Chemotherapeutic deletion of CTG repeats in lymphoblast cells from DM1 patients

Myotonic dystrophy type 1 (DM1) is caused by the expansion of a (CTG).(CAG) repeat in the DMPK gene on chromosome 19q13.3. At least 17 neurological diseases have similar genetic mutations, the expansion of DNA repeats. In most of these disorders, the disease severity is related to the length of the repeat expansion, and in DM1 the expanded repeat undergoes further elongation in somatic and germline tissues. At present, in this class of diseases, no therapeutic approach exists to prevent or slow the repeat expansion and thereby reduce disease severity or delay disease onset. We present initial results testing the hypothesis that repeat deletion may be mediated by various chemotherapeutic agents. Three lymphoblast cell lines derived from two DM1 patients treated with either ethylmethanesulfonate (EMS), mitomycin C, mitoxantrone or doxorubicin, at therapeutic concentrations, accumulated deletions following treatment. Treatment with EMS frequently prevented the repeat expansion observed during growth in culture. A significant reduction of CTG repeat length by 100-350 (CTG).(CAG) repeats often occurred in the cell population following treatment with these drugs. Potential mechanisms of drug-induced deletion are presented.     

CTG trinucleotide repeat "big jumps": large expansions, small mice

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 CTG*CAG 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.     

CTG Trinucleotide Repeat “Big Jumps”: Large Expansions, Small Mice

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 CTG•CAG 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.     

Trinucleotide repeats associated with human disease.

Triplet repeat expansion diseases (TREDs) are characterized by the coincidence of disease manifestation with amplification of d(CAG. CTG), d(CGG.CCG) or d(GAA.TTC) repeats contained within specific genes. Amplification of triplet repeats continues in offspring of affected individuals, which generally results in progressive severity of the disease and/or an earlier age of onset, phenomena clinically referred to as 'anticipation'. Recent biophysical and biochemical studies reveal that five of the six [d(CGG)n, d(CCG)n, (CAG)n, d(CTG)n and d(GAA)n] complementary sequences that are associated with human disease form stable hairpin structures. Although the triplet repeat sequences d(GAC)n and d(GTC)n also form hairpins, repeats of the double-stranded forms of these sequences are conspicuously absent from DNA sequence databases and are not anticipated to be associated with human disease. With the exception of d(GAG)n and d(GTG)n, the remaining triplet repeat sequences are unlikely to form hairpin structures at physiological salt and temperature. The details of hairpin structures containing trinucleotide repeats are summarized and discussed with respect to potential mechanisms of triplet repeat expansion and d(CGG.CCG) n methylation/demethylation.     

Involvement of the nucleotide excision repair protein UvrA in instability of CAG*CTG repeat sequences in Escherichia coli.

Several human genetic diseases have been associated with the genetic instability, specifically expansion, of trinucleotide repeat sequences such as (CTG)(n).(CAG)(n). Molecular models of repeat instability imply replication slippage and the formation of loops and imperfect hairpins in single strands. Subsequently, these loops or hairpins may be recognized and processed by DNA repair systems. To evaluate the potential role of nucleotide excision repair in repeat instability, we measured the rates of repeat deletion in wild type and excision repair-deficient Escherichia coli strains (using a genetic assay for deletions). The rate of triplet repeat deletion decreased in an E. coli strain deficient in the damage recognition protein UvrA. Moreover, loops containing 23 CTG repeats were less efficiently excised from heteroduplex plasmids after their transformation into the uvrA(-) strain. As a result, an increased proportion of plasmids containing the full-length repeat were recovered after the replication of heteroduplex plasmids containing unrepaired loops. In biochemical experiments, UvrA bound to heteroduplex substrates containing repeat loops of 1, 2, or 17 CAG repeats with a K(d) of about 10-20 nm, which is an affinity about 2 orders of magnitude higher than that of UvrA bound to the control substrates containing (CTG)(n).(CAG)(n) in the linear form. These results suggest that UvrA is involved in triplet repeat instability in cells. Specifically, UvrA may bind to loops formed during replication slippage or in slipped strand DNA and initiate DNA repair events that result in repeat deletion. These results imply a more comprehensive role for UvrA, in addition to the recognition of DNA damage, in maintaining the integrity of the genome.     

Anticipation and Instability of IT-15 (CAG)N Repeats in Parent-Offspring Pairs with Huntington Disease

Huntington disease (HD) is an autosomal dominant degenerative disorder caused by an expanded and unstable trinucleotide repeat (CAG)n in a gene (IT-15) on chromosome 4. HD exhibits genetic anticipation—earlier onset in successive generations within a pedigree. From a population-based clinical sample, we ascertained parent-offspring pairs with expanded alleles, to examine the intergenerational behavior of the trinucleotide repeat and its relationship to anticipation. We find that the change in repeat length with paternal transmission is significantly correlated with the change in age at onset between the father and offspring. When expanded triplet repeats of affected parents are separated by median repeat length, we find that the longer paternal and maternal repeats are both more unstable on transmission. However, unlike in paternal transmission, in which longer expanded repeats display greater net expansion than do shorter expanded repeats, in maternal transmission there is no mean change in repeat length for either longer or shorter expanded repeats. We also confirmed the inverse relationship between repeat length and age at onset, the higher frequency of juvenile-onset cases arising from paternal transmission, anticipation as a phenomenon of paternal transmission, and greater expansion of the trinucleotide repeat with paternal transmission. Stepwise multiple regression indicates that, in addition to repeat length of offspring, age at onset of affected parent and sex of affected parent contribute significantly to the variance in age at onset of the offspring. Thus, in addition to triplet repeat length, other factors, which could act as environmental factors, genetic factors, or both, contribute to age at onset. Our data establish that further expansion of paternal repeats within the affected range provides a biological basis of anticipation in HD.     

Genetic recombination destabilizes (CTG)n.(CAG)n repeats in E. coli

The expansion of trinucleotide repeats has been implicated in 17 neurological diseases to date. Factors leading to the instability of trinucleotide repeat sequences have thus been an area of intense interest. Certain genes involved in mismatch repair, recombination, nucleotide excision repair, and replication influence the instability of trinucleotide repeats in both Escherichia coli and yeast. Using a genetic assay for repeat deletion in E. coli, the effect of mutations in the recA, recB, and lexA genes on the rate of deletion of (CTG)n.(CAG)n repeats of varying lengths were examined. The results indicate that mutations in recA and recB, which decrease the rate of recombination, had a stabilizing effect on (CAG)n.(CTG)n repeats decreasing the high rates of deletion seen in recombination proficient cells. Thus, recombination proficiency correlates with high rates of genetic instability in triplet repeats. Induction of the SOS system, however, did not appear to play a significant role in repeat instability, nor did the presence of triplet repeats in cells turn on the SOS response. A model is suggested where deletion during exponential growth may result from attempts to restart replication when paused at triplet repeats.     

Triplet repeat DNA structures and human genetic disease: dynamic mutations from dynamic DNA

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 triplet repeat sequences. The propensity to form slipped strand DNA is proportional to the length and homogeneity of the repeat tract. The remarkable stability of slipped strand DNA may, in part, be due to loop-loop interactions facilitated by the sequence complementarity of the loops and the dynamic structure of three-way junctions formed at the loop-outs.     

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.     

Novel Triplet Repeat Containing Genes in Human Brain: Cloning, Expression, and Length Polymorphisms

Human genes containing triplet repeats may markedly expand in length and cause neuropsychiatric disease, explaining the phenomenon of anticipation (increasing severity or earlier age of onset in successive generations in a pedigree). To identify novel genes with triplet repeats, we screened a human brain cDNA library with oligonucleotide probes containing CTG or CCG triplet repeats. Fourteen of 40 clones encoded novel human genes, and 8 of these inserts have been sequenced on both strands. All contain repeats, and 5 of the 8 have 9 or more consecutive perfect repeats. All are expressed in brain. Chromosomal assignments reveal a distribution of these genes on multiple autosomes and the X-chromosome. Further, the repeat length in two of the genes is highly polymorphic, making them valuable index linkage markers. We predict that many triplet repeat-containing genes exist; screening with the CTG probe suggests approximately 50-100 genes containing this type of repeat are expressed in the human brain. Since additional disorders, such as Huntington's disease, bipolar affective disorder, and possibly others, show features of anticipation, we suggest that these novel human genes with triplet repeats are candidates for causing neuropsychiatric diseases.     

Gene conversion (recombination) mediates expansions of CTG[middle dot]CAG repeats

Genetic recombination is a robust mechanism for expanding CTG.CAG triplet repeats involved in the etiology of hereditary neurological diseases (Jakupciak, J. P., and Wells, R. D. (1999) J. Biol. Chem. 274, 23468-23479). This two-plasmid recombination system in Escherichia coli with derivatives of pUC19 and pACYC184 was used to investigate the effect of triplet repeat orientation on recombination and extent of expansions; tracts of 36, 50, 80, and 36, 100, and 175 repeats in length, respectively, in all possible permutations of length and in both orientations (relative to the unidirectional replication origins) revealed little or no effect of orientation of expansions. The extent of expansions was generally severalfold the length of the progenitor tract and frequently exceeded the combined length of the two tracts in the cotransformed plasmids. Expansions were much more frequent than deletions. Repeat tracts bearing two G-to-A interruptions (polymorphisms) within either 171- or 219-base pair tracts substantially reduced the expansions compared with uninterrupted repeat tracts of similar lengths. Gene conversion, rather than crossing over, was the recombination mechanism. Prior studies showed that DNA replication, repair, and tandem duplication also mediated genetic instabilities of the triplet repeat sequence. However, gene conversion (recombinational repair) is by far the most powerful expansion mechanism. Thus, we propose that gene conversion is the likely expansion mechanism for myotonic dystrophy, spinocerebellar ataxia type 8, and fragile X syndrome.     

New functions of Ctf18-RFC in preserving genome stability outside its role in sister chromatid cohesion

Expansion of DNA trinucleotide repeats causes at least 15 hereditary neurological diseases, and these repeats also undergo contraction and fragility. Current models to explain this genetic instability invoke erroneous DNA repair or aberrant replication. Here we show that CAG/CTG tracts are stabilized in Saccharomyces cerevisiae by the alternative clamp loader/unloader Ctf18-Dcc1-Ctf8-RFC complex (Ctf18-RFC). Mutants in Ctf18-RFC increased all three forms of triplet repeat instability--expansions, contractions, and fragility--with effect over a wide range of allele lengths from 20-155 repeats. Ctf18-RFC predominated among the three alternative clamp loaders, with mutants in Elg1-RFC or Rad24-RFC having less effect on trinucleotide repeats. Surprisingly, chl1, scc1-73, or scc2-4 mutants defective in sister chromatid cohesion (SCC) did not increase instability, suggesting that Ctf18-RFC protects triplet repeats independently of SCC. Instead, three results suggest novel roles for Ctf18-RFC in facilitating genomic stability. First, genetic instability in mutants of Ctf18-RFC was exacerbated by simultaneous deletion of the fork stabilizer Mrc1, but suppressed by deletion of the repair protein Rad52. Second, single-cell analysis showed that mutants in Ctf18-RFC had a slowed S phase and a striking G2/M accumulation, often with an abnormal multi-budded morphology. Third, ctf18 cells exhibit increased Rad52 foci in S phase, often persisting into G2, indicative of high levels of DNA damage. The presence of a repeat tract greatly magnified the ctf18 phenotypes. Together these results indicate that Ctf18-RFC has additional important functions in preserving genome stability, besides its role in SCC, which we propose include lesion bypass by replication forks and post-replication repair.     

Expansion of DNA repeats in Escherichia coli: effects of recombination and replication functions.

Duplication or expansion of directly repeated sequence elements is associated with a number of human genetic diseases. To study the mechanisms of repeat expansion, we have developed a plasmid assay in Escherichia coli. Our assay involves two simple repeats of 787 bp in length; expansion to three or more copies of the repeat can be selected by restoration of an intact tetracycline-resistance gene. Expansions occurred at relatively high rates, >10(-5), in the population. Both RecA-dependent recombination and RecA-independent slipped misalignments contributed to the observed expansion events. Mutations that impair DNA polymerase III (DnaE, DnaQ subunits) or the replication fork helicase, DnaB, stimulated both RecA-dependent and RecA-independent expansion events. In these respects, the properties of repeat expansion resemble repeat deletion and suggest that difficulties in DNA replication may trigger both classes of rearrangements. About 20% of the RecA-independent expansion events are accompanied by reciprocal sister-chromosome exchange, producing dimeric plasmids carrying one triplicated and one deleted locus. These products are explained by a model involving misaligned strands across the replication fork. This model predicts that the location of a replication stall site may govern the types of resulting rearrangements. The specific location of such a stall site can also, in theory, account for propensity towards expansion or deletion of repeat arrays. This may have relevance to trinucleotide repeat expansion in human genetic disease.     

A fragile X case with an amplification/deletion mosaic pattern

Fragile X syndrome is the most common cause of hereditary mental retardation. The FMR1 gene, which is involved in fragile X syndrome, contains a polymorphic CGG repeat, which expands in affected patients. Expanding triplet repeats have been shown to be a new type of mutation, termed "dynamic mutation", responsible for more than 12 genetic diseases. These mutations occur as multiple steps rather than as a single event. The first step leads to an unstable allele that then becomes increasingly unstable generally achieving further increases in copy or occasionally contraction. In this report, we describe a fragile X boy with both a hypermethylated full mutation and a deletion of 905 bp encompassing the CGG repeat. The upstream breakpoint is 438 bp 5' to the CGG repeat and the downstream breakpoint is 420 bp 3' of the triplet repeats. The deletion includes the ATG starting codon for translation of the FMR1 gene. This was confirmed by using FMRP immunocytochemistry both on blood smears and hair roots. The deleted region is flanked by a ccgg direct repeat next to the breakpoints; this may have had a critical role in the formation of a secondary DNA structure leading to the deletion.     

Identification of RTG2 as a modifier gene for CTG*CAG repeat instability in Saccharomyces cerevisiae

Trinucleotide repeats (TNRs) undergo frequent mutations in families affected by TNR diseases and in model organisms. Much of the instability is conferred in cis by the sequence and length of the triplet tract. Trans-acting factors also modulate TNR instability risk, on the basis of such evidence as parent-of-origin effects. To help identify trans-acting modifiers, a screen was performed to find yeast mutants with altered CTG.CAG repeat mutation frequencies. The RTG2 gene was identified as one such modifier. In rtg2 mutants, expansions of CTG.CAG repeats show a modest increase in rate, depending on the starting tract length. Surprisingly, contractions were suppressed in an rtg2 background. This creates a situation in a model system where expansions outnumber contractions, as in humans. The rtg2 phenotype was apparently specific for CTG.CAG repeat instability, since no changes in mutation rate were observed for dinucleotide repeats or at the CAN1 reporter gene. This feature sets rtg2 mutants apart from most other mutants that affect genetic stability both for TNRs and at other DNA sequences. It was also found that RTG2 acts independently of its normal partners RTG1 and RTG3, suggesting a novel function of RTG2 that helps modify CTG.CAG repeat mutation risk.     

The origin of genetic instability in CCTG repeats

CCTG tetranucleotide repeat expansion is associated with a hereditary neurological disease called myotonic dystrophy type 2 (DM2). The underlying reasons that lead to genetic instability and thus repeat expansion during DNA replication remains elusive. Here, we have shown CCTG repeats have a high propensity to form metastable hairpin and dumbbell structures using high-resolution nuclear magnetic resonance (NMR) spectroscopy. When the repeat length is equal to three, a hairpin with a two-residue CT loop is formed. In addition to the hairpin, a dumbbell structure with two CT-loops is formed when the repeat length is equal to four. Nuclear Overhauser effect (NOE) and chemical shift data reveal both the hairpin and dumbbell structures contain a flexible stem comprising a C-bulge and a T·T mismatch. With the aid of single-site mutation samples, NMR results show these peculiar structures undergo dynamic conformational exchange. In addition to the intrinsic flexibility in the stem region of these structures, the exchange process also serves as an origin of genetic instability that leads to repeat expansion during DNA replication. The structural features provide important drug target information for developing therapeutics to inhibit the expansion process and thus the onset of DM2.     

Saccharomyces cerevisiae Srs2 DNA helicase selectively blocks expansions of trinucleotide repeats

Trinucleotide repeats (TNRs) undergo frequent mutations in families afflicted with certain neurodegenerative disorders and in model organisms. TNR instability is modulated both by the repeat tract itself and by cellular proteins. Here we identified the Saccharomyces cerevisiae DNA helicase Srs2 as a potent and selective inhibitor of expansions. srs2 mutants had up to 40-fold increased expansion rates of CTG, CAG, and CGG repeats. The expansion phenotype was specific, as mutation rates at dinucleotide repeats, at unique sequences, or for TNR contractions in srs2 mutants were not altered. Srs2 is known to suppress inappropriate genetic recombination; however, the TNR expansion phenotype of srs2 mutants was largely independent of RAD51 and RAD52. Instead, Srs2 mainly functioned with DNA polymerase delta to block expansions. The helicase activity of Srs2 was important, because a point mutant lacking ATPase function was defective in blocking expansions. Purified Srs2 was substantially better than bacterial UvrD helicase at in vitro unwinding of a DNA substrate that mimicked a TNR hairpin. Disruption of the related helicase gene SGS1 did not lead to excess expansions, nor did wild-type SGS1 suppress the expansion phenotype of an srs2 strain. We conclude that Srs2 selectively blocks triplet repeat expansions through its helicase activity and primarily in conjunction with polymerase delta.     

Rapid unwinding of triplet repeat hairpins by Srs2 helicase of Saccharomyces cerevisiae

Expansions of trinucleotide repeats cause at least 15 heritable human diseases. Single-stranded triplet repeat DNA in vitro forms stable hairpins in a sequence-dependent manner that correlates with expansion risk in vivo. Hairpins are therefore considered likely intermediates during the expansion process. Unwinding of a hairpin by a DNA helicase would help protect against expansions. Yeast Srs2, but not the RecQ homolog Sgs1, blocks expansions in vivo in a manner largely dependent on its helicase function. The current study tested the idea that Srs2 would be faster at unwinding DNA substrates with an extrahelical triplet repeat hairpin embedded in a duplex context. These substrates should mimic the relevant intermediate structure thought to occur in vivo. Srs2 was faster than Sgs1 at unwinding several substrates containing triplet repeat hairpins or another structured loop. In contrast, control substrates with an unstructured loop or a Watson–Crick duplex were unwound equally well by both enzymes. Results with a fluorescently labeled, three-way junction showed that Srs2 unwinding proceeds unabated through extrahelical triplet repeats. In summary, Srs2 maintains its facile unwinding of triplet repeat hairpins embedded within duplex DNA, supporting the genetic evidence that Srs2 is a key helicase in Saccharomyces cerevisiae for preventing expansions.