Alleviating transcript insufficiency caused by Friedreich's ataxia triplet repeats.

Expanded GAA.TTC trinucleotide repeats in intron 1 of the frataxin gene cause Friedreich's ataxia (FRDA) by reducing frataxin mRNA levels. Insufficient frataxin, a nuclear encoded mitochondrial protein, leads to the progressive neurodegeneration and cardiomyopathy characteristic of FRDA. Previously we demonstrated that long GAA.TTC tracts impede transcription elongation in vitro and provided evidence that the impediment results from an intramolecular purine.purine.pyrimidine DNA triplex formed behind an advancing RNA polymerase. Our model predicts that inhibiting formation of this triplex during transcription will increase successful elongation through GAA.TTC tracts. Here we show that this is the case. Oligodeoxyribonucleotides designed to block particular types of triplex formation provide specific and concentration-dependent increases in full-length transcript. In principle, therapeutic agents that selectively interfere with triplex formation could alleviate the frataxin transcript insufficiency caused by pathogenic FRDA alleles.

The GAA*TTC triplet repeat expanded in Friedreich's ataxia impedes transcription elongation by T7 RNA polymerase in a length and supercoil dependent manner.

Large expansions of the trinucleotide repeat GAA*TTC within the first intron of the X25 (frataxin) gene cause Friedreich's ataxia, the most common inherited ataxia. Expansion leads to reduced levels of frataxin mRNA in affected individuals. Here we show that GAA*TTC tracts, in the absence of any other frataxin gene sequences, can reduce the amount of GAA-containing transcript produced in a defined in vitro transcription system. This effect is due to an impediment to elongation that forms in the GAA*TTC tract during transcription, a phenomenon that is exacerbated by both superhelical stress and increased tract length. On supercoiled templates the major truncations of the GAA-containing transcripts occur in the distal (3') end of the GAA repeat. To account for these observations we present a model in which an RNA polymerase advancing within a long GAA*TTC tract initiates the transient formation of an R*R*Y intramolecular DNA triplex. The non-template (GAA) strand folds back creating a loop in the template strand, and the polymerase is paused at the distal triplex-duplex junction.

Tetraplex formation by the progressive myoclonus epilepsy type-1 repeat: implications for instability in the repeat expansion diseases.

The repeat expansion diseases are a group of genetic disorders resulting from an increase in size or expansion of a specific array of tandem repeats. It has been suggested that DNA secondary structures are responsible for this expansion. If this is so, we would expect that all unstable repeats should form such structures. We show here that the unstable repeat that causes progressive myoclonus epilepsy type-1 (EPM1), like the repeats associated with other diseases in this category, forms a variety of secondary structures. However, EPM1 is unique in that tetraplexes are the only structures likely to form in long unpaired repeat tracts under physiological conditions.

Transcription defects induced by repeat expansion: fragile X syndrome, FRAXE mental retardation, progressive myoclonus epilepsy type 1, and Friedreich ataxia.

Fragile X mental retardation syndrome, FRAXE mental retardation, Progressive myoclonus epilepsy Type I, and Friedreich ataxia are members of a larger group of genetic disorders known as the Repeat Expansion Diseases. Unlike other members of this group, these four disorders all result from a primary defect in the initiation or elongation of transcription. In this review, we discuss current models for the relationship between the expanded repeat and the disease symptoms.

Instability of the fragile X syndrome repeat in mice: the effect of age, diet and mutations in genes that affect DNA replication, recombination and repair proficiency.

Repeat expansion diseases such as fragile X syndrome (FXS) result from increases in the size of a specific tandem repeat array. In addition to large expansions, small changes in repeat number and deletions are frequently seen in FXS pedigrees. No mouse model accurately recapitulates all aspects of this instability, particularly the occurrence of large expansions. This may be due to differences between mice and humans in CIS and/or TRANS-acting factors that affect repeat stability. The identification of such factors may help reveal the expansion mechanism and allow the development of suitable animal models for these disorders. We have examined the effect of age, dietary folate, and mutations in the Werner's syndrome helicase (WRN) and TRP53 genes on FXS repeat instability in mice. WRN facilitates replication of the FXS repeat and enhances Okazaki fragment processing, thereby reducing the incidence of processes that have been suggested to lead to expansion. p53 is a protein involved in DNA damage surveillance and repair. We find two types of repeat instability in these mice, small changes in repeat number that are seen at frequencies approaching 100%, and large deletions which occur at a frequency of about 10%. The frequency of these events was independent of WRN, p53, parental age, or folate levels. The large deletions occur at the same frequency in mice homozygous and heterozygous for the repeat suggesting that they are not the result of an interallelic recombination event. In addition, no evidence of large expansions was seen. Our data thus show that the absence of repeat expansions in mice is not due to a more efficient WRN protein or p53-mediated error correction mechanism, and suggest that these proteins, or the pathways in which they are active, may not be involved in expansion in humans either. Moreover, the fact that contractions occur in the absence of expansions suggests that these processes occur by different mechanisms.

Fragile X syndrome and Friedreich's ataxia: two different paradigms for repeat induced transcript insufficiency.

DNA repeat expansion is the genetic basis for a growing number of neurological disorders. While the largest subset of these diseases results in an increase in the length of a polyglutamine tract in the protein encoded by the affected gene, the most common form of inherited mental retardation, fragile X syndrome, and the most common inherited ataxia, Friedreich's ataxia, are both caused by expansions that are transcribed but not translated. These expansions both decrease expression of the gene in which the expanded repeat is located, but they do so by quite different mechanisms. In fragile X syndrome, CGG. CCG expansion in the 5' untranslated region of the FMR1 gene leads to hypermethylation of the repeats and the adjacent CpG-rich promoter. Methylation prevents the binding of the transcription factor alpha-Pal/NRF-1, and may indirectly affect the binding of other factors via the formation of transcriptionally silent chromatin. In Friedreich's ataxia, GAA. TTC expansion in an intron of the FRDA gene reduces expression by interfering with transcription elongation. The model that best describes the available data is transcription-driven formation of a transient purine. purine. pyrimidine DNA triplex behind an advancing RNA polymerase. This structure lassoes the RNA polymerase that caused it, trapping the enzyme on the template.

Hypercard-based data management tools for molecular biologists.

I have designed a Macintosh data management system for molecular biologists. This system, called DataMinder, can be used to store information about oligonucleotides, nucleic acid or protein sequences, recombinant DNA clones, cells, reagents and protocols. DataMinder is not limited to data storage. A number of utilities for data analysis are provided, including those for the evaluation of oligonucleotides for use as hybridization probes or primers for DNA synthesis, and a variety of sequence editing features. Context-sensitive help is available on-line. DataMinder is simple to use and to customize and allows for sharing of database information across a computer network.

NGG-triplet repeats form similar intrastrand structures: implications for the triplet expansion diseases.

Tandem repeats of certain trinucleotides show extensive intergenerational instability in humans that is associated with a class of genetic disorders known as the Triplet Expansion Diseases. This instability is thought to be a consequence of the formation of intrastrand structures, including hairpins, triplexes and tetraplexes, by the tandem repeats. I show here that CGG-repeats which are associated with this group of diseases, and AGG- and TGG-repeats which are not currently known to be, form several intrastrand structures including tetraplexes. In all cases the tetraplexes have the same overall conformation in which all the G residues are involved in G4-tetrads. CGG-repeats also form stable hairpins, but AGG- and TGG-repeats do not form hairpins of comparable stability. However, since tetraplexes can be thought of as folded hairpins, many of the properties ascribed to disease-associated triplets that form hairpins, may apply to these sequences as well. The fact that AGG- and TGG-repeats are not currently associated with any triplet expansion disease suggests either that the ability to adopt an intrastrand folded structure is not sufficient for expansion, or that other diseases associated with such triplets might remain to be identified.

Interaction of the transcription factors USF1, USF2, and alpha -Pal/Nrf-1 with the FMR1 promoter. Implications for Fragile X mental retardation syndrome.

Hypermethylation of the FMR1 promoter reduces its transcriptional activity, resulting in the mental retardation and macroorchidism characteristic of Fragile X syndrome. How exactly methylation causes transcriptional silencing is not known but is relevant if current attempts to reactivate the gene are to be successful. Understanding the effect of methylation requires a better understanding of the factors responsible for FMR1 gene expression. To this end we have identified five evolutionarily conserved transcription factor binding sites in this promoter and shown that four of them are important for transcriptional activity in neuronally derived cells. We have also shown that USF1, USF2, and alpha-Pal/Nrf-1 are the major transcription factors that bind the promoter in brain and testis extracts and suggest that elevated levels of these factors account in part for elevated FMR1 expression in these organs. We also show that methylation abolishes alpha-Pal/Nrf-1 binding to the promoter and affects binding of USF1 and USF2 to a lesser degree. Methylation may therefore inhibit FMR1 transcription not only by recruiting histone deacetylases but also by blocking transcription factor binding. This suggests that for efficient reactivation of the FMR1 promoter, significant demethylation must occur and that current approaches to gene reactivation using histone deacetylase inhibitors alone may therefore have limited effect.

The ability to form intrastrand tetraplexes is an evolutionarily conserved feature of the 3' end of L1 retrotransposons.

Mammalian genomes contain many thousands of members of a family of retrotransponsons known as L1 (or LINE-1) elements. These elements lack long terminal repeats (LTRs), and are thought to use a retroposition mechanism than differs from that of retroviruses and other LTR-containing retroelements. In order to define those regions of the L1 element that may be important for L1 retroposition, we examined the 3' untranslated regions (UTRs) of L1 elements from a diverse group of mammals. We show that while the 3' UTRs of L1 elements from different species share little if any sequence homology, they all contain a G-rich polypurine tract of variable length and sequence which can form one or more intrastrand tetraplexes. This conservation over the 100 Myr since the mammalian radiation suggests that either the G-rich motif itself or a conserved structure such as the tetraplex that can be formed by this motif is a significant structural feature of L1 elements and may play a role in their propagation.

DNA repeat expansions and human disease.

The repeat expansion diseases are genetic disorders caused by intergenerational expansions of a specific tandem DNA repeat. These disorders range from mildly to severely debilitating or fatal, and all have limited treatment options. How expansion occurs and causes disease is only now beginning to be understood. Efforts to model expansion in mice have so far met with only limited success, perhaps due to a requirement for specific cis- or trans-acting factors. In vitro studies and data from bacteria and yeast suggest that in addition to secondary structures formed by the repeats, components of the DNA replication and recombination machinery are important determinants of instability. The consequences of expansion differ depending on where in the gene the repeat tract is located, and range from reduction of transcription initiation to protein toxicity. Recent advances are beginning to make rational approaches to the development of therapies possible.

Insertion of L1 elements into sites that can form non-B DNA. Interactions of non-B DNA-forming sequences.

Three rat L1 element integration (target) sites chosen at random can adopt non-B DNA structures in vitro at normal bacterial superhelical densities. These target sites contain, respectively, short, mixed (AT)n tracts that we show can form one or more cruciforms, short (GT)n tracts, or polypurine:polypyrimidine regions. These sites share no sequence homology, and a non-B DNA structure appears to be the only feature common to them all. When the right end of the L1Rn3 element which forms a complex series of non-B DNA structures including two triplexes, and its target site which undergoes cruciform extrusion, are present on the same supercoiled molecule, they compete for available supercoil energy. The amount of non-B DNA formed at each site varies with pH, the concentration of cations, and the size of the topological domain. The implication of our findings for recombination of L1 elements and for the effect of these elements on contiguous DNA sequences is discussed.

Evidence for the wide distribution of repetitive DNA sequences in the genus Streptomyces.

Repeated DNA sequences were detected as rapidly reannealing sequences in the chromosomal DNA of 13 out of 14 Streptomyces species using either hypochromicity measurements or hydroxyapatite chromatography. These sequences made up between approximately 4% and 11% of the total DNA of these species; only in Streptomyces rimosus were repeated DNA sequences not detected. The repeated sequences fall into a number of distinct percentage G + C (%G + C) classes, many being of rather low %G + C. Analytical density ultracentrifugation of the DNA of these species indicated satellite bands of low %G + C, and high-resolution thermal denaturation profiles indicated the presence of blocks of DNA of low G + C content too. No such satellite band could be found in Streptomyces coelicolor and no low-%G + C DNA could be detected in its thermal denaturation profile. The possible relationship of this repeated DNA, an unusual occurrence in a procaryote, to genetic instability and genetic control mechanisms in Streptomyces is discussed.

Rat L (long interspersed repeated DNA) elements contain guanine-rich homopurine sequences that induce unpairing of contiguous duplex DNA.

The L family (long interspersed repeated DNA) of mobile genetic elements is a persistent feature of the mammalian genome. In rats, this family contains approximately equal to 40,000 members and accounts for approximately equal to 10% of the haploid genome. We demonstrate here that the guanine-rich homopurine stretches located at the right end of L-DNA induce oligonucleotide uptake by contiguous duplex DNA. The uptake is dependent on negative supercoiling and the length of the homopurine stretch and occurs even when the L-DNA homopurine stretches are introduced into a different DNA environment. The bound oligomer primes DNA synthesis when DNA polymerase and deoxyribonucleoside triphosphates are added, resulting in a faithful copy of the template to which the oligonucleotide had bound. The implications of this property of the L-DNA guanine-rich homopurine stretches in the amplification, recombination, and dispersal of L elements is discussed.

CGG repeats associated with DNA instability and chromosome fragility form structures that block DNA synthesis in vitro.

A large increase in the length of a CGG tandem array is associated with a number of triplet expansion diseases, including fragile X syndrome, the most common cause of heritable mental retardation in humans. Expansion results in the appearance of a fragile site on the X chromosome in the region of the CGG array. We show here that CGG repeats readily form a series of barriers to DNA synthesis in vitro. There barriers form only when the (CGG)n strand is used as the template, are K(+)-dependent, template concentration-independent, and involve hydrogen bonding between guanines. Chemical modification experiments suggest these blocks to DNA synthesis result from the formation of a series of intrastrand tetraplexes. A number of lines of evidence suggest that both triplet expansion and chromosome fragility are the result of replication defects. Our data are discussed in the light of such evidence.

The structure of the guanine-rich polypurine:polypyrimidine sequence at the right end of the rat L1 (LINE) element.

We report here that the 64-base pair (bp) guanine-rich polypurine:polypyrimidine tract derived from the right end of the rat long interspersed DNA element is reactive in a supercoil-dependent manner with a variety of chemical probes of non-B DNA structure. At pH 5.0 in the presence of Mg2+, part of the sequence (position 10-40) forms the following two types of triplexes: a G.G.C triplex, and an unusual C.G.C triplex. The latter structure is much more prevalent than the former and is unusual in that the resultant free purine strand forms a hairpin loop. In the absence of Mg2+ the G.G.C triplex disappears and the amount of C.G.C triplex is diminished, and at pH 7.5 in the presence or absence of Mg2+, little or no triplex is observed. Deletion of the 24-bp region just 3' of the triplex-forming region greatly reduces the amount of triplex formed. In this region, which includes an 18-bp polypurine:polypyrimidine sequence, both strands exhibit a moderate symmetric reactivity with the chemical probes tested, independent of pH and Mg2+. The implications of this structurally complex region for the properties of the rat L1 element are discussed.

The effect of inhibitors of DNA repair on the genetic instability of Streptomyces cattleya.

Various streptomycetes show well defined instabilities that do not appear to be attributable to plasmid loss. The unstable phenotype, in many cases, arises at frequencies too high to be explained by point mutations. The frequency of instability can be enhanced by UV irradiation. Two major repair systems have been found in Escherichia coli: the 'error-free' system which is inhibited by caffeine and the 'error-prone' system which is inhibited by arsenite. Using spores of Streptomyces cattleya NRRL 8057 and the virulent actinophage VC11 we have shown that a caffeine inhibitable, host mediated UV repair system is active in spores during early development. Some evidence was also found for the presence of an arsenite inhibitable UV repair system. The caffeine inhibitable UV repair system was found to be involved in the induction of genetic instability in S. cattleya. The arsenite system may be implicated in the repair of such events. Genetic instability was also induced by single strand breaks in DNA caused by 32P.

The development and use of a DNA polymerase arrest assay for the evaluation of parameters affecting intrastrand tetraplex formation.

We show here that a K+-dependent block to DNA synthesis is a sensitive and specific indicator of intrastrand tetraplex formation that can be used, both to identify sequences with tetraplex-forming potential and to examine parameters that affect tetraplex formation. We show that tetraplex formation is determined by a complex combination of factors including the size and base composition of its constituent loops and stems. In the process of carrying out this study we have found that the number of sequences with the ability to form tetraplexes is larger than previously thought, and that such sequences are ubiquitous in eukaryote genomes.

DNA secondary structures and the evolution of hypervariable tandem arrays.

Tandem repeats are ubiquitous in nature and constitute a major source of genetic variability in populations. This variability is associated with a number of genetic disorders in humans including triplet expansion diseases such as Fragile X syndrome and Huntington's disease. The mechanism responsible for the variability/instability of these tandem arrays remains contentious. We show here that formation of secondary structures, in particular intrastrand tetraplexes, is an intrinsic property of some of the more unstable arrays. Tetraplexes block DNA polymerase progression and may promote instability of tandem arrays by increasing the likelihood of reiterative strand slippage. In the course of doing this work we have shown that some of these tetraplexes involve unusual base interactions. These interactions not only generate tetraplexes with novel properties but also lead us to conclude that the number of sequences that can form stable tetraplexes might be much larger than previously thought.