Processing of double-R-loops in (CAG)·(CTG) and C9orf72 (GGGGCC)·(GGCCCC) repeats causes instability.

R-loops, transcriptionally-induced RNA:DNA hybrids, occurring at repeat tracts (CTG)n, (CAG)n, (CGG)n, (CCG)n and (GAA)n, are associated with diseases including myotonic dystrophy, Huntington's disease, fragile X and Friedreich's ataxia. Many of these repeats are bidirectionally transcribed, allowing for single- and double-R-loop configurations, where either or both DNA strands may be RNA-bound. R-loops can trigger repeat instability at (CTG)·(CAG) repeats, but the mechanism of this is unclear. We demonstrate R-loop-mediated instability through processing of R-loops by HeLa and human neuron-like cell extracts. Double-R-loops induced greater instability than single-R-loops. Pre-treatment with RNase H only partially suppressed instability, supporting a model in which R-loops directly generate instability by aberrant processing, or via slipped-DNA formation upon RNA removal and its subsequent aberrant processing. Slipped-DNAs were observed to form following removal of the RNA from R-loops. Since transcriptionally-induced R-loops can occur in the absence of DNA replication, R-loop processing may be a source of repeat instability in the brain. Double-R-loop formation and processing to instability was extended to the expanded C9orf72 (GGGGCC)·(GGCCCC) repeats, known to cause amyotrophic lateral sclerosis and frontotemporal dementia, providing the first suggestion through which these repeats may become unstable. These findings provide a mechanistic basis for R-loop-mediated instability at disease-associated repeats.

Fragile sites, chromosomal lesions, tandem repeats, and disease.

Expanded tandem repeat DNAs are associated with various unusual chromosomal lesions, despiralizations, multi-branched inter-chromosomal associations, and fragile sites. Fragile sites cytogenetically manifest as localized gaps or discontinuities in chromosome structure and are an important genetic, biological, and health-related phenomena. Common fragile sites (∼230), present in most individuals, are induced by aphidicolin and can be associated with cancer; of the 27 molecularly-mapped common sites, none are associated with a particular DNA sequence motif. Rare fragile sites ( ≳ 40 known), ≤ 5% of the population (may be as few as a single individual), can be associated with neurodevelopmental disease. All 10 molecularly-mapped folate-sensitive fragile sites, the largest category of rare fragile sites, are caused by gene-specific CGG/CCG tandem repeat expansions that are aberrantly CpG methylated and include FRAXA, FRAXE, FRAXF, FRA2A, FRA7A, FRA10A, FRA11A, FRA11B, FRA12A, and FRA16A. The minisatellite-associated rare fragile sites, FRA10B, FRA16B, can be induced by AT-rich DNA-ligands or nucleotide analogs. Despiralized lesions and multi-branched inter-chromosomal associations at the heterochromatic satellite repeats of chromosomes 1, 9, 16 are inducible by de-methylating agents like 5-azadeoxycytidine and can spontaneously arise in patients with ICF syndrome (Immunodeficiency Centromeric instability and Facial anomalies) with mutations in genes regulating DNA methylation. ICF individuals have hypomethylated satellites I-III, alpha-satellites, and subtelomeric repeats. Ribosomal repeats and subtelomeric D4Z4 megasatellites/macrosatellites, are associated with chromosome location, fragility, and disease. Telomere repeats can also assume fragile sites. Dietary deficiencies of folate or vitamin B12, or drug insults are associated with megaloblastic and/or pernicious anemia, that display chromosomes with fragile sites. The recent discovery of many new tandem repeat expansion loci, with varied repeat motifs, where motif lengths can range from mono-nucleotides to megabase units, could be the molecular cause of new fragile sites, or other chromosomal lesions. This review focuses on repeat-associated fragility, covering their induction, cytogenetics, epigenetics, cell type specificity, genetic instability (repeat instability, micronuclei, deletions/rearrangements, and sister chromatid exchange), unusual heritability, disease association, and penetrance. Understanding tandem repeat-associated chromosomal fragile sites provides insight to chromosome structure, genome packaging, genetic instability, and disease.

Disease-associated repeat instability and mismatch repair.

Expanded tandem repeat sequences in DNA are associated with at least 40 human genetic neurological, neurodegenerative, and neuromuscular diseases. Repeat expansion can occur during parent-to-offspring transmission, and arise at variable rates in specific tissues throughout the life of an affected individual. Since the ongoing somatic repeat expansions can affect disease age-of-onset, severity, and progression, targeting somatic expansion holds potential as a therapeutic target. Thus, understanding the factors that regulate this mutation is crucial. DNA repair, in particular mismatch repair (MMR), is the major driving force of disease-associated repeat expansions. In contrast to its anti-mutagenic roles, mammalian MMR curiously drives the expansion mutations of disease-associated (CAG)·(CTG) repeats. Recent advances have broadened our knowledge of both the MMR proteins involved in disease repeat expansions, including: MSH2, MSH3, MSH6, MLH1, PMS2, and MLH3, as well as the types of repeats affected by MMR, now including: (CAG)·(CTG), (CGG)·(CCG), and (GAA)·(TTC) repeats. Mutagenic slipped-DNA structures have been detected in patient tissues, and the size of the slip-out and their junction conformation can determine the involvement of MMR. Furthermore, the formation of other unusual DNA and R-loop structures is proposed to play a key role in MMR-mediated instability. A complex correlation is emerging between tissues showing varying amounts of repeat instability and MMR expression levels. Notably, naturally occurring polymorphic variants of DNA repair genes can have dramatic effects upon the levels of repeat instability, which may explain the variation in disease age-of-onset, progression and severity. An increasing grasp of these factors holds prognostic and therapeutic potential.