Conformational and migrational dynamics of slipped-strand DNA three-way junctions containing trinucleotide repeats.
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ABSTRACT:
SUBMITTER: Hu T
PROVIDER: S-EPMC7794359 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The disease-associated expansion of (CTG)*(CAG) repeats is likely to involve slipped-strand DNAs. There are two types of slipped DNAs (S-DNAs): slipped homoduplex S-DNAs are formed between two strands having the same number of repeats; and heteroduplex slipped intermediates (SI-DNAs) are formed between two strands having different numbers of repeats. We present the first characterization of S-DNAs formed by disease-relevant lengths of (CTG)*(CAG) repeats which contained all predicted components including slipped-out repeats and slip-out junctions, where two arms of the three-way junction were composed of complementary paired repeats. In S-DNAs multiple short slip-outs of CTG or CAG repeats occurred throughout the repeat tract. Strikingly, in SI-DNAs most of the excess repeats slipped-out at preferred locations along the fully base-paired Watson-Crick duplex, forming defined three-way slip-out junctions. Unexpectedly, slipped-out CAG and slipped-out CTG repeats were predominantly in the random-coil and hairpin conformations, respectively. Both the junctions and the slip-outs could be recognized by DNA metabolizing proteins: only the strand with the excess repeats was hypersensitive to cleavage by the junction-specific T7 endonuclease I, while slipped-out CAG was preferentially bound by single-strand binding protein. An excellent correlation was observed for the size of the slip-outs in S-DNAs and SI-DNAs with the size of the tract length changes observed in quiescent and proliferating tissues of affected patients-suggesting that S-DNAs and SI-DNAs are mutagenic intermediates in those tissues, occurring during error-prone DNA metabolism and replication fork errors.
| S-EPMC137136 | biostudies-literature
Project description:Expansions of (CTG)·(CAG) repeated DNAs are the mutagenic cause of 14 neurological diseases, likely arising through the formation and processing of slipped-strand DNAs. These transient intermediates of repeat length mutations are formed by out-of-register mispairing of repeat units on complementary strands. The three-way slipped-DNA junction, at which the excess repeats slip out from the duplex, is a poorly understood feature common to these mutagenic intermediates. Here, we reveal that slipped junctions can assume a surprising number of interconverting conformations where the strand opposite the slip-out either is fully base paired or has one or two unpaired nucleotides. These unpaired nucleotides can also arise opposite either of the nonslipped junction arms. Junction conformation can affect binding by various structure-specific DNA repair proteins and can also alter correct nick-directed repair levels. Junctions that have the potential to contain unpaired nucleotides are repaired with a significantly higher efficiency than constrained fully paired junctions. Surprisingly, certain junction conformations are aberrantly repaired to expansion mutations: misdirection of repair to the non-nicked strand opposite the slip-out leads to integration of the excess slipped-out repeats rather than their excision. Thus, slipped-junction structure can determine whether repair attempts lead to correction or expansion mutations.
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Project description:DNA trinucleotide repeats (TRs) can exhibit dynamic expansions by integer numbers of trinucleotides that lead to neurodegenerative disorders. Strand slipped hairpins during DNA replication, repair and/or recombination may contribute to TR expansion. Here, we combine single-molecule FRET experiments and molecular dynamics studies to elucidate slipping dynamics and conformations of (CAG)n TR hairpins. We directly resolve slipping by predominantly two CAG units. The slipping kinetics depends on the even/odd repeat parity. The populated states suggest greater stability for 5'-AGCA-3' tetraloops, compared with alternative 5'-CAG-3' triloops. To accommodate the tetraloop, even(odd)-numbered repeats have an even(odd) number of hanging bases in the hairpin stem. In particular, a paired-end tetraloop (no hanging TR) is stable in (CAG)n = even, but such situation cannot occur in (CAG)n = odd, where the hairpin is "frustrated'' and slips back and forth between states with one TR hanging at the 5' or 3' end. Trinucleotide interrupts in the repeating CAG pattern associated with altered disease phenotypes select for specific conformers with favorable loop sequences. Molecular dynamics provide atomic-level insight into the loop configurations. Reducing strand slipping in TR hairpins by sequence interruptions at the loop suggests disease-associated variations impact expansion mechanisms at the level of slipped hairpins.
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Project description:Slipped-strand DNAs, formed by out-of-register mispairing of repeat units on complementary strands, were proposed over 55 years ago as transient intermediates in repeat length mutations, hypothesized to cause at least 40 neurodegenerative diseases. While slipped-DNAs have been characterized in vitro, evidence of slipped-DNAs at an endogenous locus in biologically relevant tissues, where instability varies widely, is lacking. Here, using an anti-DNA junction antibody and immunoprecipitation, we identify slipped-DNAs at the unstable trinucleotide repeats (CTG)n•(CAG)n of the myotonic dystrophy disease locus in patient brain, heart, muscle and other tissues, where the largest expansions arise in non-mitotic tissues such as cortex and heart, and are smallest in the cerebellum. Slipped-DNAs are shown to be present on the expanded allele and in chromatinized DNA. Slipped-DNAs are present as clusters of slip-outs along a DNA, with each slip-out having 1-100 extrahelical repeats. The allelic levels of slipped-DNA containing molecules were significantly greater in the heart over the cerebellum (relative to genomic equivalents of pre-IP input DNA) of a DM1 individual; an enrichment consistent with increased allelic levels of slipped-DNA structures in tissues having greater levels of CTG instability. Surprisingly, this supports the formation of slipped-DNAs as persistent mutation products of repeat instability, and not merely as transient mutagenic intermediates. These findings further our understanding of the processes of mutation and genetic variation.
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Project description:The virulence and pathogenicity of bacterial pathogens are related to their adaptability to changing environments. One process enabling adaptation is based on minor changes in genome sequence, as small as a few base pairs, within segments of genome called simple sequence repeats (SSRs) that consist of multiple copies of a short sequence (from one to several nucleotides), repeated in series. SSRs are found in eukaryotes as well as prokaryotes, and length variation in them occurs at frequencies up to a million-fold higher than bacterial point mutations through the process of slipped strand mispairing (SSM) by DNA polymerase during replication. The characterization of SSR length by standard sequencing methods is complicated by the appearance of length variation introduced during the sequencing process that obscures the lower abundance repeat number variants in a population. Here we report a computational approach to correct for sequencing process-induced artifacts, validated for tetranucleotide repeats by use of synthetic constructs of fixed, known length. We apply this method to a laboratory culture of Histophilus somni, prepared from a single colony, and demonstrate that the culture consists of populations of distinct sequence phase and length variants at individual tetranucleotide SSR loci.
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Project description:The three-way junctions contained in X-ray structures of folded RNAs have been compiled and analyzed. Three-way junctions with two helices approximately coaxially stacked can be divided into three main families depending on the relative lengths of the segments linking the three Watson-Crick helices. Each family has topological characteristics with some conservation in the non-Watson-Crick pairs within the linking segments as well as in the types of contacts between the segments and the helices. The most populated family presents tertiary interactions between two helices as well as extensive shallow/minor groove contacts between a linking segment and the third helix. On the basis of the lengths of the linking segments, some guidelines could be deduced for choosing a topology for a three-way junction on the basis of a secondary structure. Examples and prediction bas'ed on those rules are discussed.
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Project description:The standard slipped-strand mispairing (SSM) model for the formation of variable number tandem repeats (VNTRs) proposes that a few tandem repeats, produced by chance mutations, provide the "raw material" for VNTR expansion. However, this model is unlikely to explain the formation of VNTRs with long motifs (e.g., minisatellites), because the likelihood of a tandem repeat forming by chance decreases rapidly as the length of the repeat motif increases. Phylogenetic reconstruction of the birth of a mitochondrial (mt) DNA minisatellite in guppies suggests that VNTRs with long motifs can form as a consequence of SSM at noncontiguous repeats. VNTRs formed in this manner have motifs longer than the noncontiguous repeat originally formed by chance and are flanked by one unit of the original, noncontiguous repeat. SSM at noncontiguous repeats can therefore explain the birth of VNTRs with long motifs and the "imperfect" or "short direct" repeats frequently observed adjacent to both mtDNA and nuclear VNTRs.
| S-EPMC1461170 | biostudies-other
Project description:Trinucleotide repeat (TNR) expansions are present in a wide range of genes involved in several neurological disorders, being directly involved in the molecular mechanisms underlying pathogenesis through modulation of gene expression and/or the function of the RNA or protein it encodes. Structural and functional information on the role of TNR sequences in RNA and protein is crucial to understand the effect of TNR expansions in neurodegeneration. Therefore, this review intends to provide to the reader a structural and functional view of TNR and encoded homopeptide expansions, with a particular emphasis on polyQ expansions and its role at inducing the self-assembly, aggregation and functional alterations of the carrier protein, which culminates in neuronal toxicity and cell death. Detail will be given to the Machado-Joseph Disease-causative and polyQ-containing protein, ataxin-3, providing clues for the impact of polyQ expansion and its flanking regions in the modulation of ataxin-3 molecular interactions, function, and aggregation.
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Project description:The etiology of a large class of inherited neurological diseases is founded on hairpin structures adopted by repeated DNA sequences, and this folding is determined by base sequence and DNA context. Using single substitutions of adenine with 2-aminopurine, we show that intrastrand folding in repeated CAG trinucleotides is also determined by the number of repeats. This isomeric analogue has a fluorescence quantum yield that varies strongly with solvent exposure, thereby distinguishing particular DNA motifs. Prior studies demonstrated that (CAG)(8) alone favors a stem-loop hairpin, yet the same sequence adopts an open loop conformation in a three-way junction. This comparison suggests that repeat folding is disrupted by base pairing in the duplex arms and by purine-purine mismatches in the repeat stem. However, these perturbations are overcome in longer CAG repeats, as demonstrated by studies of isolated and integrated forms of (CAG)(15). The oligonucleotide alone forms a symmetrically folded hairpin with looplike properties exhibited by the relatively high emission intensities from a modification in the central eighth repeat and with stemlike properties evident from the relatively low emission intensities from peripheral modifications. Significantly, these hairpin properties are retained when (CAG)(15) is integrated into a duplex. Intrastrand folding by (CAG)(15) in the three-way junction contrasts with the open loop adopted by (CAG)(8) in the analogous context. This distinction suggests that cooperative interactions in longer repeat tracts overwhelm perturbations to reassert the natural folding propensity. Given that anomalously long repeats are the genetic basis of a large class of inherited neurological diseases, studies with (CAG)-based three-way junctions suggest that their secondary structure is a key factor in the length-dependent manifestation and progression of such diseases.
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Project description:Toehold-mediated strand displacement is widely used to construct and operate DNA nanodevices. Cooperative regulation of strand displacement with diverse factors is pivotal in the design and construction of functional and dynamic devices. Herein, a cofactor-assisted three-way DNA junction-driven strand displacement strategy was reported, which could tune the reaction kinetics by the collaboration of DNA and other types of stimulus. This strategy is responsive to various inputs by incorporation of the specific sequence into the three-way junction structure. Specifically, the cooperation of multiple factors changes the conformation of the specific domain and promotes the reaction. To demonstrate the strategy, adenosine triphosphate (ATP), HG2+, and pH were used as cofactors to modulate the displacement reaction. The electrophoresis and fluorescence experiments showed that the cooperative regulation of the strand displacement reaction could be achieved by diverse factors using this strategy. The proposed strategy provides design flexibility for dynamic DNA devices and may have potential in biosensing and biocomputing.
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