non-B DNA Conformations Research Articles

Mutations at BCL11B Exon 4 Associated with T Cell Acute Lymphoblastic Leukemia Are Facilitated by AID and Formation of Non-B DNA Conformations.

One of the primary reasons behind the pathogenesis of T cell acute lymphoblastic leukemia (T-ALL) is the deregulation of the transcription factor BCL11B. The exon 4 of BCL11B harbors several driver mutations, which abolishes its DNA-binding ability. The high frequency of C > T or G > A conversion in close vicinity of AID (Activation-induced cytidine deaminase)-hotspot motifs in the deregulated gene prompted us to investigate the role of AID in BCL11B mutagenesis. Our results reveal that AID is expressed in T-ALL patient-derived cells, binds to BCL11B fragile region (FR) in exon 4 of T cells invivo, and generates a signature mutation pattern in this region. The mutation frequency in BCL11B FR could be modulated upon overexpression of the AID gene in the knockout background, further suggesting the involvement of AID in BCL11B mutagenesis. Importantly, various lines of experimentation reveal that BCL11B FR could fold into parallel G-quadruplex, triplex, and hairpin structures, which could act as a replication/transcription block, causing mutagenesis. Thus, our results suggest that AID binds to BCL11B exon 4 due to non-B DNA formation, causing U:G mismatches or replication blocks, which, when repaired erroneously, generates deleterious mutations, resulting in loss of functionality of BCL11B, and thus becomes the cause of T-ALL.

Distinguishing Isomeric Cyclobutane Thymidine Dimers by Ion Mobility and Tandem Mass Spectrometry.

Irradiation of the major conformation of duplex DNA found in cells (B form) produces cyclobutane pyrimidine dimers (CPDs) from adjacent pyrimidines in a head-to-head orientation (syn) with the C5 substituents in a cis stereochemistry. These CPDs have crucial implications in skin cancer. Irradiation of G-quadruplexes and other non-B DNA conformations in vitro produces, however, CPDs between nonadjacent pyrimidines in nearby loops with syn and head-to-tail orientations (anti) with both cis and trans stereochemistry to yield a mixture of six possible isomers of the T=T dimer. This outcome is further complicated by formation of mixtures of nonadjacent CPDs of C=T, T=C, and C=C, and successful analysis depends on development of specific and sensitive methods. Toward meeting this need, we investigated whether ion mobility mass spectrometry (IMMS) and MS/MS can distinguish the cis,syn and trans,anti T=T CPDs. Ion mobility can afford baseline separation and give relative mobilities that are in accord with predicted cross sections. Complementing this ability to distinguish isomers is MS/MS collisional activation where fragmentation also distinguishes the two isomers and confirms conclusions drawn from ion mobility analysis. The observations offer early support that ion mobility and MS/MS can enable the distinction of DNA photoproduct isomers.

Conserved microRNAs and Flipons Shape Gene Expression during Development by Altering Promoter Conformations.

The classical view of gene regulation draws from prokaryotic models, where responses to environmental changes involve operons regulated by sequence-specific protein interactions with DNA, although it is now known that operons are also modulated by small RNAs. In eukaryotes, pathways based on microRNAs (miR) regulate the readout of genomic information from transcripts, while alternative nucleic acid structures encoded by flipons influence the readout of genetic programs from DNA. Here, we provide evidence that miR- and flipon-based mechanisms are deeply connected. We analyze the connection between flipon conformation and the 211 highly conserved human miR that are shared with other placental and other bilateral species. The direct interaction between conserved miR (c-miR) and flipons is supported by sequence alignments and the engagement of argonaute proteins by experimentally validated flipons as well as their enrichment in promoters of coding transcripts important in multicellular development, cell surface glycosylation and glutamatergic synapse specification with significant enrichments at false discovery rates as low as 10-116. We also identify a second subset of c-miR that targets flipons essential for retrotransposon replication, exploiting that vulnerability to limit their spread. We propose that miR can act in a combinatorial manner to regulate the readout of genetic information by specifying when and where flipons form non-B DNA (NoB) conformations, providing the interactions of the conserved hsa-miR-324-3p with RELA and the conserved hsa-miR-744 with ARHGAP5 genes as examples.

The Effect of HMGB1 Protein and Its Truncated Form on the Expression of RAGE Variants in Breast Cancer Cells

HMGB1 protein is a DNA binding nuclear protein. Its properties to bind different non-B DNA conformations and also to bend linear DNA implicate the protein in many essential cellular processes as DNA replication, repair, transcription, remodelling, etc. HMGB1 plays important role outside the cell as it is passively released from necrotic cells and actively from apoptotic ones and binds its specific receptor for advanced glycation end products RAGE. HMGB1/RAGE interactin is implicated in various diseases including cancer. Different soluble RAGE forms were reported whose functional role is to serve as a decoy for the ligands and in this way to block the signalling pathway. How the ligands regulate the production of RAGE variants is a subject of great scientific interest. We studied the effect of HMGB1 and its truncated form lacking the C- terminus on the expression of full-length (flRAGE) and soluble RAGE (sRAGE) in breast cancer cell lines: MCF7 represents a hormone dependent cancer with better prognosis and MDA-MB-231 – hormone independent with substantial invasive capacity. HMGB1 stimulates the total RAGE expression in both breast cancer cells but in MCF7 the ratio changes and is in favour of the membrane fraction. The absence of the C-tail of HMGB1 provokes comparable changes in RAGE production in MCF7 cells as the whole HMGB1 molecule. In MDA-MB- 231cell line the total amount of RAGE was slightly affected but it is entirely represented by the membrane form and the soluble one is in negligible amounts.

Non-B DNA conformations analysis through molecular dynamics simulations

BackgroundNon-B DNA conformations are molecular structures that do not follow the canonical DNA double helix. Mutagenetic instability in nuclear and mitochondrial DNA (mtDNA) genomes has been associated with simple non-B DNA conformations, as hairpins or more complex structures, as G-quadruplexes. One of these structures is Structure A, a cloverleaf-like non-B conformation predicted for a 93-nt (nucleotide) stretch of the mtDNA control region 5′-peripheral domain. Structure A is embedded in a hot spot for the 3′ end of human mtDNA deletions revealing its importance in influencing the mutational instability of the mtDNA genome. MethodsTo better characterize Structure A, we predicted its 3D conformation using state-of-art methods and algorithms. The methodologic workflow consisted in the prediction of non-B conformations using molecular dynamics simulations. The conservation scores of alignments of the Structure A region in humans, primates, and mammals, was also calculated. ResultsOur results show that these computational methods are able to measure the stability of non-B conformations by using the level of base pairing during molecular dynamics. Structure A showed high stability and low flexibility correlated with high conservation scores in mammalian, more specifically in primate lineages. ConclusionsWe showed that 3D non-B conformations can be predicted and characterized by our methodology. This allowed the in-depth analysis of the structure A, and the main results showed the structure remains stable during the simulations. General significanceThe fine-scale atomic molecular determination of this type of non-B conformation opens the way to perform computational molecular studies that can show their involvement in mtDNA cellular mechanisms.

Satellitome comparison of two oedipodine grasshoppers highlights the contingent nature of satellite DNA evolution

BackgroundThe full catalog of satellite DNA (satDNA) within a same genome constitutes the satellitome. The Library Hypothesis predicts that satDNA in relative species reflects that in their common ancestor, but the evolutionary mechanisms and pathways of satDNA evolution have never been analyzed for full satellitomes. We compare here the satellitomes of two Oedipodine grasshoppers (Locusta migratoria and Oedaleus decorus) which shared their most recent common ancestor about 22.8 Ma ago.ResultsWe found that about one third of their satDNA families (near 60 in every species) showed sequence homology and were grouped into 12 orthologous superfamilies. The turnover rate of consensus sequences was extremely variable among the 20 orthologous family pairs analyzed in both species. The satDNAs shared by both species showed poor association with sequence signatures and motives frequently argued as functional, except for short inverted repeats allowing short dyad symmetries and non-B DNA conformations. Orthologous satDNAs frequently showed different FISH patterns at both intra- and interspecific levels. We defined indices of homogenization and degeneration and quantified the level of incomplete library sorting between species.ConclusionsOur analyses revealed that satDNA degenerates through point mutation and homogenizes through partial turnovers caused by massive tandem duplications (the so-called satDNA amplification). Remarkably, satDNA amplification increases homogenization, at intragenomic level, and diversification between species, thus constituting the basis for concerted evolution. We suggest a model of satDNA evolution by means of recursive cycles of amplification and degeneration, leading to mostly contingent evolutionary pathways where concerted evolution emerges promptly after lineages split.

G-quadruplexes in the mitochondrial genome - a cause for instability.

Accumulation of mutations such as deletions in mitochondrial DNA is associated with ageing, cancer and human genetic disorders. These deletions are often flanked by GC-skewed sequence motifs that can potentially fold into secondary non-B DNA conformations. G-quadruplexes are emerging as key initiators of mitochondrial genomic instability. In this issue, Dahal etal provide an insilico analysis of sequence motifs that can fold into altered DNA structures in mitochondrial genomic regions that contain frequent deletions. They show the formation of five G-quadruplexes near such frequent breakpoints using biochemical and biophysical approaches invitro and more importantly inside mammalian cells. Comment on: https://doi.org/10.1111/febs.16113.

Development of a Molecularly Stable Gene Therapy Vector for the Treatment of RPGR-Associated X-Linked Retinitis Pigmentosa.

In a screen of 1,000 consecutively ascertained families, we recently found that mutations in the gene RPGR are the third most common cause of all inherited retinal disease. As the two most frequent disease-causing genes, ABCA4 and USH2A, are far too large to fit into clinically relevant adeno-associated virus (AAV) vectors, RPGR is an obvious early target for AAV-based ocular gene therapy. In generating plasmids for this application, we discovered that those containing wild-type RPGR sequence, which includes the highly repetitive low complexity region ORF15, were extremely unstable (i.e., they showed consistent accumulation of genomic changes during plasmid propagation). To develop a stable RPGR gene transfer vector, we used a bioinformatics approach to identify predicted regions of genomic instability within ORF15 (i.e., potential non-B DNA conformations). Synonymous substitutions were made in these regions to reduce the repetitiveness and increase the molecular stability while leaving the encoded amino acid sequence unchanged. The resulting construct was subsequently packaged into AAV serotype 5, and the ability to drive transcript expression and functional protein production was demonstrated via subretinal injection in rat and pull-down assays, respectively. By making synonymous substitutions within the repetitive region of RPGR, we were able to stabilize the plasmid and subsequently generate a clinical-grade gene transfer vector (IA-RPGR). Following subretinal injection in rat, we demonstrated that the augmented transcript was expressed at levels similar to wild-type constructs. By performing in vitro pull-down experiments, we were able to show that IA-RPGR protein product retained normal protein binding properties (i.e., analysis revealed normal binding to PDE6D, INPP5E, and RPGRIP1L). In summary, we have generated a stable RPGR gene transfer vector capable of producing functional RPGR protein, which will facilitate safety and toxicity studies required for progression to an Investigational New Drug application.

Emerging Role of G-quadruplex DNA as Target in Anticancer Therapy.

DNA has represented the most exploited target for the development of anticancer agents. It is now established that DNA may assume a variety of non-B conformations. This evidence has generated a total novel wave of interest in DNA as a cancer-associated target, since its distinct non-B structures may be regarded as sites for selective therapeutic intervention. G-quadruplexes are peculiar non-B DNA conformations that may form within guaninerich nucleic acid sequences. They are generated by a core of two or more vertically stacked G-quartets (i.e., the square planar arrangement of four guanine residues) held together by intervening loops of variable length. The evidence that G-quadruplexes are highly polymorphic and overrepresented within human genome points out at such non-B DNA conformations as druggable sites amenable of targeting by small molecules. In the present paper we will provide a concise overview on the emerging role of G-quadruplex structures forming within telomeres, gene promoters and mitochondrial DNA as a promising therapeutic target in cancer. In this context, a variety of small molecules has been documented to have excellent G-quadruplex binding/stabilizing properties and to exert good antiproliferative and antitumor activity in several in vitro and in vivo models of human cancers. Pieces of evidence indicate that targeting G-quadruplexes may represent an innovative and fascinating approach for the therapeutic management of the neoplastic disease. However, several issues still need to be addressed both at chemical and biological level before G-quadruplex-interacting molecules will turn out into effective therapeutic agents. Nevertheless, this has been an exciting, though sometime subdued, field of research over the last century. The continued improvements in methodologies and the development of specific tools will contribute not only to achieve the design and development of potentially novel anticancer approaches but also to deepen our knowledge of G-quadruplex biology and, consequently, of cancer at molecular level.

Detection of cis- and trans-acting Factors in DNA Structure-Induced Genetic Instability Using In silico and Cellular Approaches.

Sequences that can adopt alternative DNA structures (i.e., non-B DNA) are very abundant in mammalian genomes, and recent studies have revealed many important biological functions of non-B DNA structures in chromatin remodeling, DNA replication, transcription, and genetic instability. Here, we provide results from an in silico web-based search engine coupled with cell-based experiments to characterize the roles of non-B DNA conformations in genetic instability in eukaryotes. The purpose of this article is to illustrate strategies that can be used to identify and interrogate the biological roles of non-B DNA structures, particularly on genetic instability. We have included unpublished data using a short H-DNA-forming sequence from the human c-MYC promoter region as an example, and identified two different mechanisms of H-DNA-induced genetic instability in yeast and mammalian cells: a DNA replication-related model of mutagenesis; and a replication-independent cleavage model. Further, we identified candidate proteins involved in H-DNA-induced genetic instability by using a yeast genetic screen. A combination of in silico and cellular methods, as described here, should provide further insight into the contributions of non-B DNA structures in biological functions, genetic evolution, and disease development.

Impact of alternative DNA structures on DNA damage, DNA repair, and genetic instability.

Repetitive genomic sequences can adopt a number of alternative DNA structures that differ from the canonical B-form duplex (i.e. non-B DNA). These non-B DNA-forming sequences have been shown to have many important biological functions related to DNA metabolic processes; for example, they may have regulatory roles in DNA transcription and replication. In addition to these regulatory functions, non-B DNA can stimulate genetic instability in the presence or absence of DNA damage, via replication-dependent and/or replication-independent pathways. This review focuses on the interactions of non-B DNA conformations with DNA repair proteins and how these interactions impact genetic instability.

Microhomology-Mediated Mechanisms Underlie Non-Recurrent Disease-Causing Microdeletions of the FOXL2 Gene or Its Regulatory Domain

Genomic disorders are often caused by recurrent copy number variations (CNVs), with nonallelic homologous recombination (NAHR) as the underlying mechanism. Recently, several microhomology-mediated repair mechanisms—such as microhomology-mediated end-joining (MMEJ), fork stalling and template switching (FoSTeS), microhomology-mediated break-induced replication (MMBIR), serial replication slippage (SRS), and break-induced SRS (BISRS)—were described in the etiology of non-recurrent CNVs in human disease. In addition, their formation may be stimulated by genomic architectural features. It is, however, largely unexplored to what extent these mechanisms contribute to rare, locus-specific pathogenic CNVs. Here, fine-mapping of 42 microdeletions of the FOXL2 locus, encompassing FOXL2 (32) or its regulatory domain (10), serves as a model for rare, locus-specific CNVs implicated in genetic disease. These deletions lead to blepharophimosis syndrome (BPES), a developmental condition affecting the eyelids and the ovary. For breakpoint mapping we used targeted array-based comparative genomic hybridization (aCGH), quantitative PCR (qPCR), long-range PCR, and Sanger sequencing of the junction products. Microhomology, ranging from 1 bp to 66 bp, was found in 91.7% of 24 characterized breakpoint junctions, being significantly enriched in comparison with a random control sample. Our results show that microhomology-mediated repair mechanisms underlie at least 50% of these microdeletions. Moreover, genomic architectural features, like sequence motifs, non-B DNA conformations, and repetitive elements, were found in all breakpoint regions. In conclusion, the majority of these microdeletions result from microhomology-mediated mechanisms like MMEJ, FoSTeS, MMBIR, SRS, or BISRS. Moreover, we hypothesize that the genomic architecture might drive their formation by increasing the susceptibility for DNA breakage or promote replication fork stalling. Finally, our locus-centered study, elucidating the etiology of a large set of rare microdeletions involved in a monogenic disorder, can serve as a model for other clustered, non-recurrent microdeletions in genetic disease.

Two sequential cleavage reactions on cruciform DNA structures cause palindrome-mediated chromosomal translocations

Gross chromosomal rearrangements (GCRs), such as translocations, deletions or inversions, are often generated by illegitimate repair between two DNA breakages at regions with nucleotide sequences that might potentially adopt a non-B DNA conformation. We previously established a plasmid-based model system that recapitulates palindrome-mediated recurrent chromosomal translocations in humans, and demonstrated that cruciform DNA conformation is required for the translocation-like rearrangements. Here we show that two sequential reactions that cleave the cruciform structures give rise to the translocation: GEN1-mediated resolution that cleaves diagonally at the four-way junction of the cruciform and Artemis-mediated opening of the subsequently formed hairpin ends. Indeed, translocation products in human sperm reveal the remnants of this two-step mechanism. These two intrinsic pathways that normally fulfil vital functions independently, Holliday-junction resolution in homologous recombination and coding joint formation in rearrangement of antigen-receptor genes, act upon the unusual DNA conformation in concert and lead to a subset of recurrent GCRs in humans.

Mitochondrial DNA deletions are associated with non-B DNA conformations

Mitochondrial DNA (mtDNA) deletions are a primary cause of mitochondrial disease and are believed to contribute to the aging process and to various neurodegenerative diseases. Despite strong observational and experimental evidence, the molecular basis of the deletion process remains obscure. In this study, we test the hypothesis that the primary cause of mtDNA vulnerability to breakage resides in the formation of non-B DNA conformations, namely hairpin, cruciform and cloverleaf-like elements. Using the largest database of human mtDNA deletions built thus far (753 different cases), we show that site-specific breakage hotspots exist in the mtDNA. Furthermore, we discover that the most frequent deletion breakpoints occur within or near predicted structures, a result that is supported by data from transgenic mice with mitochondrial disease. There is also a significant association between the folding energy of an mtDNA region and the number of breakpoints that it harbours. In particular, two clusters of hairpins (near the D-loop 3′-terminus and the L-strand origin of replication) are hotspots for mtDNA breakage. Consistent with our hypothesis, the highest number of 5′- and 3′-breakpoints per base is found in the highly structured tRNA genes. Overall, the data presented in this study suggest that non-B DNA conformations are a key element of the mtDNA deletion process.

Premutation huntingtin allele adopts a non-B conformation and contains a hot spot for DNA damage

The expansion of a CAG trinucleotide repeat (TNR) sequence has been linked to several neurological disorders, for example, Huntington’s disease (HD). In HD, healthy individuals have 5–35 CAG repeats. Those with 36–39 repeats have the premutation allele, which is known to be prone to expansion. In the disease state, greater than 40 repeats are present. Interestingly, the formation of non-B DNA conformations by the TNR sequence is proposed to contribute to the expansion. Here we provide the first structural and thermodynamic analysis of a premutation length TNR sequence. Using chemical probes of nucleobase accessibility, we found that similar to (CAG) 10, the premutation length sequence (CAG) 36 forms a stem-loop hairpin and contains a hot spot for DNA damage. Additionally, calorimetric analysis of a series of (CAG) n sequences, that includes repeat tracts in both the healthy and premutation ranges, reveal that thermodynamic stability increases linearly with the number of repeats. Based on these data, we propose that while non-B conformations can be formed by TNR tracts found in both the healthy and premutation allele, only sequences containing at least 36 repeats have sufficient thermodynamic stability to contribute to expansion.

The polypyrimidine/polypurine motif in the mouse mu opioid receptor gene promoter is a supercoiling-regulatory element

The mu opioid receptor (MOR) is the principle molecular target of opioid analgesics. The polypyrimidine/polypurine (PPy/u) motif enhances the activity of the MOR gene promoter by adopting a non-B DNA conformation. Here, we report that the PPy/u motif regulates the processivity of torsional stress, which is important for endogenous MOR gene expression. Analysis by topoisomerase assays, S1 nuclease digests, and atomic force microscopy showed that, unlike homologous PPy/u motifs, the position- and orientation-induced structural strains to the mouse PPy/u element affect its ability to perturb the relaxation activity of topoisomerase, resulting in polypurine strand-nicked and catenated DNA conformations. Raman spectrum microscopy confirmed that mouse PPy/u containing-plasmid DNA molecules under the different structural strains have a different configuration of ring bases as well as altered Hoogsteen hydrogen bonds. The mouse MOR PPy/u motif drives reporter gene expression fortyfold more effectively in the sense orientation than in the antisense orientation. Furthermore, mouse neuronal cells activate MOR gene expression in response to the perturbations of topology by topoisomerase inhibitors, whereas human cells do not. These results suggest that, interestingly among homologous PPy/u motifs, the mouse MOR PPy/u motif dynamically responds to torsional stress and consequently regulates MOR gene expression in vivo.

Complete ascertainment of intragenic copy number mutations (CNMs) in the CFTR gene and its implications for CNM formation at other autosomal loci

Over the last 20 years since the discovery of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, more than 1,600 different putatively pathological CFTR mutations have been identified. Until now, however, copy number mutations (CNMs) involving the CFTR gene have not been methodically analyzed, resulting almost certainly in the underascertainment of CFTR gene duplications compared with deletions. Here, high-resolution array comparative genomic hybridization (averaging one interrogating probe every 95 bp) was used to analyze the entire length of the CFTR gene (189 kb) in 233 cystic fibrosis chromosomes lacking conventional mutations. We succeeded in identifying five duplication CNMs that would otherwise have been refractory to analysis. Based upon findings from this and other studies, we propose that deletion and duplication CNMs in the human autosomal genome are likely to be generated in the proportion of approximately 2-3:1. We further postulate that intragenic gene duplication CNMs in other disease loci may have been routinely underascertained. Finally, our analysis of +/-20 bp flanking each of the 40 CFTR breakpoints characterized at the DNA sequence level provide support for the emerging concept that non-B DNA conformations in combination with specific sequence motifs predispose to both recurring and nonrecurring genomic rearrangements.

Predictable suppression of gene expression by 5′-UTR-based RNA quadruplexes

Four-stranded DNA and RNA quadruplexes or G4 motifs are non-B DNA conformations that are presumed to form in vivo, although only few explicit evidence has been reported. Using bioinformatics the presence of putative DNA G-quadruplexes within critical promoter regions has been demonstrated and a regulatory role in transcription has been suspected. However, in genomic DNA the presence of the complementary strand interferes with the potential to form a quadruplex motif. Contrarily RNA G4 motifs have no such limitation and consequently strong interference with gene expression is suspected. Nevertheless, experimental evidence is scarce. Here we show a well-defined structure–function relationship of synthetic quadruplex sequences in 5′-UTRs in multiple mammalian cell-lines. We establish a universal ‘translational suppressor’ effect of these motifs on gene expression at the translational level and show for the first time that specific features such as loop-length and the number of ‘GGG’-repeats further determine the suppressive impact. Moreover, a consistent and predictable repression of gene expression is observed for naturally occurring RNA G4 motifs, augmenting the functional relevance of these unusual nucleic acid structures.

Structure-Dependent DNA Damage and Repair in a Trinucleotide Repeat Sequence

Triplet repeat sequences, such as CAG/CTG, expand in the human genome to cause several neurological disorders. As part of the expansion process the formation of non-B DNA conformations by the repeat sequence has previously been proposed. Furthermore, the base excision repair enzyme 7,8-dihydro-8-oxoguanine glycosylase (OGG1) has recently been implicated in the repeat expansion [Kovtun, I. V., Liu, Y., Bjoras, M., Klugland, A., Wilson, S. H., and McMurray, C. T. (2007) Nature 447, 447-452]. In this work we have found that the non-B conformation adopted by (CAG)(10), a hairpin, is hypersusceptible to DNA damage relative to the (CAG)(10)/(CTG)(10) duplex and, in particular, that a hot spot for DNA damage exists. Specifically, we find that a single guanine in the loop of the hairpin is susceptible to modification by peroxynitrite. Interestingly, we find that human OGG1 (hOGG1) is able to excise 7,8-dihydro-8-oxoguanine (8-oxoG) from the loop of a hairpin substrate, albeit with a marked decrease in efficiency relative to duplex substrates; the hOGG1 enzyme removes 8-oxoG from the loop of a hairpin with a rate that is approximately 700-fold slower than that observed for DNA duplex. Thus, while damage is preferentially generated in the loop of the hairpin, DNA repair is less efficient. These observed structure-dependent patterns of DNA damage and repair may contribute to the OGG1-dependent mechanism of trinucleotide repeat expansion.

Discovery of the Role of Non-B DNA Structures in Mutagenesis and Human Genomic Disorders

Discovery of the Role of Non-B DNA Structures in Mutagenesis and Human Genomic Disorders