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 MutSβ 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.

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.

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

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.

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.

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.

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.

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.

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).

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.

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.

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.

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.

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-).

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.

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.

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.

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.

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.

Long homopurine*homopyrimidine sequences are characteristic of genes expressed in brain and the pseudoautosomal region.

Homo(purine*pyrimidine) sequences (R*Y tracts) with mirror repeat symmetries form stable triplexes that block replication and transcription and promote genetic rearrangements. A systematic search was conducted to map the location of the longest R*Y tracts in the human genome in order to assess their potential function(s). The 814 R*Y tracts with > or =250 uninterrupted base pairs were preferentially clustered in the pseudoautosomal region of the sex chromosomes and located in the introns of 228 annotated genes whose protein products were associated with functions at the cell membrane.

Roles of double-strand breaks, nicks, and gaps in stimulating deletions of CTG.CAG repeats by intramolecular DNA repair.

A series of plasmids harboring CTG.CAG repeats with double-strand breaks (DSB), single-strand nicks, or single-strand gaps (15 or 30 nucleotides) within the repeat regions were used to determine their capacity to induce genetic instabilities. These plasmids were introduced into Escherichia coli in the presence of a second plasmid containing a sequence that could support homologous recombination repair between the two plasmids.

Increased negative superhelical density in vivo enhances the genetic instability of triplet repeat sequences.

The influence of negative superhelical density on the genetic instabilities of long GAA.TTC, CGG.CCG, and CTG.

Advances in mechanisms of genetic instability related to hereditary neurological diseases.

Substantial progress has been realized in the past several years in our understanding of the molecular mechanisms responsible for the expansions and deletions (genetic instabilities) of repeating tri-, tetra- and pentanucleotide repeating sequences associated with a number of hereditary neurological diseases. These instabilities occur by replication, recombination and repair processes, probably acting in concert, due to slippage of the DNA complementary strands relative to each other. The biophysical properties of the folded-back repeating sequence strands play a critical role in these instabilities.