Flexible DNA: genetically unstable CTG.CAG and CGG.CCG from human hereditary neuromuscular disease genes

The properties of duplex CTG.CAG and CGG.CCG, which are involved in the etiology of several hereditary neurodegenerative diseases, were investigated by a variety of methods, including circularization kinetics, apparent helical repeat determination, and polyacrylamide gel electrophoresis. The bending moduli were 1.13 x 10(-19) erg.cm for CTG and 1.27 x 10(-19) erg.cm for CGG, approximately 40% less than for random B-DNA. Also, the persistence lengths of the triplet repeat sequences were approximately 60% the value for random B-DNA. However, the torsional moduli and the helical repeats were 2.3 x 10(-19) erg.cm and 10.4 base pairs (bp)/turn for CTG and 2.4 x 10(-19) erg.cm and 10.3 bp/turn for CGG, respectively, all within the range for random B-DNA. Determination of the apparent helical repeat by the band shift assay indicated that the writhe of the repeats was different from that of random B-DNA. In addition, molecules of 224-245 bp in length (64-71 triplet repeats) were able to form topological isomers upon cyclization. The low bending moduli are consistent with predictions from crystallographic variations in slide, roll, and tilt. No unpaired bases or non-B-DNA structures could be detected by chemical and enzymatic probe analyses, two-dimensional agarose gel electrophoresis, and immunological studies. Hence, CTG and CGG are more flexible and highly writhed than random B-DNA and thus would be expected to act as sinks for the accumulation of superhelical density.     

Small slipped register genetic instabilities in Escherichia coli in triplet repeat sequences associated with hereditary neurological diseases

Genetic instability investigations on three triplet repeat sequences (TRS) involved in human hereditary neurological diseases (CTG.CAG, CGG.CCG, and GAA.TTC) revealed a high frequency of small expansions or deletions in 3-base pair registers in Escherichia coli. The presence of G to A polymorphisms in the CTG.CAG sequences served as reporters for the size and location of these instabilities. For the other two repeat sequences, length determinations confirmed the conclusions found for CTG.CAG. These studies were conducted in strains deficient in methyl-directed mismatch repair or nucleotide excision repair in order to investigate the involvement of these postreplicative processes in the genetic instabilities of these TRS. The observation that small and large instabilities for (CTG.CAG)175 fall into distinct size classes (1-8 repeats and approximate multiples of 41 repeats, respectively) leads to the conclusion that more than one DNA instability process is involved. The slippage of the complementary strands of the TRS is probably responsible for the small deletions and expansions in methyl-directed mismatch repair-deficient and nucleotide excision repair-deficient cells. A model is proposed to explain the observed instabilities via strand misalignment, incision, or excision, followed by DNA synthesis and ligation. This slippage-repair mechanism may be responsible for the small expansions in type 1 hereditary neurological diseases involving polyglutamine expansions. Furthermore, these observations may relate to the high frequency of small deletions versus a lower frequency of large instabilities observed in lymphoblastoid cells from myotonic dystrophy patients.     

Search for unstable DNA in schizophrenia families with evidence for genetic anticipation

Evidence for genetic anticipation has recently become an important subject of research in clinical psychiatric genetics. Renewed interest in anticipation was evoked by molecular genetic findings of a novel type of mutation termed "unstable DNA." The unstable DNA model can be construed as the "best fit" for schizophrenia twin and family epidemiological data. We have performed a large-scale Southern blot hybridization, asymmetrical PCR-based, and repeat expansion-detection screening for (CAG)n/(CTG)n and (CCG)n/(CGG)n expansions in eastern Canadian schizophrenia multiplex families demonstrating genetic anticipation. There were no differences in (CAG)n/(CTG)n and (CCG)n/(CGG)n pattern distribution either between affected and unaffected individuals or across generations. Our findings do not support the hypothesis that large (CAG)n/(CTG)n or (CCG)n/(CGG)n expansions are the major etiologic factor in schizophrenia. A separate set of experiments directed to the analysis of small (30-130 trinucleotides), Huntington disease-type expansions in individual genes is required in order to fully exclude the presence of (CAG)n/(CTG)n- or (CCG)n/(CGG)n-type unstable mutation.     

Biomedical advances in developmental psychology: The case of fragile X syndrome.

Fragile X syndrome, the most common inherited cause of mental retardation, is caused by an abnormal gene on the bottom end of the X chromosome. Discovered and sequenced in 1991, it is called the Fragile X Mental Retardation-1 (FMR-1) gene. Mutations in the FMR-1 gene include small expansions with a CGG (a specific sequence of the nucleotides) repetitive sequence that repeats from 50 to 200 times (the premutation) and the full mutation that involves a CGG repeat sequence that is greater than 200. In the full mutation, the FMR-1 gene is usually methylated, turning off the gene so that no protein is produced. Mutations within the FMR- I gene can cause a spectrum of learning difficulties ranging from mild problems to severe mental retardation. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Helix bending as a factor in protein/DNA recognition

Normal vectors perpendicular to individual base pairs are a powerful tool for studying the bending behavior of B-DNA, both in the form of normal vector plots and in matrices that list angles between vectors for all possible base pair combinations. A new analysis program, FREEHELIX, has been written for this purpose, and applied to 86 examples of sequence-specific protein/DNA complexes whose coordinates are on deposit in the Nucleic Acid Data Base. Bends in this sample of 86 structures almost invariably follow from roll angles between adjacent base pairs; tilt makes no net contribution. Roll in a direction compressing the broad major groove is much more common than that which compresses the minor groove. Three distinct types of B-DNA bending are observed, each with a different molecular origin: (1) Localized kinking is produced by large roll at single steps or at two steps separated by one turn of helix. (2) Smooth, planar curvature is produced by positive and negative roll angles spaced a half-turn apart, with random side-to-side zigzag roll at intermediate points, rather than a tilt contribution that might have been expected theoretically. (3) Three-dimensional writhe results from significant roll angles at a continuous series of steps. Writhe need not change the overall direction of helix axis, if it is continued indefinitely or for an integral number of helical turns. A-DNA itself can be formally considered as possessing uniform, continuous writhe that yields no net helix bending. Smooth curvature is the most intricate deformation of the three, and is least common. Writhe is the simplest deformation and is most common; indeed, a low level of continuous writhe is the normal condition of an otherwise unbent B-DNA helix of general sequence. With one exception, every example of major kinking in this sample of 86 structures involves a pyrimidine-purine step: C-A/T-G, T-A, or C-G. Purine-purine steps, especially A-A, show the least tendency toward roll deformations.     

Heterogeneity of DM kinase repeat expansion in different fetal tissues and further expansion during cell proliferation in vitro: evidence for a casual involvement of methyl-directed DNA mismatch repair in triplet repeat stability

We have analysed the mitotic behaviour of expanded CTG repeats in somatic tissues and cultured somatic cells from myotonic dystrophy (DM) fetuses using indirect and direct methods. Heterogeneity of expansions between fetal tissues was demonstrated in a 16 week old fetus whereas there was no evidence for such a somatic heterogeneity in a 13 week old fetus. Dilution plating of cultured cells from an adult patient and a fetus resulted in isolation of clones showing single expanded restriction fragments when the donor showed a heterogeneous smear of expansions or a single expanded fragment. During proliferation in vitro to 45 doublings, DM cells experienced highly synchronous further repeat expansion which first became evident at approximately 15 cell generations and reached a plateau of maximum expansion at approximately 200 days. When mathematically expressed as a function of culture time the dynamics of expansion of restriction fragments followed a sigmoid curve. This unstable behaviour of CTG repeat expansions in DM was compared to the mitotically stable patterns of full mutation in fragile X fetal tissues and led to the hypothesis that methylation of CpGs within the repeat sequence is a stabilizing factor of largely expanded CGG and GCC repeats allowing for efficient methyl-directed strand-specific DNA mismatch repair.     

Conformational properties of DNA dodecamers containing four tandem repeats of the CNG triplets

We studied DNA dodecamers (CAG)4, (CCG)4, (CGG)4 and (CTG)4by CD spectroscopy and polyacrylamide gel electrophoresis. Each dodecamer adopted several ordered conformers which denatured in a cooperative way. Stability of the conformers depended on the dodecamer concentration, ionic strength, temperature and pH. The dodecamers, having a pyrimidine base in the triplet center, generated foldbacks at low ionic strength whose stem conformations were governed by the GC pairs. At high salt, (CCG)4 isomerized into a peculiar association of two strands. The association was also promoted by high oligonucleotide concentrations. No similar behavior was exhibited by (CTG)4. At low salt, (CGG)4 coexisted in two bimolecular conformers whose populations were strongly dependent on the ionic strength. In addition, (CGG)4 associated into a tetraplex at acidic pH. A tetraplex was even observed at neutral pH if the (CGG)4 concentration was sufficiently high. (CAG)4 was very stable in a monomolecular conformer similar to the known extremely stable foldback of the (GCGAAGC) heptamer. Nevertheless, even this very stable conformer disappeared if (CTG)4 was added to the solution of (CAG)4. Association of the complementary strands was also strongly preferred to the particular strand conformations by the other couple, (CCG)4 and (CGG)4.     

A theory of thermal fluctuations in DNA miniplasmids

A recent analysis of the normal modes of vibration of a ring formed by bringing together and sealing, with or without the addition of twist, the ends of rods that are straight when stress free is taken as the basis for a theory of the statistical thermodynamics of a canonical ensemble of DNA minicircles with specified linking number difference deltaLk and number N of base pairs. It is assumed that N corresponds to a circumference in the range of one or two persistence lengths. For such an ensemble, the theory yields an expression for the average writhe (Wr), which can be employed to calculate the free energy, entropy, and enthalpy of supercoiling, deltaGsc, deltaSsc, and deltaHsc. The results obtained for the dependence of deltaGsc on deltaLk and N are in accord with experimental observations of equilibrium distributions of topoisomers of plasmids with N approximately 200 bp.     

Mental status and fragile X expression in relation to FMR-1 gene mutation

The fragile X mental retardation syndrome is caused by unstable expansion of a CGG repeat in the FMR-1 gene. Clinical expression is associated with a large expansion of the CGG repeat. The mutation in the FMR-1 gene and the cytogenetic expression of the fragile site at Xq27.3 have been studied in 52 fragile X male patients. The percentage of the cytogenetic expression of the fragile site at Xq27.3 positively correlates with the mean size of the full mutation in the FMR-1 gene (p < 0.0001) irrespective of the presence of additional premutation alleles. We noted a less frequent occurrence of additional premutation alleles in adult patients compared with juveniles, suggesting a continued mitotic instability in life. Additionally, the level of mental retardation has been ascertained in 35 patients using the Stanford-Binet or Terman-Merrill test of general intelligence. The presence of a full mutation in the FMR-1 gene seemed decisive for the occurrence of mental impairment in the patient. No correlation is observed between the degree of mental retardation and the size of the full mutation. The degree of mental retardation seemed not to be influenced by the presence of premutation alleles in part of the cells in addition to a full mutation. One patient is described with the 'Prader-Willi-like' subphenotype of the fragile X syndrome, showing a deletion in the FMR-1 gene in a part of his cells in addition to a full mutation.     

Screening for CGG trinucleotide repeat expansion in the fragile X mental retardation 1 gene in schizophrenic patients

Patients diagnosed using DSM-III-R criteria as having schizophrenia and other related disorders (n = 128) were assessed for CGG trinucleotide repeat expansion in the fragile X mental retardation 1 (FMR-1) gene. One subject, a woman with schizophreniform disorder, was found to have a premutation of the gene. Her case report is given. The present investigation supports the view that mutation or premutation of the FMR-1 gene is not of importance for the aetiology of the vast majority of schizophrenic patients.     

Effect of nitrates and nitrites on small intestine

Models for the disease-associated expansion of (CTG)n.(CAG)n, (CGG)n.(CCG)n, and (GAA)n.(TTC)n trinucleotide repeats involve alternative DNA structures formed during DNA replication, repair and recombination. These repeat sequences are inherently flexible and can form a variety of hairpins, intramolecular triplexes, quadruplexes, and slipped-strand structures that may be important intermediates and result in their genetic instability.     

Binding of actinomycin D to DNA oligomers of CXG trinucleotide repeats

Actinomycin D (ACTD) binding propensities of DNA with CXG trinucleotide repeats were investigated using oligomers of the form d[AT(CXG)n = 2-4AT] and their corresponding heteroduplexes, where X = A, C, G, or T. These oligonucleotides contain -CXGCXG-, -CXGCXGCXG-, and -CXGCXGCXGCXG- units that can form homoduplexes containing one, two, and three GpC binding sites, respectively, with flanking X/X mismatches. The corresponding heteroduplexes contain these same sites with flanking Watson-Crick base pairs. It was found that oligomers with X = G exhibit weak ACTD affinities whereas those with X not equal to G and n = 3 exhibit unusually strong ACTD binding affinities with binding constants ranging from 2.3 x 10(7) to 3.3 x 10(7) M-1 and binding densities of approximately 1 drug molecule/strand (or 2/duplex). These binding affinities are considerably higher than those of their shorter and longer counterparts and are about 2- and 10-fold stronger than the corresponding CAG.CTG and CGG.CCG heteroduplexes, respectively. The CTG-containing oligomer d[AT(CTG)3AT] stands out as unique in having its ACTD dissociation kinetics being dominated by a strikingly slow process with a characteristic time of 205 min at 20 degrees C, which is 100-fold slower than d[AT(CAG)3AT], nearly 10-fold slower than the corresponding heteroduplex, and considerably slower than d[AT(CTG)2AT] (63 min) and d[AT(CTG)4AT] (16 min). The faster dissociation rate of the n = 4 oligomer compared to its n = 2 counterpart is in apparent contrast with the observed 10-fold stronger ACTD binding affinity of the former. It was also found that d[AT(CCG)3AT] exhibits the slowest dissociation rate of the CGG/CCG series, being more than an order of magnitude slower than that of its heteroduplex (tau slow of 43 vs 2 min). The finding that a homoduplex d[AT-CXG-CXG-CXG-AT]2 can bind two ACTD molecules tightly is significant since it was thought unlikely for two consecutive GpC sites separated by a single T/T mismatch to do so.     

Bcl-2 overexpression blocks activation of the death protease CPP32/Yama/apopain

Females who are affected by fragile X syndrome (FXS) can have significant physical, neuropsychological and emotional involvement. This study was designed to explore the relationships between these three domains and to learn how the degree of involvement in each of these phenotypic areas relates to molecular parameters including CGG repeat length and activation ratio (the proportion of normal FMR1 alleles on the active X chromosome). Three groups of females were studied: 35 women who grew up in a fragile X family but do not carry an FMR1 mutation, 92 women with a premutation, and 29 women with a full mutation. Correlations between neurocognitive, physical and emotional traits were calculated for each of the three groups. Within the full mutation group significant correlations were seen between schizotypal traits and full scale IQ. The Lie scale was significantly correlated with the physical findings index. The activation ratio correlated significantly with the measure of executive function (r = .50, P = .01). There was a trend toward correlations of activation ratio with the physical index score, outer ear prominence and IQ. CGG repeat number significantly correlated only with the physical index (r = .44, P = .01). Thus, activation ratio may be the more pertinent molecular parameter in full mutation women in determining the degree of cognitive and physical phenotypic involvement.     

Expansion and deletion of triplet repeat sequences in Escherichia coli occur on the leading strand of DNA replication

Expansions and deletions of triplet repeat sequences that cause human hereditary neurological diseases were previously suggested to be mediated by the formation of DNA hairpins on the lagging strand during replication. The replication properties of CTG.CAG, CGG.CCG, and TTC.GAA repeats were studied in Escherichia coli using an in vivo phagemid system as a model for continuous leading strand synthesis. The repeats were substantially deleted when the CTG, CGG, and GAA repeats were the templates for rolling circle replication from the f1 phage origin. The deletions may be mediated by hairpins formed by these repeat tracts. The distributions of the deletion products of the CTG.CAG and CGG.CCG tracts indicated that hairpins of discrete sizes mediate deletions during complementary strand synthesis. Deletions during rolling circle synthesis are caused by larger hairpins of specific sizes. Thus, most deletion products were of defined lengths, suggesting a preference for specific hairpin intermediates. Small expansions of the CTG.CAG and CGG.CCG repeats were also observed, presumably due to the formation of CTG and CGG hairpins on the nascent complementary strand. Since rolling circle replication has been established in vitro as a model for leading strand synthesis, we conclude that triplet repeat instability can also occur on the leading strand of DNA replication.     

Relative stability of a minimal CTG repeat expansion in a large kindred with myotonic dystrophy

The genetic basis for myotonic dystrophy (DM) is a CTG trinucleotide repeat expansion. The number of CTG repeats commonly increases in affected individuals of successive generations, in association with anticipation. We identified a large DM family in which multiple members had minimal CTG repeat expansions, and in which the number of CTG repeats remained in the minimally expanded range through at least three, and possibly four, generations. This relative stability of minimal CTG repeat expansions may help to maintain the DM mutation in the population.     

Triplet repeats form secondary structures that escape DNA repair in yeast

Several human neurodegenerative diseases result from expansion of CTG/CAG or CGG/CCG triplet repeats. The finding that single-stranded CNG repeats form hairpin-like structures in vitro has led to the hypothesis that DNA secondary structure formation is an important component of the expansion mechanism. We show that single-stranded DNA loops containing 10 CTG/CAG or CGG/CCG repeats are inefficiently repaired during meiotic recombination in Saccharomyces cerevisiae. Comparisons of the repair of DNA loops with palindromic and nonpalindromic sequences suggest that this inefficient repair reflects the ability of these sequences to form hairpin structures in vivo.     

Somatic instability of the myotonic dystrophy (CTG)n repeat during human fetal development

Myotonic dystrophy is characterised by the striking level of somatic heterogeneity seen between and within tissues of the same patient, which probably accounts for a significant proportion of the pleiotropy associated with this disorder. The congenital form of the disease is associated with the largest (CTG)n repeat expansions. We have investigated the timing of instability of myotonic dystrophy (CTG)n repeats in a series of congenitally affected fetuses and neonates. We find that during the first trimester the repeat is apparently stable and that instability only becomes detectable during the second and third trimesters. In our series repeat instability is apparent only after 13 weeks gestational age and before 16 weeks. The appearance of heterogeneity shows some tissue specificity, with heart most commonly having the largest expansion. The degree of heterogeneity is not correlated with initial expansion size as gauged by chorionic villus and blood (CTG)n repeat sizes.     

Myotonic dystrophy phenotype without expansion of (CTG)n repeat: an entity distinct from proximal myotonic myopathy (PROMM)?

Myotonic dystrophy (DM) is associated with an expansion of an unstable (CTG)n repeat in the 3' untranslated region of the DM protein kinase (DMPK) gene on chromosome 19q13.3. We studied six patients from two families who showed no expansions of the repeat, in spite of their clinical diagnosis of DM. These patients had multi-systemic manifestations that were distinguishable from those seen in other myotonic disorders, including proximal myotonic myopathy (PROMM). In one additional family, two symptomatic members showed no expanded (CTG)n repeats, while their affected relatives had the expanded repeats. DM haplotype analysis failed to exclude the DMPK locus as a possible site of mutation in each family; however, DMPK mRNA levels were normal. We conclude that a mutation(s) other than the expanded (CTG)n repeat can cause the DM phenotype. The mutation(s) in these families remain(s) to be mapped and characterized.     

Stability of intrastrand hairpin structures formed by the CAG/CTG class of DNA triplet repeats associated with neurological diseases

Expansions of trinucleotide repeats in DNA, a novel source of mutations associated with human disease, may arise by DNA replication slippage initiated by hairpin folding of primer or template strands containing such repeats. To evaluate the stability of single-strand folding by repeating triplets of DNA bases, thermal melting profiles of (CAG)10, (CTG)10, (GAC)10 and (GTC)10 strands are determined at low and physiological salt concentrations, and measurements of melting temperature and enthalpy change are made in each case. Comparisons are made to strands with three times as many repeats, (CAG)30 and (CTG)30. Evidence is presented for stable intrastrand folding by the CAG/CTG class of triplet repeats. Relative to the GAC/GTC class not associated with disease, the order of folding stability is found to be CTG > GAC approximately = CAG > GTC for 10 repeats. Surprisingly, the folds formed by 30 repeats of CTG or CAG have no higher melting temperature and are only 40% more stable in free energy than those formed by 10 repeats. This finding suggests that triplet expansions with higher repeat number may result from the formation of more folded structures with similar stability rather than fewer but longer folds of greater stability.     

Cloning, characterization, and properties of seven triplet repeat DNA sequences

Several neuromuscular and neurodegenerative diseases are caused by genetically unstable triplet repeat sequences (CTG.CAG, CGG.CCG, or AAG.CTT) in or near the responsible genes. We implemented novel cloning strategies with chemically synthesized oligonucleotides to clone seven of the triplet repeat sequences (GTA.TAC, GAT.ATC, GTT.AAC, CAC.GTG, AGG.CCT, TCG.CGA, and AAG.CTT), and the adjoining paper (Ohshima, K., Kang, S., Larson, J. E., and Wells, R. D.(1996) J. Biol. Chem. 271, 16784-16791) describes studies on TTA.TAA. This approach in conjunction with in vivo expansion studies in Escherichia coli enabled the preparation of at least 81 plasmids containing the repeat sequences with lengths of approximately 16 up to 158 triplets in both orientations with varying extents of polymorphisms. The inserts were characterized by DNA sequencing as well as DNA polymerase pausings, two-dimensional agarose gel electrophoresis, and chemical probe analyses to evaluate the capacity to adopt negative supercoil induced non-B DNA conformations. AAG.CTT and AGG.CCT form intramolecular triplexes, and the other five repeat sequences do not form any previously characterized non-B structures. However, long tracts of TCG.CGA showed strong inhibition of DNA synthesis at specific loci in the repeats as seen in the cases of CTG.CAG and CGG.CCG (Kang, S., Ohshima, K., Shimizu, M., Amirhaeri, S., and Wells, R. D.(1995) J. Biol. Chem. 270, 27014-27021). This work along with other studies (Wells, R. D.(1996) J. Biol. Chem. 271, 2875-2878) on CTG.CAG, CGG.CCG, and TTA.TAA makes available long inserts of all 10 triplet repeat sequences for a variety of physical, molecular biological, genetic, and medical investigations. A model to explain the reduction in mRNA abundance in Friedreich's ataxia based on intermolecular triplex formation is proposed.