DNA triplet repeat expansion and mismatch repair.

DNA mismatch repair is a conserved antimutagenic pathway that maintains genomic stability through rectification of DNA replication errors and attenuation of chromosomal rearrangements. Paradoxically, mutagenic action of mismatch repair has been implicated as a cause of triplet repeat expansions that cause neurological diseases such as Huntington disease and myotonic dystrophy. This mutagenic process requires the mismatch recognition factor MutSß and the MutLα (and/or possibly MutLγ) endonuclease, and is thought to be triggered by the transient formation of unusual DNA structures within the expanded triplet repeat element. This review summarizes the current knowledge of DNA mismatch repair involvement in triplet repeat expansion, which encompasses in vitro biochemical findings, cellular studies, and various in vivo transgenic animal model experiments. We present current mechanistic hypotheses regarding mismatch repair protein function in mediating triplet repeat expansions and discuss potential therapeutic approaches targeting the mismatch repair pathway.

Expanded complexity of unstable repeat diseases.

Unstable repeat diseases (URDs) share a common mutational phenomenon of changes in the copy number of short, tandemly repeated DNA sequences. More than 20 human neurological diseases are caused by instability, predominantly, expansion of microsatellite sequences. Changes in the repeat size initiate a cascade of pathological processes, frequently characteristic of a unique disease or a small subgroup of the URDs. Understanding of both the mechanism of repeat instability and molecular consequences of the repeat expansions is critical to developing successful therapies for these diseases. Recent technological breakthroughs in whole genome, transcriptome and proteome analyses will almost certainly lead to new discoveries regarding the mechanisms of repeat instability, the pathogenesis of URDs, and will facilitate development of novel therapeutic approaches. The aim of this review is to give a general overview of unstable repeats diseases, highlight the complexities of these diseases, and feature the emerging discoveries in the field.

Scott: an 11-year-old boy with repetitive lying.

CASE: Scott, an 11-year-old boy in the fifth grade, is brought to his pediatrician, Dr. Lewis, by his maternal grandparents with the principle concern that "he lies constantly." Scott lived with his maternal grandparents since he was 2 years old, and they have full custody. His mother and father had serious substance abuse problems. The grandparents provide a stable home for Scott and his 15-year-old sister. Scott has had no contact with his mother in more than 6 years and sees his father infrequently. During the last visit with his father, he was so inebriated that he was thrown out of the movie theatre and barely avoided several car accidents on the way home. He left the children at the curb of their home and made them promise that they would lie to their grandparents about the reasons for the early return. Scott was diagnosed with attention-deficit hyperactivity disorder (ADHD) in second grade. Methylphenidate (36 mg) provides improvement in attention and concentration. His grandfather describes Scott as highly unpredictable. When he is the "good Jake," he is eager to help, polite, and caring. When Scott gets behind in school or is avoiding his chores and assignments, he lies by saying that he got it all done, even though he knows his grandfather will discover the lie and punish him. When confronted with reports from school, Scott often lies and may develop more elaborate confabulatory stories. His grandfather admits that he becomes irate at these moments. He responds by removing Scott's privileges. When he planned to take Scott to see his favorite sport team in the playoffs, Scott was caught in a lie the day of his departure. His grandfather offered him a chance to fess up, pay a small price in extra chores, and save the trip. Scott stubbornly refused to admit that he lied and lost the trip. His grandfather worries that Scott has no "moral compass." He takes things that do not belong to him and violates household curfew rules. He has never been physically aggressive or has never stole items from a store. He takes his sister's CD player or his grandfather's cell phone even when he has been told not to. He will then lie that he did not take it. Even when it is pulled out of his backpack, he will say he did not put it in there. His grandfather is a businessman with high moral integrity. He loves his grandson and is eager to help him. He asks Dr. Lewis what they should do about Scott's persistent lying.

R loops stimulate genetic instability of CTG.CAG repeats.

Transcription stimulates the genetic instability of trinucleotide repeat sequences. However, the mechanisms leading to transcription-dependent repeat length variation are unclear. We demonstrate, using biochemical and genetic approaches, that the formation of stable RNA.DNA hybrids enhances the instability of CTG.CAG repeat tracts. In vitro transcribed CG-rich repeating sequences, unlike AT-rich repeats and nonrepeating sequences, form stable, ribonuclease A-resistant structures. These RNA.DNA hybrids are eliminated by ribonuclease H treatment. Mutation in the rnhA1 gene that decreases the activity of ribonuclease HI stimulates the instability of CTG.CAG repeats in E. coli. Importantly, the effect of ribonuclease HI depletion on repeat instability requires active transcription. We also showed that transcription-dependent CTG.CAG repeat instability in human cells is stimulated by siRNA knockdown of RNase H1 and H2. In addition, we used bisulfite modification, which detects single-stranded DNA, to demonstrate that the nontemplate DNA strand at transcribed CTG.CAG repeats remains partially single-stranded in human genomic DNA, thus indicating that it is displaced by an RNA.DNA hybrid. These studies demonstrate that persistent hybrids between the nascent RNA transcript and the template DNA strand at CTG.CAG tracts promote instability of DNA trinucleotide repeats.

Gene conversion causing human inherited disease: evidence for involvement of non-B-DNA-forming sequences and recombination-promoting motifs in DNA breakage and repair.

A variety of DNA sequence motifs including inverted repeats, minisatellites, and the chi recombination hotspot, have been reported in association with gene conversion in human genes causing inherited disease. However, no methodical statistically based analysis has been performed to formalize these observations. We have performed an in silico analysis of the DNA sequence tracts involved in 27 nonoverlapping gene conversion events in 19 different genes reported in the context of inherited disease. We found that gene conversion events tend to occur within (C+G)- and CpG-rich regions and that sequences with the potential to form non-B-DNA structures, and which may be involved in the generation of double-strand breaks that could, in turn, serve to promote gene conversion, occur disproportionately within maximal converted tracts and/or short flanking regions. Maximal converted tracts were also found to be enriched (P<0.01) in a truncated version of the chi-element (a TGGTGG motif), immunoglobulin heavy chain class switch repeats, translin target sites and several novel motifs including (or overlapping) the classical meiotic recombination hotspot, CCTCCCCT. Finally, gene conversions tend to occur in genomic regions that have the potential to fold into stable hairpin conformations. These findings support the concept that recombination-inducing motifs, in association with alternative DNA conformations, can promote recombination in the human genome.

Non-B DNA conformations as determinants of mutagenesis and human disease.

Repetitive DNA motifs may fold into non-B DNA structures, including cruciforms/hairpins, triplexes, slipped conformations, quadruplexes, and left-handed Z-DNA, thereby representing chromosomal targets for DNA repair, recombination, and aberrant DNA synthesis leading to repeat expansion or genomic rearrangements associated with neurodegenerative and genomic disorders. Hairpins and quadruplexes also determined the relative abundances of simple sequence repeats (SSR) in vertebrate genomes, whereas strong base stacking has permitted the expansion of purine.pyrimidine-rich SSR during evolutionary time. SSR are enriched in regulatory and cancer-related gene classes, where they have been actively recruited to participate in both gene and protein functions. SSR polymorphic alleles in the population are associated with cancer susceptibility, including within genes that appear to share regulatory circuits involving reactive oxygen species.

Mutation spectra in fragile X syndrome induced by deletions of CGG*CCG repeats.

The fragile X syndrome results from expansions as well as deletions of the repeating CGG.CCG DNA sequence in the 5'-untranslated region of the FMR1 gene on the X chromosome. The relative frequency of disease cases promoted by these two types of mutations cannot be ascertained at present because the routine clinical assay monitors only expansions. At least 30 articles have been reviewed that document the involvement of deletions of part or all of the CGG.CCG repeats along with varying extents of DNA flanking regions as well as very small mutations including single base pair changes. Studies of deletion mutants of CGG.CCG tracts in Escherichia coli plasmids revealed a similar spectrum of mutagenic products. The triplet repeat tract in a non-B conformation is the mutagen, not the sequence per se in the right-handed B helix. Hence, molecular investigations in a simple model organism may generate useful initial information toward therapeutic strategies for this disease.

Abundance and length of simple repeats in vertebrate genomes are determined by their structural properties.

Microsatellites are abundant in vertebrate genomes, but their sequence representation and length distributions vary greatly within each family of repeats (e.g., tetranucleotides). Biophysical studies of 82 synthetic single-stranded oligonucleotides comprising all tetra- and trinucleotide repeats revealed an inverse correlation between the stability of folded-back hairpin and quadruplex structures and the sequence representation for repeats > or =30 bp in length in nine vertebrate genomes. Alternatively, the predicted energies of base-stacking interactions correlated directly with the longest length distributions in vertebrate genomes. Genome-wide analyses indicated that unstable sequences, such as CAG:CTG and CCG:CGG, were over-represented in coding regions and that micro/minisatellites were recruited in genes involved in transcription and signaling pathways, particularly in the nervous system. Microsatellite instability (MSI) is a hallmark of cancer, and length polymorphism within genes can confer susceptibility to inherited disease. Sequences that manifest the highest MSI values also displayed the strongest base-stacking interactions; analyses of 62 tri- and tetranucleotide repeat-containing genes associated with human genetic disease revealed enrichments similar to those noted for micro/minisatellite-containing genes. We conclude that DNA structure and base-stacking determined the number and length distributions of microsatellite repeats in vertebrate genomes over evolutionary time and that micro/minisatellites have been recruited to participate in both gene and protein function.

DNA triplexes and Friedreich ataxia.

Friedreich ataxia, the most common inherited ataxia, is caused by the transcriptional silencing of the FXN gene, which codes for the 210 amino acid frataxin, a mitochondrial protein involved in iron-sulfur cluster biosynthesis. The expansion of the GAA x TTC tract in intron 1 to as many as 1700 repeats elicits the transcriptional silencing by the formation of non-B DNA structures (triplexes or sticky DNA), the formation of a persistent DNA x RNA hybrid, or heterochromatin formation. The triplex (sticky DNA) adopted by the long repeat sequence also elicits profound mutagenic, genetic instability, and recombination behaviors. Early stage therapeutic investigations involving polyamides or histone deacetylase inhibitors are being pursued. Friedreich ataxia may be one of the most thoroughly studied hereditary neurological disease from a pathophysiological standpoint.

Fragile X repeats are potent inducers of complex, multiple site rearrangements in flanking sequences in Escherichia coli.

(CGG.CCG)n repeats induce the formation of complex, multiple site rearrangements and/or gross deletions in flanking DNA sequences in Escherichia coli plasmids. DNA sequence analyses of mutant clones revealed the influence of (a) the length (24, 44 or 73 repeats), (b) the orientation of the CGG.CCG region relative to the unidirectional origin, and (c) its transcription status. Complex rearrangements had occurred in the mutant clones since some products contained deletions, inversions and insertions and some products had only gross deletions. Furthermore, the CGG.CCG repeats repeatedly induced, up to 22 times, the formation of identical (to the bp) mutagenic products indicating the powerful nature of the complex processes involved. Also, the mutations were bidirectional from the CGG.CCG tract. The healed junctions had CG-rich microhomologies of 1-6bp, CG-rich regions and putative cruciforms and slipped structures. Hence, the fragile X syndrome mutagenic spectrum has been found, at least in part, in our model system.

Advancements in the pathophysiology of Friedreich's Ataxia and new prospects for treatments.

On November 9-12, 2006, the Friedreich's Ataxia Research Alliance (FARA) and the National Institutes of Health (NIH) hosted the Third International Friedreich's Ataxia (FRDA) Scientific Conference at the NIH in Bethesda, Maryland, highlighting the exciting research leading now to a variety of clinical trials that show promise of effective treatments for this devastating disorder. Nearly 150 leading FRDA scientists from around the world discussed their new insights and findings. The presence of six pharmaceutical and biotechnology companies underscored the importance of the public-private partnership that has grown in the past years. Some of these companies are already involved in advancing promising drug compounds into clinical trials, while others are eager to help take newer discoveries through drug development and into subsequent clinical trials. National Institute of Neurological Disorders and Stroke (NINDS) Director Dr. Story Landis noted in her opening remarks for the conference that there was a "palpable sense of energy, excitement, and enthusiasm" over the scientific progress made since the FRDA gene was discovered over 10 years ago.

Non-B DNA conformations, mutagenesis and disease.

Recent discoveries have revealed that simple repeating DNA sequences, which are known to adopt non-B DNA conformations (such as triplexes, cruciforms, slipped structures, left-handed Z-DNA and tetraplexes), are mutagenic. The mutagenesis is due to the non-B DNA conformation rather than to the DNA sequence per se in the orthodox right-handed Watson-Crick B-form. The human genetic consequences of these non-B structures are approximately 20 neurological diseases, approximately 50 genomic disorders (caused by gross deletions, inversions, duplications and translocations), and several psychiatric diseases involving polymorphisms in simple repeating sequences. Thus, the convergence of biochemical, genetic and genomic studies has demonstrated a new paradigm implicating the non-B DNA conformations as the mutagenesis specificity determinants, not the sequences as such.

Double-strand breaks in the myotonic dystrophy type 1 and the fragile X syndrome triplet repeat sequences induce different types of mutations in DNA flanking sequences in Escherichia coli.

The putative role of double-strand breaks (DSBs) created in vitro by restriction enzyme cleavage in or near CGG*CCG or CTG*CAG repeat tracts on their genetic instabilities, both within the repeats and in their flanking sequences, was investigated in an Escherichia coli plasmid system. DSBs at TRS junctions with the vector generated a large number of mutagenic events in flanking sequences whereas DSBs within the repeats elicited no similar products. A substantial enhancement in the number of mutants was caused by transcription of the repeats and by the absence of recombination functions (recA-, recBC-). Surprisingly, DNA sequence analyses on mutant clones revealed the presence of only single deletions of 0.4-1.6 kb including the TRS and the flanking sequence from plasmids originally containing (CGG*CCG)43 but single, double and multiple deletions as well as insertions were found for plasmids originally containing (CTG*CAG)n (where n = 43 or 70). Non-B DNA structures (slipped structures with loops, cruciforms, triplexes and tetraplexes) as well as microhomologies are postulated to participate in the recombination and/or repair processes.

DNA sequence-specific polyamides alleviate transcription inhibition associated with long GAA.TTC repeats in Friedreich's ataxia.

The DNA abnormality found in 98% of Friedreich's ataxia (FRDA) patients is the unstable hyperexpansion of a GAA.TTC triplet repeat in the first intron of the frataxin gene. Expanded GAA.TTC repeats result in decreased transcription and reduced levels of frataxin protein in affected individuals. Beta-alanine-linked pyrrole-imidazole polyamides bind GAA.TTC tracts with high affinity and disrupt the intramolecular DNA.DNA-associated region of the sticky-DNA conformation formed by long GAA.TTC repeats. Fluorescent polyamide-Bodipy conjugates localize in the nucleus of a lymphoid cell line derived from a FRDA patient. The synthetic ligands increase transcription of the frataxin gene in cell culture, resulting in increased levels of frataxin protein. DNA microarray analyses indicate that a limited number of genes are significantly affected in FRDA cells. Polyamides may increase transcription by altering the DNA conformation of genes harboring long GAA.TTC repeats or by chromatin opening.

The involvement of non-B DNA structures in gross chromosomal rearrangements.

Non-B DNA conformations adopted by certain types of DNA sequences promote genetic instabilities, especially gross rearrangements including translocations. We conclude the following: (a) slipped (hairpin) structures, cruciforms, triplexes, tetraplexes and i-motifs, and left-handed Z-DNA are formed in chromosomes and elicit profound genetic consequences via recombination-repair, (b) repeating sequences, probably in their non-B conformations, cause gross genomic rearrangements (translocations, deletions, insertions, inversions, and duplications), and (c) these rearrangements are the genetic basis for numerous human diseases including polycystic kidney disease, adrenoleukodystrophy, follicular lymphomas, and spermatogenic failure.

Sticky DNA: in vivo formation in E. coli and in vitro association of long GAA*TTC tracts to generate two independent supercoiled domains.

The expanded GAA*TTC repeat sequence associated with Friedreich's ataxia (FRDA) adopts non-B DNA structures, (triplexes and sticky DNA). Sticky DNA is formed in plasmids by the association of two long GAA*TTC tracts at lengths that are found in the sequence of the frataxin gene in patients. Most FRDA patients have expanded GAA*TTC repeats (up to 1700 triplets), which inhibit the transcription of the gene, thus diminishing the synthesis of frataxin, a mitochondrial protein involved in iron-sulfur cluster biogenesis. Negative supercoiling and MgCl(2) (or MnCl(2)) are required to stabilize sticky DNA (a dumbbell-shaped structure) in plasmids with a pair of repeat tracts where n> or =60 in the direct repeat orientation in vitro. Since the triplet repeat sequences (TRS) were symmetrically positioned in the plasmids and because a number of unique restriction sites were present in the vector, studies were conducted to evaluate the influence of selectively linearizing one or the other supercoiled domains created by the DNA*DNA associated region, i.e. the stable complex at the pair of TRS's. The two domains behave independently, thus confirming the association of the two tracts and the dumbbell-shaped plasmid in our model for sticky DNA. Linking number investigations were performed on a family of plasmids harboring different lengths (30, 60, or 176 repeats), orientations and number of tracts (one or two) of a GAA*TTC repeat in Escherichia coli to evaluate the in vivo role, if any, of sticky DNA. Unexpectedly, this non-B DNA conformation elicited the formation of a TRS-length dependent change in the global topology of the plasmids, indicative of an apparent compression of the primary helices. Thus, linking number determinations confirm that sticky DNA has an important consequence in vivo.

Non-B DNA conformations formed by long repeating tracts of myotonic dystrophy type 1, myotonic dystrophy type 2, and Friedreich's ataxia genes, not the sequences per se, promote mutagenesis in flanking regions.

The expansions of long repeating tracts of CTG.CAG, CCTG.CAGG, and GAA.TTC are integral to the etiology of myotonic dystrophy type 1 (DM1), myotonic dystrophy type 2 (DM2), and Friedreich's ataxia (FRDA). Essentially all studies on the molecular mechanisms of this expansion process invoke an important role for non-B DNA conformations which may be adopted by these repeat sequences. We have directly evaluated the role(s) of the repeating sequences per se, or of the non-B DNA conformations formed by these sequences, in the mutagenic process. Studies in Escherichia coli and three types of mammalian (COS-7, CV-1, and HEK-293) fibroblast-like cells revealed that conditions which promoted the formation of the non-B DNA structures enhanced the genetic instabilities, both within the repeat sequences and in the flanking sequences of up to approximately 4 kbp. The three strategies utilized included: the in vivo modulation of global negative supercoil density using topA and gyrB mutant E. coli strains; the in vivo cleavage of hairpin loops, which are an obligate consequence of slipped-strand structures, cruciforms, and intramolecular triplexes, by inactivation of the SbcC protein; and by genetic instability studies with plasmids containing long repeating sequence inserts that do, and do not, adopt non-B DNA structures in vitro. Hence, non-B DNA conformations are critical for these mutagenesis mechanisms.

DM2 CCTG*CAGG repeats are crossover hotspots that are more prone to expansions than the DM1 CTG*CAG repeats in Escherichia coli.

Myotonic dystrophy type 2 (DM2) is caused by the extreme expansion of the repeating tetranucleotide CCTG*CAGG sequence from <30 repeats in normal individuals to approximately 11,000 for the full mutation in certain patients. This repeat is in intron 1 of the zinc finger protein 9 gene on chromosome 3q21. Since prior work demonstrated that CTG*CAG and GAA*TTC triplet repeats (responsible for DM1 and Friedreich's ataxia, respectively) can expand by genetic recombination, we investigated the capacity of the DM2 tetranucleotide repeats to also expand during this process. Both gene conversion and unequal crossing over are attractive mechanisms to effect these very large expansions. (CCTG*CAGG)n (where n=30, 75, 114 or 160) repeats showed high recombination crossover frequencies (up to 27-fold higher than the non-repeating control) in an intramolecular plasmid system in Escherichia coli. Furthermore, a distinct orientation effect was observed where orientation II (CAGG on the leading strand template) was more prone to recombine. Expansions of up to double the length of the tetranucleotide repeats were found. Also, the repeating tetranucleotide sequence was more prone to expansions (to give lengths longer than a single repeating tract) than deletions as observed for the CTG*CAG and GAA*TTC repeats. We determined that the DM2 tetranucleotide repeats showed a lower thermodynamic stability when compared to the DM1 trinucleotide repeats, which could make them better targets for DNA repair events, thus explaining their expansion-prone behavior. Genetic studies in SOS-repair mutants revealed high frequencies of recombination crossovers although the SOS-response itself was not induced. Thus, the genetic instabilities of the CCTG*CAGG repeats may be mediated by a recombination-repair mechanism that is influenced by DNA structure.