58 Packaging trinucleotide repeats: the effect of CAG/CTG repeats on nucleosome formation

In neurological diseases such as fragile X syndrome, spinal and bulbar muscular atrophy, myotonic dystrophy, and Huntington’s disease, the molecular basis of pathogenicity is the presence of an expanded trinucleotide repeat (TNR) tract (Ashley & Warren, 1995). TNRs implicated in many of these diseases are composed of CAG/CTG repeats. For example, in healthy individuals 5–35, CAG/CTG TNR repeats are present in the huntingtin gene. However, individuals with 40 or greater repeats will develop Huntington’s disease (Andrew et al., 1993). We are particularly interested in how these TNR sequences are packaged in chromatin. Recent evaluations of CAG/CTG TNR sequences in our laboratory have demonstrated that the repeats increase the propensity for the DNA sequences to incorporate into nucleosomes, where nucleosomes represent the minimal unit of packaging in chromatin (Volle & Delaney, 2012). In this work, we are interested in determining the minimum number of CAG/CTG repeats required to confer a significant increase in nucleosome incorporation relative to sequences that lack the TNR sequence. By defining the changes imposed on these fundamental interactions by the presence of a CAG/CTG repeat tract, we will gain insight into the possible interactions that allow for the expansion of these TNR tracts.     

Instability of CAG and CTG trinucleotide repeats in Saccharomyces cerevisiae.

A quantitative genetic assay was developed to monitor alterations in tract lengths of trinucleotide repeat sequences in Saccharomyces cerevisiae. Insertion of (CAG)50 or (CTG)50 repeats into a promoter that drives expression of the reporter gene ADE8 results in loss of expression and white colony color. Contractions within the trinucleotide sequences to repeat lengths of 8 to 38 restore functional expression of the reporter, leading to red colony color. Reporter constructs including (CAG)50 or (CTG)50 repeat sequences were integrated into the yeast genome, and the rate of red colony formation was measured. Both orientations yielded high rates of instability (4 x 10(-4) to 18 x 10(-4) per cell generation). Instability depended on repeat sequences, as a control harboring a randomized (C,A,G)50 sequence was at least 100-fold more stable. PCR analysis of the trinucleotide repeat region indicated an excellent correlation between change in color phenotype and reduction in length of the repeat tracts. No preferential product sizes were observed. Strains containing disruptions of the mismatch repair gene MSH2, MSH3, or PMS1 or the recombination gene RAD52 showed little or no difference in rates of instability or distributions of products, suggesting that neither mismatch repair nor recombination plays an important role in large contractions of trinucleotide repeats in yeast.     

In vitro repair of DNA hairpins containing various numbers of CAG/CTG trinucleotide repeats

Expansion of CAG/CTG trinucleotide repeats (TNRs) in humans is associated with a number of neurological and neurodegenerative disorders including Huntington's disease. Increasing evidence suggests that formation of a stable DNA hairpin within CAG/CTG repeats during DNA metabolism leads to TNR instability. However, the molecular mechanism by which cells recognize and repair CAG/CTG hairpins is largely unknown. Recent studies have identified a novel DNA repair pathway specifically removing (CAG)(n)/(CTG)(n) hairpins, which is considered a major mechanism responsible for TNR instability. The hairpin repair (HPR) system targets the repeat tracts for incisions in the nicked strand in an error-free manner. To determine the substrate spectrum of the HPR system and its ability to process smaller hairpins, which may be the intermediates for CAG/CTG expansions, we constructed a series of CAG/CTG hairpin heteroduplexes containing different numbers of repeats (from 5 to 25) and examined their repair in human nuclear extracts. We show here that although repair efficiencies differ slightly among these substrates, removal of the individual hairpin structures all involve endonucleolytic incisions within the repeat tracts in the nicked DNA strand. Analysis of the repair intermediates defined specific incision sites for each substrate, which were all located within the repeat regions. Mismatch repair proteins are not required for, nor do they inhibit, the processing of smaller hairpin structures. These results suggest that the HPR system ensures CAG/CTG stability primarily by removing various sizes of (CAG)(n)/(CTG)(n) hairpin structures during DNA metabolism.     

Length-dependent energetics of (CTG)n and (CAG)n trinucleotide repeats

Trinucleotide repeats are involved in a number of debilitating diseases such as myotonic dystrophy. Twelve to seventy-five base-long (CTG)_n oligodeoxynucleotides were analysed using a combination of biophysical [UV-absorbance, circular dichroism and differential scanning calorimetry (DSC)] and biochemical methods (non-denaturing gel electrophoresis and enzymatic footprinting). All oligomers formed stable intramolecular structures under near physiological conditions with a melting temperature that was only weakly dependent on oligomer length. Thermodynamic analysis of the denaturation process by UV-melting and calorimetric experiments revealed an unprecedented length-dependent discrepancy between the enthalpy values deduced from model-dependent (UV-melting) and model-independent (calorimetry) experiments. Evidence for non-zero molar heat capacity changes was also derived from the analysis of the Arrhenius plots and DSC profiles. Such behaviour is analysed in the framework of an intramolecular ‘branched-hairpin’ model, in which long CTG oligomers do not fold into a simple long hairpin–stem intramolecular structure, but allow the formation of several independent folding units of unequal stability. We demonstrate that, for sequences ranging from 12 to 25 CTG repeats, an intramolecular structure with two loops is formed which we will call ‘bis-hairpin’. Similar results were also found for CAG oligomers, suggesting that this observation may be extended to various trinucleotide repeats-containing sequences.     

Contractions and expansions of CAG/CTG trinucleotide repeats occur during ectopic gene conversion in yeast,by a MUS81-independent mechanism

CAG/CTG trinucleotide repeat tracts expand and contract at a high rate during gene conversion in Saccharomyces cerevisiae. In order to characterize the mechanism responsible for such rearrangements, we built an experimental system based on the use of the rare cutter endonuclease I-SceI, to study the fate of trinucleotide repeat tracts during meiotic or mitotic (allelic or ectopic) gene conversion. After double-strand break (DSB) induced meiotic recombination, (CAG)(98) and (CAG)(255) are rearranged in 5% and 52% of the gene conversions, respectively, with similar proportions of contractions and expansions. No evidence of a meiotic hot spot activity associated with trinucleotide repeats could be found. When gene conversion is induced by a DSB during mitotic growth of the cells, no rearrangement of the repeat tracts is detected when the donor sequence is allelic to the recipient site of the DSB. However, when the donor sequence is at an ectopic location, frequent contractions and expansions of the repeat tract are found. No crossing-over associated with gene conversion could be detected. Mutants for the MUS81 gene, involved in the resolution of recombination intermediates, show a frequency of rearrangements identical with that of the wild-type strain. We concluded that trinucleotide repeat rearrangements occur frequently during ectopic but not during allelic recombination, by a mechanism that does not require crossover formation.     

Double-strand break repair can lead to high frequencies of deletions within short CAG/CTG trinucleotide repeats

Trinucleotide repeats undergo contractions and expansions in humans, leading in some cases to fatal neurological disorders. The mechanism responsible for these large size variations is unknown, but replication-slippage events are often suggested as a possible source of instability. We constructed a genetic screen that allowed us to detect spontaneous expansions/contractions of a short trinucleotide repeat in yeast. We show that deletion of RAD27, a gene involved in the processing of Okazaki fragments, increases the frequency of contractions tenfold. Repair of a chromosomal double-strand break (DSB) using a trinucleotide repeat-containing template induces rearrangements of the repeat with a frequency 60 times higher than the natural rate of instability of the same repeat. Our data suggest that both gene conversion and single-strand annealing are major sources of trinucleotide repeat rearrangements. Received: 8 January 1999 / Accepted: 17 March 1999     

Identification and mapping of 26 human testis mRNAs containing CAG/CTG repeats

Various types of pathologies, including neurodegenerative diseases, as well as different types of neoplasia, are related to genes exhibiting simple tandem repeat instabilities. In order to seek for new candidate genes for such disorders, we screened 4.10⁶ human testis cDNAs for CAG- and CTG-containing clones. Among 910 positive clones, we characterized 109 cDNAs corresponding to 26 independent mRNAs. Fourteen of these mRNAs represent new genes. The corresponding clones contain between 3 and 19 consecutive CAG or CTG triplets. We assigned 15 out of these 26 genes to 14 different human chromosomes. These genes represent new potential candidates for diseases associated with CAG or CTG repeat mutations. Received: 5 March 1998 / Accepted: 6 May 1998     

Replication and expansion of trinucleotide repeats in yeast

The mechanisms of trinucleotide repeat expansions, underlying more than a dozen hereditary neurological disorders, are yet to be understood. Here we looked at the replication of (CGG)(n) x (CCG)(n) and (CAG)(n) x (CTG)(n) repeats and their propensity to expand in Saccharomyces cerevisiae. Using electrophoretic analysis of replication intermediates, we found that (CGG)(n) x (CCG)(n) repeats significantly attenuate replication fork progression. Replication inhibition for this sequence becomes evident at as few as approximately 10 repeats and reaches a maximal level at 30 to 40 repeats. This is the first direct demonstration of replication attenuation by a triplet repeat in a eukaryotic system in vivo. For (CAG)(n) x (CTG)(n) repeats, on the contrary, there is only a marginal replication inhibition even at 80 repeats. The propensity of trinucleotide repeats to expand was evaluated in a parallel genetic study. In wild-type cells, expansions of (CGG)(25) x (CCG)(25) and (CAG)(25) x (CTG)(25) repeat tracts occurred with similar low rates. A mutation in the large subunit of the replicative replication factor C complex (rfc1-1) increased the expansion rate for the (CGG)(25) repeat approximately 50-fold but had a much smaller effect on the expansion of the (CTG)(25) repeat. These data show dramatic sequence-specific expansion effects due to a mutation in the lagging strand DNA synthesis machinery. Together, the results of this study suggest that expansions are likely to result when the replication fork attempts to escape from the stall site.     

CAG trinucleotide RNA repeats interact with RNA-binding proteins.

Genes associated with several neurological diseases are characterized by the presence of an abnormally long trinucleotide repeat sequence. By way of example, Huntington's disease (HD), is characterized by selective neuronal degeneration associated with the expansion of a polyglutamine-encoding CAG tract. Normally, this CAG tract is comprised of 11-34 repeats, but in HD it is expanded to > 37 repeats in affected individuals. The mechanism by which CAG repeats cause neuronal degeneration is unknown, but it has been speculated that the expansion primarily causes abnormal protein functioning, which in turn causes HD pathology. Other mechanisms, however, have not been ruled out. Interactions between RNA and RNA-binding proteins have previously been shown to play a role in the expression of several eukaryotic genes. Herein, we report the association of cytoplasmic proteins with normal length and extended CAG repeats, using gel shift and UV crosslinking assays. Cytoplasmic protein extracts from several rat brain regions, including the striatum and cortex, sites of neuronal degeneration in HD, contain a 63-kD RNA-binding protein that specifically interacts with these CAG-repeat sequences. These protein-RNA interactions are dependent on the length of the CAG repeat, with longer repeats binding substantially more protein. Two CAG repeat-binding proteins are present in human cortex and striatum; one comigrates with the rat protein at 63 kD, while the other migrates at 49 kD. These data suggest mechanisms by which RNA-binding proteins may be involved in the pathological course of trinucleotide repeat-associated neurological diseases.     

Small-molecule ligand induces nucleotide flipping in (CAG)n trinucleotide repeats

DNA trinucleotide repeats, particularly CXG, are common within the human genome. However, expansion of trinucleotide repeats is associated with a number of disorders, including Huntington disease, spinobulbar muscular atrophy and spinocerebellar ataxia. In these cases, the repeat length is known to correlate with decreased age of onset and disease severity. Repeat expansion of (CAG)n, (CTG)n and (CGG)n trinucleotides may be related to the increased stability of alternative DNA hairpin structures consisting of CXG-CXG triads with X-X mismatches. Small-molecule ligands that selectively bound to CAG repeats could provide an important probe for determining repeat length and an important tool for investigating the in vivo repeat extension mechanism. Here we report that napthyridine-azaquinolone (NA, 1) is a ligand for CAG repeats and can be used as a diagnostic tool for determining repeat length. We show by NMR spectroscopy that binding of NA to CAG repeats induces the extrusion of a cytidine nucleotide from the DNA helix.     

Selectable system for monitoring the instability of CTG/CAG triplet repeats in mammalian cells

Despite substantial progress in understanding the mechanism by which expanded CTG/CAG trinucleotide repeats cause neurodegenerative diseases, little is known about the basis for repeat instability itself. By taking advantage of a novel phenomenon, we have developed a selectable assay to detect contractions of CTG/CAG triplets. When inserted into an intron in the APRT gene or the HPRT minigene, long tracts of CTG/CAG repeats (more than about 33 repeat units) are efficiently incorporated into mRNA as a new exon, thereby rendering the encoded protein nonfunctional, whereas short repeat tracts do not affect the phenotype. Therefore, contractions of long repeats can be monitored in large cell populations, by selecting for HPRT(+) or APRT(+) clones. Using this selectable system, we determined the frequency of spontaneous contractions and showed that treatments with DNA-damaging agents stimulate repeat contractions. The selectable system that we have developed provides a versatile tool for the analysis of CTG/CAG repeat instability in mammalian cells. We also discuss how the effect of long CTG/CAG repeat tracts on splicing may contribute to the progression of polyglutamine diseases.     

Mismatch repair blocks expansions of interrupted trinucleotide repeats in yeast

Disease-causing expansions of trinucleotide repeats (TNRs) can occur very frequently. In contrast, expansions are rare if the TNR is interrupted (imperfect). The molecular mechanism stabilizing interrupted alleles and thereby preventing disease has been elusive. We show that mismatch repair is the major stabilizing force for interrupted TNRs in Saccharomyces cerevisiae. Interrupted alleles expand much more often when mismatch repair is blocked by mutation or by poorly corrected mispairs. These results suggest that interruptions lead to mismatched expansion precursors. In normal cells, expansions are prevented in trans by mismatch repair, which coexcises the mismatches plus the aberrant, TNR-mediated secondary structure that otherwise resists removal. This study indicates a novel role for mismatch repair in mutation avoidance and, potentially, in disease prevention.     

Genetic instabilities in (CTG.CAG) repeats occur by recombination.

The expansion of triplet repeat sequences (TRS) associated with hereditary neurological diseases is believed from prior studies to be due to DNA replication. This report demonstrates that the expansion of (CTG.CAG)(n) in vivo also occurs by homologous recombination as shown by biochemical and genetic studies. A two-plasmid recombination system was established in Escherichia coli with derivatives of pUC19 (harboring the ampicillin resistance gene) and pACYC184 (harboring the tetracycline resistance gene). The derivatives contained various triplet repeat inserts ((CTG.CAG), (CGG.CCG), (GAA.TTC), (GTC.GAC), and (GTG.CAC)) of different lengths, orientations, and extents of interruptions and a control non-repetitive sequence. The availability of the two drug resistance genes and of several unique restriction sites on the plasmids enabled rigorous genetic and biochemical analyses. The requirements for recombination at the TRS include repeat lengths >30, the presence of CTG.CAG on both plasmids, and recA and recBC. Sequence analyses on a number of DNA products isolated from individual colonies directly demonstrated the crossing-over and expansion of the homologous CTG.CAG regions. Furthermore, inversion products of the type [(CTG)(13)(CAG)(67)].[(CTG)(67)(CAG)(13)] were isolated as the apparent result of "illegitimate" recombination events on intrahelical pseudoknots. This work establishes the relationships between CTG.CAG sequences, multiple fold expansions, genetic recombination, formation of new recombinant DNA products, and the presence of both drug resistance genes. Thus, if these reactions occur in humans, unequal crossing-over or gene conversion may also contribute to the expansions responsible for anticipation associated with several hereditary neurological syndromes.