Mutation spectra in fragile X syndrome induced by deletions of CGG*CCG repeats

The fragile X syndrome results from expansions as well as deletions of the repeating CGG.CCG DNA sequence in the 5'-untranslated region of the FMR1 gene on the X chromosome. The relative frequency of disease cases promoted by these two types of mutations cannot be ascertained at present because the routine clinical assay monitors only expansions. At least 30 articles have been reviewed that document the involvement of deletions of part or all of the CGG.CCG repeats along with varying extents of DNA flanking regions as well as very small mutations including single base pair changes. Studies of deletion mutants of CGG.CCG tracts in Escherichia coli plasmids revealed a similar spectrum of mutagenic products. The triplet repeat tract in a non-B conformation is the mutagen, not the sequence per se in the right-handed B helix. Hence, molecular investigations in a simple model organism may generate useful initial information toward therapeutic strategies for this disease.     

Role of replication and CpG methylation in fragile X syndrome CGG deletions in primate cells

Instability of the fragile X CGG repeat involves both maternally derived expansions and deletions in the gametes of full-mutation males. It has also been suggested that the absence of aberrant CpG methylation may enhance repeat deletions through an unknown process. The effect of CGG tract length, DNA replication direction, location of replication initiation, and CpG methylation upon CGG stability were investigated using an SV40 primate replication system. Replication-dependant deletions with 53 CGG repeats were observed when replication was initiated proximal to the repeat, with CGG as the lagging-strand template. When we initiated replication further from the repeat, while maintaining CGG as the lagging-strand template or using CCG as the lagging-strand template, significant instability was not observed. CpG methylation of the unstable template stabilized the repeat, decreasing both the frequency and the magnitude of deletion events. Furthermore, CpG methylation slowed the efficiency of replication for all templates. Interestingly, replication forks displayed no evidence of a block at the CGG repeat tract, regardless of replication direction or CpG methylation status. Templates with 20 CGG repeats were stable under all circumstances. These results reveal that CGG deletions occur during replication and are sensitive to replication-fork dynamics, tract length, and CpG methylation.     

Replication and expansion of trinucleotide repeats in yeast

The mechanisms of trinucleotide repeat expansions, underlying more than a dozen hereditary neurological disorders, are yet to be understood. Here we looked at the replication of (CGG)(n) x (CCG)(n) and (CAG)(n) x (CTG)(n) repeats and their propensity to expand in Saccharomyces cerevisiae. Using electrophoretic analysis of replication intermediates, we found that (CGG)(n) x (CCG)(n) repeats significantly attenuate replication fork progression. Replication inhibition for this sequence becomes evident at as few as approximately 10 repeats and reaches a maximal level at 30 to 40 repeats. This is the first direct demonstration of replication attenuation by a triplet repeat in a eukaryotic system in vivo. For (CAG)(n) x (CTG)(n) repeats, on the contrary, there is only a marginal replication inhibition even at 80 repeats. The propensity of trinucleotide repeats to expand was evaluated in a parallel genetic study. In wild-type cells, expansions of (CGG)(25) x (CCG)(25) and (CAG)(25) x (CTG)(25) repeat tracts occurred with similar low rates. A mutation in the large subunit of the replicative replication factor C complex (rfc1-1) increased the expansion rate for the (CGG)(25) repeat approximately 50-fold but had a much smaller effect on the expansion of the (CTG)(25) repeat. These data show dramatic sequence-specific expansion effects due to a mutation in the lagging strand DNA synthesis machinery. Together, the results of this study suggest that expansions are likely to result when the replication fork attempts to escape from the stall site.     

DNA polymerase III proofreading mutants enhance the expansion and deletion of triplet repeat sequences in Escherichia coli

The influence of mutations in the 3' to 5' exonucleolytic proofreading epsilon-subunit of Escherichia coli DNA polymerase III on the genetic instabilities of the CGG.CCG and the CTG.CAG repeats that cause human hereditary neurological diseases was investigated. The dnaQ49(ts) and the mutD5 mutations destabilize the CGG.CCG repeats. The distributions of the deletion products indicate that slipped structures containing a small number of repeats in the loop mediate the deletion process. The CTG.CAG repeats were destabilized by the dnaQ49(ts) mutation by a process mediated by long hairpin loop structures (>/=5 repeats). The mutD5 mutator strain stabilized the (CTG.CAG)(175) tract, which contained two interruptions. Since the mutD5 mutator strain has a saturated mismatch repair system, the stabilization is probably an indirect effect of the nonfunctional mismatch repair system in these strains. Shorter uninterrupted tracts expand readily in the mutD5 strain, presumably due to the greater stability of long CTG.CAG tracts (>100 repeats) in this strain. When parallel studies were conducted in minimal medium, where the mutD5 strain is defective in exonucleolytic proofreading but has a functional MMR system, both CTG.CAG and CGG.CCG repeats were destabilized, showing that the proofreading activity is essential for maintaining the integrity of TRS tracts. Thus, we conclude that the expansion and deletion of triplet repeats are enhanced by mutations that reduce the fidelity of replication.     

The hidden side of unstable DNA repeats: Mutagenesis at a distance

Structure-prone DNA repeats are common components of genomic DNA in all kingdoms of life. In humans, these repeats are linked to genomic instabilities that result in various hereditary disorders, including many cancers. It has long been known that DNA repeats are not only highly polymorphic in length but can also cause chromosomal fragility and stimulate gross chromosomal rearrangements, i.e., deletions, duplications, inversions, translocations and more complex shuffles. More recently, it has become clear that inherently unstable DNA repeats dramatically elevate mutation rates in surrounding DNA segments and that these mutations can occur up to ten kilobases away from the repetitive tract, a phenomenon we call repeat-induced mutagenesis (RIM). This review describes experimental data that led to the discovery and characterization of RIM and discusses the molecular mechanisms that could account for this phenomenon.     

In vitro and in vivo analysis of transcription within the replication region of plasmid pIP501

Summary The tobacco (Nicotiana tabacum) nuclear genome contains long tracts of DNA (i.e. in excess of 18 kb) with high sequence homology to the tobacco plastid genome. Five lambda clones containing these nuclear DNA sequences encompass more than one-third of the tobacco plastid genome. The absolute size of these five integrants is unknown but potentially includes uninterrupted sequences that are as large as the plastid genome itself. An additional sequence was cloned consisting of both nuclear and plastid-derived DNA sequences. The nuclear component of the clone is part of a family of repeats, which are present in about 400 locations in the nuclear genome. The homologous sequences present in chromosomal DNA were very similar to those of the corresponding sequences in the plastid genome. However significant sequence divergence, including base substitutions, insertions and deletions of up to 41 bp, was observed between these nuclear sequences and the plastid genome. Associated with the larger deletions were sequence motifs suggesting that processes such as DNA replication slippage and excision of hairpin loops may have been involved in deletion formation.     

Spontaneous deletion formation at the aprt locus of hamster cells: the presence of short sequence homologies and dyad symmetries at deletion termini.

To examine the factors governing the generation of DNA sequence rearrangements in mammalian somatic cells, we have cloned and sequenced novel junctions produced by six spontaneous deletion mutations at the aprt locus of Chinese hamster ovary cells. Our analyses indicate that these rearrangements were produced by non-homologous recombinational events occurring between short (2-7 bp) sequence repeats at the two termini of the deletion which leave one copy of the repeat in the mutant gene. Certain tri- and tetranucleotides recur at the deletion termini, suggesting that these may possibly be a recognition sequence for an enzyme involved in the event. No other gene structural alterations were found at the novel junctions or in neighbouring sequences. The deletions are not randomly distributed over the aprt gene; four termini clustered in a 40-bp sequence. This region of aprt is unusual as it contains both significant stretches of dyad symmetry which could potentially form stable DNA secondary structures and short direct repeats. Regions of dyad symmetry were also found at at least one terminus of all the deletions. In view of the similar properties of this set of deletions, possible mechanisms for the formation of this type of gene rearrangement are considered.     

A small molecule regulates hairpin structures in d(CGG) trinucleotide repeats

Unusual expansion of trinucleotide repeats has been identified as a common mechanism of hereditary neurodegenerative diseases. Although the actual mechanism of repeat expansion remains uncertain, trinucleotide repeat instability may be related to the increased stability of an alternative DNA hairpin structure formed in the repeat sequences. Here we report that a synthetic ligand naphthyridine carbamate dimer (NCD) selectively bound to and stabilized an intra-stranded hairpin structure in CGG repeat sequences. The NCD-CGG hairpin complex was a stable structure that efficiently interfered with DNA replication by Taq DNA polymerase. Considering the sequence preference of NCD, the use of NCD would be valuable to investigate the genetic instabilities of CGG/CCG repeat sequences in human genomes.     

Comprehensive identification of mutations induced by heavy‐ion beam irradiation in Arabidopsis thaliana

Heavy‐ion beams are widely used for mutation breeding and molecular biology. Although the mutagenic effects of heavy‐ion beam irradiation have been characterized by sequence analysis of some restricted chromosomal regions or loci, there have been no evaluations at the whole‐genome level or of the detailed genomic rearrangements in the mutant genomes. In this study, using array comparative genomic hybridization (array‐CGH) and resequencing, we comprehensively characterized the mutations in Arabidopsis thaliana genomes irradiated with Ar or Fe ions. We subsequently used this information to investigate the mutagenic effects of the heavy‐ion beams. Array‐CGH demonstrated that the average number of deleted areas per genome were 1.9 and 3.7 following Ar‐ion and Fe‐ion irradiation, respectively, with deletion sizes ranging from 149 to 602 180 bp; 81% of the deletions were accompanied by genomic rearrangements. To provide a further detailed analysis, the genomes of the mutants induced by Ar‐ion beam irradiation were resequenced, and total mutations, including base substitutions, duplications, in/dels, inversions, and translocations, were detected using three algorithms. All three resequenced mutants had genomic rearrangements. Of the 22 DNA fragments that contributed to the rearrangements, 19 fragments were responsible for the intrachromosomal rearrangements, and multiple rearrangements were formed in the localized regions of the chromosomes. The interchromosomal rearrangements were detected in the multiply rearranged regions. These results indicate that the heavy‐ion beams led to clustered DNA damage in the chromosome, and that they have great potential to induce complicated intrachromosomal rearrangements. Heavy‐ion beams will prove useful as unique mutagens for plant breeding and the establishment of mutant lines.     

Werner syndrome protein suppresses the formation of large deletions during the replication of human telomeric sequences

Werner syndrome (WS) is a disorder characterized by features of premature aging and increased cancer that is caused by loss of the RecQ helicase WRN. Telomeres consisting of duplex TTAGGG repeats in humans protect chromosome ends and sustain cellular proliferation. WRN prevents the loss of telomeres replicated from the G-rich strand, which can form secondary G-quadruplex (G4) structures. Here, we dissected WRN roles in the replication of telomeric sequences by examining factors inherent to telomeric repeats, such as G4 DNA, independently from other factors at chromosome ends that can also impede replication. For this we used the supF shuttle vector (SV) mutagenesis assay. We demonstrate that SVs with [TTAGGG]₆ sequences are stably replicated in human cells, and that the repeats suppress the frequency of large deletions despite G4 folding potential. WRN depletion increased the supF mutant frequency for both the telomeric and non-telomeric SVs, compared with the control cells, but this increase was much greater (27-fold) for telomeric SVs. The higher SV mutant frequencies in WRN-deficient cells were primarily due to an increase in large sequence deletions and rearrangements. However, WRN depletion caused a more dramatic increase in deletions and rearrangements arising within the telomeric SV (70-fold), compared with non-telomeric SV (8-fold). Our results indicate that WRN prevents large deletions and rearrangements during replication, and that this role is particularly important in templates with telomeric sequence. This provides a possible explanation for increased telomere loss in WS cells.     

Alpha-galactosidase A gene rearrangements causing Fabry disease. Identification of short direct repeats at breakpoints in an Alu-rich gene

Fabry disease, an inborn error of glycosphingolipid catabolism, results from mutations in the X-linked gene encoding the lysosomal enzyme, alpha-galactosidase A (EC 3.2.1.22). Six alpha-galactosidase A gene rearrangements that cause Fabry disease were investigated to assess the role of Alu repetitive elements and short direct and/or inverted repeats in the generation of these germinal mutations. The breakpoints of five partial gene deletions and one partial gene duplication were determined by either cloning and sequencing the mutant gene from an affected hemizygote, or by polymerase chain reaction amplifying and sequencing the genomic region containing the novel junction. Although the alpha-galactosidase A gene contains 12 Alu repetitive elements (representing approximately 30% of the 12-kilobase (kb) gene or approximately 1 Alu/1.0 kb), only one deletion resulted from an Alu-Alu recombination. The remaining five rearrangements involved illegitimate recombinational events between short direct repeats of 2 to 6 base pairs (bp) at the deletion or duplication breakpoints. Of these rearrangements, one had a 3' short direct repeat within an Alu element, while another was unusual having two deletions of 1.7 kb and 14 bp separated by a 151-bp inverted sequence. These findings suggested that slipped mispairing or intrachromosomal exchanges involving short direct repeats were responsible for the generation of most of these gene rearrangements. There were no inverted repeat sequences or alternating purine-pyrimidine regions which may have predisposed the gene to these rearrangements. Intriguingly, the tetranucleotide CCAG and the trinucleotide CAG (or their respective complements, CTGG and CTG) occurred within or adjacent to the direct repeats at the 5' breakpoints in three and four of the five alpha-galactosidase A gene rearrangements, respectively, suggesting a possible functional role in these illegitimate recombinational events. These studies indicate that short direct repeats are important in the formation of gene rearrangements, even in human genes like alpha-galactosidase A that are rich in Alu repetitive elements.     

Werner syndrome protein suppresses the formation of large deletions during the replication of human telomeric sequences

Werner syndrome (WS) is a disorder characterized by features of premature aging and increased cancer that is caused by loss of the RecQ helicase WRN. Telomeres consisting of duplex TTAGGG repeats in humans protect chromosome ends and sustain cellular proliferation. WRN prevents the loss of telomeres replicated from the G-rich strand, which can form secondary G-quadruplex (G4) structures. Here, we dissected WRN roles in the replication of telomeric sequences by examining factors inherent to telomeric repeats, such as G4 DNA, independently from other factors at chromosome ends that can also impede replication. For this we used the supF shuttle vector (SV) mutagenesis assay. We demonstrate that SVs with [TTAGGG]₆ sequences are stably replicated in human cells, and that the repeats suppress the frequency of large deletions despite G4 folding potential. WRN depletion increased the supF mutant frequency for both the telomeric and non-telomeric SVs, compared with the control cells, but this increase was much greater (27-fold) for telomeric SVs. The higher SV mutant frequencies in WRN-deficient cells were primarily due to an increase in large sequence deletions and rearrangements. However, WRN depletion caused a more dramatic increase in deletions and rearrangements arising within the telomeric SV (70-fold), compared with non-telomeric SV (8-fold). Our results indicate that WRN prevents large deletions and rearrangements during replication, and that this role is particularly important in templates with telomeric sequence. This provides a possible explanation for increased telomere loss in WS cells.     

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.     

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.     

Enhanced Deletion Formation by Aberrant DNA Replication in Escherichia Coli

Repeated genes and sequences are prone to genetic rearrangements including deletions. We have investigated deletion formation in Escherichia coli strains mutant for various replication functions. Deletion was selected between 787 base pair tandem repeats carried either on a ColE1-derived plasmid or on the E. coli chromosome. Only mutations in functions associated with DNA Polymerase III elevated deletion rates in our assays. Especially large increases were observed in strains mutant in dnaQ, the ε editing subunit of Pol III, and dnaB, the replication fork helicase. Mutations in several other functions also altered deletion formation: the α polymerase (dnaE), the γ clamp loader complex (holC, dnaX), and the β clamp (dnaN) subunits of Pol III and the primosomal proteins, dnaC and priA. Aberrant replication stimulated deletions through several pathways. Whereas the elevation in dnaB strains was mostly recA- and lexA-dependent, that in dnaQ strains was mostly recA- and lexA-independent. Deletion product analysis suggested that slipped mispairing, producing monomeric replicon products, may be preferentially increased in a dnaQ mutant and sister-strand exchange, producing dimeric replicon products, may be elevated in dnaE mutants. We conclude that aberrant Polymerase III replication can stimulate deletion events through several mechanisms of deletion and via both recA-dependent and independent pathways.     

Structural basis for triplet repeat disorders: a computational analysis

MOTIVATION: Over a dozen major degenerative disorders, including myotonic distrophy, Huntington's disease and fragile X syndrome, result from unstable expansions of particular trinucleotides. Remarkably, only some of all the possible triplets, namely CAG/CTG, CGG/CCG and GAA/TTC, have been associated with the known pathological expansions. This raises some basic questions at the DNA level. Why do particular triplets seem to be singled out? What is the mechanism for their expansion and how does it depend on the triplet itself? Could other triplets or longer repeats be involved in other diseases? RESULTS: Using several different computational models of DNA structure, we show that the triplets involved in the pathological repeats generally fall into extreme classes. Thus, CAG/CTG repeats are particularly flexible, whereas GCC, CGG and GAA repeats appear to display both flexible and rigid (but curved) characteristics depending on the method of analysis. The fact that (1) trinucleotide repeats often become increasingly unstable when they exceed a length of approximately 50 repeats, and (2) repeated 12-mers display a similar increase in instability above 13 repeats, together suggest that approximately 150 bp is a general threshold length for repeat instability. Since this is about the length of DNA wrapped up in a single nucleosome core particle, we speculate that chromatin structure may play an important role in the expansion mechanism. We furthermore suggest that expansion of a dodecamer repeat, which we predict to have very high flexibility, may play a role in the pathogenesis of the neurodegenerative disorder multiple system atrophy (MSA). CONTACT: pfbaldi@ics.uci.edu, yves@netid.com, brunak@cbs.dtu.dk, gorm@cbs.dtu.dk.     

Molecular characterisation of camptothecin-induced mutations at the hprt locus in Chinese hamster cells

The capacity of the topoisomerase I inhibitor camptothecin (CPT) to induce single locus mutations at the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene and the DNA changes underlying induced mutations were analysed in Chinese hamster ovary cells. Camptothecin treatments increased hprt mutations up to 50-fold over the spontaneous levels at highly cytotoxic doses. Genomic DNA was isolated from 6-thioguanine resistant clones and subjected to multiplex PCR to screen for gross alterations in the gene structure. The molecular analysis revealed that deletion mutants represented 80% of the analysed clones, including total hprt deletion, multiple and single exon deletions. Furthermore, a fraction of the analysed clones showed deletions of more than one exon that were characterised by the absence of non-contiguous exons. These data show that single locus mutations induced by camptothecin are characterised by large deletions or complex rearrangements rather than single base substitutions and suggest that the recombinational repair of camptothecin-induced strand breaks at replication fork may be involved in the generations of these alterations at the chromatin structure level.