclization, consistent with a flexible helix. These results suggest that tracts of (CTG)·(CAG) repeats are inherently flexible. In addition, our results suggest that the unusual rapid electrophoretic mobility of CTG- or CGG-containing DNA may be a consequence of an extended flexible DNA chain.

Alternative structures in duplex DNA formed within the trinucleotide repeats of the myotonic dystrophy and fragile X loci.

Most models proposed to explain the disease-associated expansion of (CTG)n.(CAG)n and (CGG)n.(CCG)n trinucleotide repeats include the formation of slipped strand DNA structures during replication; however, physical evidence for these alternative DNA secondary structures has not been reported. Using cloned fragments from the myotonic dystrophy (DM) and fragile X syndrome (FRAXA) loci containing normal, premutation, and full mutation lengths of repeats, we report the formation of novel alternative DNA secondary structures that map within the repeat tracts during reannealing of complementary strands, containing equal lengths of repeats, into linear duplex DNA molecules. Linear duplex DNA molecules containing these alternative DNA secondary structures are characterized by reduced electrophoretic mobilities in polyacrylamide gels. These alternative secondary structures are stable at physiological ionic strengths and to temperatures up to at least 55 degrees C. Following reduplexing, the CAG strand of the (CTG)n.(CAG)n repeats is preferentially sensitive to mung bean nuclease, suggesting the presence of single-stranded DNA in the alternative DNA structure. For (CTG)17, which is a repeat length found in normal individuals, less than 3% of the DNA molecules formed alternative DNA structures upon reduplexing. DNA molecules containing (CTG)50 or (CTG)255, which represent premutation and full mutation lengths of triplet repeats, respectively, formed a heterogeneous population of alternative DNA structures in >50% of DNA molecules. The complexity of the structures formed increased with the length of the triplet repeat. The relationship between repeat length and the propensity of formation and complexity of the novel structures correlates with the effect of repeat length on genetic instability in human diseases. These are the first results consistent with the existence of slipped strand DNA structures. The potential involvement of these structures, which we term S-DNA, in the gentic instability of triplet repeats is discussed.