Non‐B DNA Structure and Mutations Causing Human Genetic Disease


Besides the canonical right‐handed double helix, biologically important noncanonical deoxyribonucleic acid (DNA) secondary structures have been characterised, including quadruplexes, triplexes, slipped/hairpins, Z‐DNA and cruciforms, collectively termed non‐B DNA. Formation of non‐B DNA is mediated by repetitive sequence motifs, such as G‐rich sequences, purine/pyrimidine tracts, direct (tandem) repeats, alternating purine–pyrimidines and inverted repeats, respectively. Such repeats are abundant in the human genome and non‐B DNA occurs at specific genomic locations, supporting a role in gene regulation, RNA translation and protein function. Repetitive motifs are also found at sites of chromosomal alterations associated with both human genetic disease and cancer. Characterised by an inherent capacity to expand spontaneously, such sequences not only cause >30 neurological diseases but may also contribute to disease susceptibility. The formation of non‐B DNA structures is believed to promote genomic alterations by impeding efficient and error‐free DNA replication, transcription and repair.

Key Concepts

  • The structure of DNA is polymorphic as well as its sequence; besides canonical right‐handed double helix (B‐DNA), repetitive sequences can also adopt alternative (non‐B DNA) conformations such as quadruplexes, triplexes, slipped/hairpins, Z‐DNA and cruciforms.
  • Repetitive DNA sequences are found at particular locations within many human genes, suggesting that they can affect transcription and encode homopolymeric amino acid runs that are important for protein–protein and protein–DNA/RNA interactions.
  • G4 and Z‐DNA structures have been detected in cells through specific antibodies, mostly in correspondence of actively transcribed genes and, in the case of G4, at telomeres.
  • Copy number variation (CNV) is a form of genetic alteration that, by involving thousands of loci in the genome, contributes to human individuality.
  • Repetitive sequences capable of forming non‐B DNA are found at sites of chromosomal breaks, CNVs and other rearrangements such as translocations, deletions and gene conversion events, which can contribute to human genetic disease and cancer.
  • The recurrent translocation t(22;11) events associated with Emanuel syndrome are mediated by cruciform structures that occur at inverted repeats.
  • Tandem repeats (microsatellites) may expand within gene sequences, contributing to more than 30 neurological diseases; present in a variable number in genes in the population, they may contribute to human disease susceptibility.
  • An increasing number of enzymes are being reported that resolve non‐B DNA and RNA structures, and whose mutations lead to genomic instability and human disease; however, in some cases, the recognition of non‐B DNA structures is the cause of genetic instability.
  • lncRNAs repress gene expression by forming triplex structures with their target duplex DNA.
  • Non‐B DNA structures stimulate mutations via mechanisms that alter DNA synthesis, transcription and repair.

Keywords: non‐B DNA; microsatellites; copy number variation (CNV); triplet repeat diseases; translocations; DNA repair; double strand breaks (DSB); gene expression regulation; cancer genomes; repeat‐associated non‐AUG (RAN) translation

Figure 1. Non‐B DNA (deoxyribonucleic acid) structures formed by genomic repetitive sequences. (a) Most common non‐B DNA conformations, ribbon models of helical folding, repetitive motifs requirement and example of sequences. Centre dot, Watson–Crick hydrogen bond interactions; x,y, nucleotides in the spacer between repeats; L, lateral loop; D, diagonal loop and CR, chain reversal loop. For cruciform DNA, an extended conformation is shown. For triplex DNA, a 3′ RRY isomer is depicted in which the 3′‐half of the purine‐rich strand folds back to form the Hoogsteen‐bound third strand. For quadruplex DNA, an idealised structure is drawn to highlight the loop characteristics and the relative orientation of the syn and anti N‐glycosidic configurations. (b) RRY base triplets showing the Hoogsteen‐bound base (left). Thymine can be incorporated into RRY triplexes because of the symmetry of the carbonyl groups. (c) YRY base triplets showing the Hoogsteen‐bound pyrimidine and the stabilisation afforded by cytosine protonation. (d) G‐tetrad.
Figure 2. Cruciform‐mediated chromosomal t(11;22) translocation and the Emanuel syndrome. The PATRR sequences on human chromosome 11 (green) and 22 (black) are proposed to fold into large cruciform structures at some frequency during gametogenesis and be cleaved at the single‐stranded tips, resulting in double‐strand breaks (left insets). The broken chromosomal ends (middle) join aberrantly, yielding the derivative chromosomes der(11) and der(22) (right). Occasional inheritance of der(22), in addition to a normal karyotype, is responsible for the Emanuel syndrome in the offspring.
Figure 3. Triplet‐repeat expansion alters mRNA (messenger ribonucleic acid) function. (a) In DM1, CTG expansion in the 3′‐UTR (untranslated region) of the DMPK gene causes the ensuing mRNA to fold into a large and stable double‐stranded hairpin stabilised by U · U and G · C base pairs, which recruits muscle blind‐like (Drosophila) (MBNL1), a mediator of pre‐mRNA alternative splicing regulation. CUG‐hairpins also stimulate CUG RNA‐binding protein 1 (CUGBP1) hyperphosphorylation and stabilisation, which alter several events related to alternative splicing, mRNA translation and mRNA decay. (b) Sequestration of MBNL1 and CUGBP1 activation shift alternative splicing programs from the adult stage towards embryonic‐specific patterns, including activation of exon 5 inclusion of cardiac isoforms of TNNT2 (cTNT) during heart remodelling, exclusion of exon 11 in the insulin receptor (IR) pre‐mRNA and inclusion of stop‐containing exon in chloride channel 1 transcripts. Adapted from Lee and Cooper 2009 with kind permission © the Biochemical Society, Portland Press Ltd.
Figure 4. Gain‐of‐function by expanded G4 DNA‐forming repeats at the C9orf72 locus. (a) The C9orf72 gene is transcribed from two alternative transcription start sites (TSSs; Ex 1b and 1a) in three gene isoforms. The G4C2 repeat is located in intron 1 (white boxes, UTRs; gray boxes, coding exons; thin lines, introns) on the nontranscribed strand (thus, it is present on the sense RNA strand) between Ex 1a and 1b. (b) Normal alleles containing two to eight G4‐forming repeats are transcribed normally (top). In expanded alleles (bottom) transcription is reduced. The nontranscribed strand forms an ‘island’ of antiparallel G4 structures. The transcribed strand yields sense RNA with multiple parallel G4 DNA structures. Antisense transcription also takes place through the island, yielding antisense RNAs with potential secondary structure‐forming sequences. (c) The aberrant transcripts sequester RNA‐binding proteins, forming nuclear protein‐RNA foci (left); they also bind nucleolin in the nucleoli, where they prevent biogenesis of new ribosomal ribonucleic acids (rRNA; right). (d) Once transported to the cytoplasm, both sense and antisense transcripts undergo RAN translation in all three possible reading frames, thereby producing dipeptide repeat proteins (DPR) prone to aggregation in the cytoplasm, nucleus and the nucleolus. Reproduced with permission from Simone et al. 2015 © John Wiley and Sons.
Figure 5. Translocation and deletion breakpoints occur near non‐B DNA‐forming sequences in cancer genomes. (a) Schematic of a 1‐kb interval (bin) with the site of rearrangement (breakpoint) at the centre and 500 bps of flanking DNA sequence. The genomic location at each breakpoint identified in cancer patients by high‐throughput whole‐genome DNA sequencing and resolved at bp resolution was first mapped to the human reference genome, and 500 bps on either side of each breakpoint were sought for the occurrence of non‐B DNA‐forming sequences. (b) Number of triplex DNA‐forming repeats. (c) Number of inverted repeats. (d) Number of direct (tandem) repeats. (e) Number of G4 DNA‐forming repeats. (f) Number of Z‐DNA‐forming repeats. Contr1, 20 222 randomly generated sites throughout the human genome; Trans, 19 947 chromosomal translocation breakpoints; Delet, 46 365 deletion breakpoints. In most cases, the number of non‐B DNA‐forming repeats peaked at the breakpoint position, implying their involvement in triggering DNA strand breaks that may have elicited the genomic rearrangements. Reproduced with permission from Bacolla et al. 2016 © Oxford University Press.


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Further Reading

Bacolla A, Wang G and Vasquez KM (2015) New perspectives on DNA and RNA triplexes as effectors of biological activity. PLoS Genetics 11: e1005696.

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Wang G and Vasquez KM (2009) Models for chromosomal replication‐independent non‐B DNA structure‐induced genetic instability. Molecular Carcinogenesis 48: 286–298.

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Bacolla, Albino, Cooper, David N, Vasquez, Karen M, and Tainer, John A(Jan 2018) Non‐B DNA Structure and Mutations Causing Human Genetic Disease. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1002/9780470015902.a0022657.pub2]