Identification of Trinucleotide Repeat Ligands with a FRET Melting Assay (original) (raw)
Related papers
The American Journal of Human Genetics, 2003
Gene-specific CTG/CAG repeat expansion is associated with at least 14 human diseases, including myotonic dystrophy type 1 (DM1). Most of our understanding of trinucleotide instability is from nonhuman models, which have presented mixed results, supporting replication errors or processes independent of cell division as causes. Nevertheless, the mechanism occurring at the disease loci in patient cells is poorly understood. Using primary fibroblasts derived from a fetus with DM1, we have shown that spontaneous expansion of the diseased (CTG) 216 allele occurred in proliferating cells but not in quiescent cells. Expansions were "synchronous," with mutation frequencies approaching 100%. Furthermore, cells were treated with agents known to alter DNA synthesis but not to directly damage DNA. Inhibiting replication initiation with mimosine had no effect upon instability. Inhibiting both leading-and lagging-strand synthesis with aphidicolin or blocking only lagging strand synthesis with emetine significantly enhanced CTG expansions. It was striking that only the expanded DM1 allele was altered, leaving the normal allele, (CTG) 12 , and other repeat loci unaffected. Standard and small-pool polymerase chain reaction revealed that inhibitors enhanced the magnitude of short expansions in most cells threefold, whereas 11%-25% of cells experienced gains of 122-170 repeats, to sizes of (CTG) 338-(CTG) 386. Similar results were observed for an adult DM1 cell line. Our results support a role for the perturbation of replication fork dynamics in DM1 CTG expansions within patient fibroblasts. This is the first report that repeat-length alterations specific to a disease allele can be modulated by exogenously added compounds.
The complex pathology of trinucleotide repeats
Current Opinion in Cell Biology, 1997
The expansion of trinucleotide repeat sequences has now been shown to be the underlying cause of at least ten human disorders. Unifying features among these diseases include the unstable behavior of the triplet repeat during germline transmission when the length of the repeat exceeds a critical value. However, the trinucleotide repeat disorders can be divided into two distinct groups. Type I disorders involve the expansion of CAG repeats, which encode an expanded polyglutamine, inserted into the open-reading frame of a gene that is usually quite broadly expressed. Recently, mouse models for type I disorders have been developed and the basis of pathology is under study, both in these models and through biochemical and cell biological approaches. The type II disorders involve repeat expansions in noncoding regions of genes. The mechanisms by which these repeat expansions lead to pathology may be quite diverse.
Biochemistry, 1996
Most models proposed to explain the disease-associated expansion of (CTG) n ‚(CAG) n and (CGG) n ‚(CCG) n trinucleotide repeats include the formation of slipped strand DNA structures during replication; however, physical evidence for these alternative DNA secondary structures has not been reported. Using cloned fragments from the myotonic dystrophy (DM) and fragile X syndrome (FRAXA) loci containing normal, premutation, and full mutation lengths of repeats, we report the formation of novel alternative DNA secondary structures that map within the repeat tracts during reannealing of complementary strands, containing equal lengths of repeats, into linear duplex DNA molecules. Linear duplex DNA molecules containing these alternative DNA secondary structures are characterized by reduced electrophoretic mobilities in polyacrylamide gels. These alternative secondary structures are stable at physiological ionic strengths and to temperatures up to at least 55°C. Following reduplexing, the CAG strand of the (CTG) n ‚(CAG) n repeats is preferentially sensitive to mung bean nuclease, suggesting the presence of single-stranded DNA in the alternative DNA structure. For (CTG) 17 , which is a repeat length found in normal individuals, less than 3% of the DNA molecules formed alternative DNA structures upon reduplexing. DNA molecules containing (CTG) 50 or (CTG) 255 , which represent premutation and full mutation lengths of triplet repeats, respectively, formed a heterogeneous population of alternative DNA structures in >50% of DNA molecules. The complexity of the structures formed increased with the length of the triplet repeat. The relationship between repeat length and the propensity of formation and complexity of the novel structures correlates with the effect of repeat length on genetic instability in human diseases. These are the first results consistent with the existence of slipped strand DNA structures. The potential involvement of these structures, which we term S-DNA, in the genetic instability of triplet repeats is discussed.
The mechanism of disease-associated trinucleotide repeat length variation may involve slippage of the triplet-containing strand at the replication fork, generating a slipped-strand DNA structure. We recently reported formation in vitro of slipped-strand DNA (S-DNA) structures when DNAs containing triplet repeat blocks of myotonic dystrophy or fragile X diseases were melted and allowed to reanneal to form duplexes. Here additional evidence is presented that is consistent with the existence of S-DNA structures. We demonstrate that S-DNA structures can form between two complementary strands containing equal numbers of repeats. In addition, we show that both the propensity for S-DNA formation and the structural complexity of S-DNAs formed increase with increasing repeat length. S-DNA structures were also analyzed by electron microscopy, confirming that the two strands are slipped out of register with respect to each other and confirming the structural polymorphism expected within long tracts of trinucleotide repeats. For (CTG) 50 ·(CAG) 50 two distinct populations of slipped structures have been identified: those involving ≤10 repeats per slippage, which appear as bent/kinked DNA molecules, and those involving >10 repeats, which have multiple loops or hairpins indicative of complex alternative DNA secondary structures.
Features of trinucleotide repeat instability in vivo
Cell Research, 2008
Unstable repeats are associated with various types of cancer and have been implicated in more than 40 neurodegenerative disorders. Trinucleotide repeats are located in non-coding and coding regions of the genome. Studies of bacteria, yeast, mice and man have helped to unravel some features of the mechanism of trinucleotide expansion. Looped DNA structures comprising trinucleotide repeats are processed during replication and/or repair to generate deletions or expansions. Most in vivo data are consistent with a model in which expansion and deletion occur by different mechanisms. In mammals, microsatellite instability is complex and appears to be influenced by genetic, epigenetic and developmental factors.
Triplet repeat DNA structures and human genetic disease: dynamic mutations from dynamic DNA
Journal of Biosciences, 2002
Fourteen genetic neurodegenerative diseases and three fragile sites have been associated with the expansion of (CTG)n•(CAG)n, (CGG)n•(CCG)n, or (GAA)n•(TTC)n repeat tracts. Different models have been proposed for the expansion of triplet repeats, most of which presume the formation of alternative DNA structures in repeat tracts. One of the most likely structures, slipped strand DNA, may stably and reproducibly form within
CTG Trinucleotide Repeat “Big Jumps”: Large Expansions, Small Mice
PLoS Genetics, 2007
Trinucleotide repeat expansions are the genetic cause of numerous human diseases, including fragile X mental retardation, Huntington disease, and myotonic dystrophy type 1. Disease severity and age of onset are critically linked to expansion size. Previous mouse models of repeat instability have not recreated large intergenerational expansions (''big jumps''), observed when the repeat is transmitted from one generation to the next, and have never attained the very large tract lengths possible in humans. Here, we describe dramatic intergenerational CTGCAG repeat expansions of several hundred repeats in a transgenic mouse model of myotonic dystrophy type 1, resulting in increasingly severe phenotypic and molecular abnormalities. Homozygous mice carrying over 700 trinucleotide repeats on both alleles display severely reduced body size and splicing abnormalities, notably in the central nervous system. Our findings demonstrate that large intergenerational trinucleotide repeat expansions can be recreated in mice, and endorse the use of transgenic mouse models to refine our understanding of triplet repeat expansion and the resulting pathogenesis. Citation: Gomes-Pereira M, Foiry L, Nicole A, Huguet A, Junien C, et al. (2007) CTG trinucleotide repeat ''big jumps'': Large expansions, small mice. PLoS Genet 3(4): e52.
BMC Molecular Biology, 2007
Background: Trinucleotide instability is a hallmark of degenerative neurological diseases like Huntington's disease, some forms of spinocerebellar ataxia and myotonic dystrophy type 1 (DM1). To investigate the effect of cell type and cell state on the behavior of the DM1 CTG•CAG repeat, we studied a knock-in mouse model for DM1 at different time points during ageing and followed how repeat fate in cells from liver and pancreas is associated with polyploidization and changes in nuclearity after the onset of terminal differentiation. Results: After separation of liver hepatocytes and pancreatic acinar cells in pools with 2n, 4n or 8n DNA, we analyzed CTG•CAG repeat length variation by resolving PCR products on an automated PAGE system. We observed that somatic CTG•CAG repeat expansion in our DM1 mouse model occurred almost uniquely in the fraction of cells with high cell nuclearity and DNA ploidy and aggravated with aging. Conclusion: Our findings suggest that post-replicative and terminal-differentiation events, coupled to changes in cellular DNA content, form a preconditional state that influences the control of DNA repair or recombination events involved in trinucleotide expansion in liver hepatocytes and pancreatic acinar cells.
Nucleic acids research, 2003
The molecular basis of the myotonic dystrophy type 1 is the expansion of a CTG repeat at the DMPK locus. The expanded disease-associated repeats are unstable in both somatic and germ lines, with a high tendency towards expansion. The rate of expansion is directly related to the size of the pathogenic allele, increasing the size heterogeneity with age. It has also been suggested that additional factors, including as yet unidentified environmental factors, might affect the instability of the expanded CTG repeats to account for the observed CTG size dynamics over time. To investigate the effect of environmental factors in the CTG repeat instability, three lymphoblastoid cell lines were established from two myotonic dystrophy patients and one healthy individual, and parallel cultures were concurrently expanded in the presence or absence of the mutagenic chemical mitomycin C for a total of 12 population doublings. The new alleles arising along the passages were analysed by radioactive sm...