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

a b s t r a c t 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

Coupling Transcriptional State to Large-Scale Repeat Expansions in Yeast

SummaryExpansions of simple DNA repeats cause numerous hereditary disorders in humans. Replication, repair, and transcription are implicated in the expansion process, but their relative contributions are yet to be distinguished. To separate the roles of replication and transcription in the expansion of Friedreich’s ataxia (GAA)n repeats, we designed two yeast genetic systems that utilize a galactose-inducible GAL1 promoter but contain these repeats in either the transcribed or nontranscribed region of a selectable cassette. We found that large-scale repeat expansions can occur in the lack of transcription. Induction of transcription strongly elevated the rate of expansions in both systems, indicating that active transcriptional state rather than transcription through the repeat per se affects this process. Furthermore, replication defects increased the rate of repeat expansions irrespective of transcriptional state. We present a model in which transcriptional state, linked to the nucleosomal density of a region, acts as a modulator of large-scale repeat expansions

Effects of Friedreich's ataxia (GAA)(n)·(TTC)(n) repeats on RNA synthesis and stability

Expansions of (GAA)(n) repeats within the first intron of the frataxin gene reduce its expression, resulting in a hereditary neurodegenerative disorder, Friedreich's ataxia. While it is generally believed that expanded (GAA)(n) repeats block transcription elongation, fine mechanisms responsible for gene repression are not fully understood. To follow the effects of (GAA)(n)·(TTC)(n) repeats on gene expression, we have chosen E. coli as a convenient model system. (GAA)(n)·(TTC)(n) repeats were cloned into bacterial plasmids in both orientations relative to a promoter, and their effects on transcription and RNA stability were evaluated both in vitro and in vivo. Expanded (GAA)(n) repeats in the sense strand for transcription caused a significant decrease in the mRNA levels in vitro and in vivo. This decrease was likely due to the tardiness of the RNA polymerase within expanded (GAA)(n) runs but was not accompanied by the enzyme's dissociation and premature transcription termination. Unexpectedly, positioning of normal- and carrier-size (TTC)(n) repeats into the sense strand for transcription led to the appearance of RNA transcripts that were truncated within those repetitive runs in vivo. We have determined that these RNA truncations are consistent with cleavage of the full-sized mRNAs at (UUC)(n) runs by the E. coli degradosome

Large-scale expansions of Friedreich's ataxia GAA•TTC repeats in an experimental human system: role of DNA replication and prevention by LNA-DNA oligonucleotides and PNA oligomers

Friedreich's ataxia (FRDA) is caused by expansions of GAA•TTC repeats in the first intron of the human FXN gene that occur during both intergenerational transmissions and in somatic cells. Here we describe an experimental system to analyze large-scale repeat expansions in cultured human cells. It employs a shuttle plasmid that can replicate from the SV40 origin in human cells or be stably maintained in S. cerevisiae utilizing ARS4-CEN6. It also contains a selectable cassette allowing us to detect repeat expansions that accumulated in human cells upon plasmid transformation into yeast. We indeed observed massive expansions of GAA•TTC repeats, making it the first genetically tractable experimental system to study large-scale repeat expansions in human cells. Further, GAA•TTC repeats stall replication fork progression, while the frequency of repeat expansions appears to depend on proteins implicated in replication fork stalling, reversal, and restart. Locked nucleic acid (LNA)-DNA mixmer oligonucleotides and peptide nucleic acid (PNA) oligomers, which interfere with triplex formation at GAA•TTC repeats in vitro, prevented the expansion of these repeats in human cells. We hypothesize, therefore, that triplex formation by GAA•TTC repeats stall replication fork progression, ultimately leading to repeat expansions during replication fork restart

Reduced local mutation density in regulatory DNA of cancer genomes is linked to DNA repair

Carcinogenesis and neoplastic progression are mediated by the accumulation of somatic mutations. Here we report that the local density of somatic mutations in cancer genomes is highly reduced specifically in accessible regulatory DNA defined by DNase I hypersensitive sites. This reduction is independent of any known factors influencing somatic mutation density and is observed in diverse cancer types, suggesting a general mechanism. By analyzing individual cancer genomes1, we show that the reduced local mutation density within regulatory DNA is linked to intact global genome repair machinery, with nearly complete abrogation of the hypomutation phenomenon in individual cancers that possess mutations in multiple nucleotide excision repair components. Together, our results connect chromatin structure, gene regulation and cancer-associated somatic mutation

A Defective mRNA Cleavage and Polyadenylation Complex Facilitates Expansions of Transcribed (GAA) n Repeats Associated with Friedreich’s Ataxia

Expansions of microsatellite repeats are responsible for numerous hereditary diseases in humans, including myotonic dystrophy and Friedreich's ataxia. Whereas the length of an expandable repeat is the main factor determining disease inheritance, recent data point to genomic trans modifiers that can impact the likelihood of expansions and disease progression. Detection of these modifiers may lead to understanding and treating repeat expansion diseases. Here, we describe a method for the rapid, genome-wide identification of trans modifiers for repeat expansion in a yeast experimental system. Using this method, we found that missense mutations in the endoribonuclease subunit (Ysh1) of the mRNA cleavage and polyadenylation complex dramatically increase the rate of (GAA) n repeat expansions but only when they are actively transcribed. These expansions correlate with slower transcription elongation caused by the ysh1 mutation. These results reveal an interplay between RNA processing and repeat-mediated genome instability, confirming the validity of our approach. Keywords: genome instability; repeat expansion; RNA polyadenylation; RNA processing; transcription-replication conflicts; Friedreich’s ataxia; DNA double-strand breaks; trans-modifiers of repeat expansions; genetic screen; whole-genome sequencin

Chromosome fragility at GAA tracts in yeast depends on repeat orientation and requires mismatch repair

Expansion of triplex-forming GAA/TTC repeats in the first intron of FXN gene results in Friedreich's ataxia. Besides FXN, there are a number of other polymorphic GAA/TTC loci in the human genome where the size variations thus far have been considered to be a neutral event. Using yeast as a model system, we demonstrate that expanded GAA/TTC repeats represent a threat to eukaryotic genome integrity by triggering double-strand breaks and gross chromosomal rearrangements. The fragility potential strongly depends on the length of the tracts and orientation of the repeats relative to the replication origin, which correlates with their propensity to adopt triplex structure and to block replication progression. We show that fragility is mediated by mismatch repair machinery and requires the MutSb and endonuclease activity of MutLa. We suggest that the mechanism of GAA/TTC-induced chromosomal aberrations defined in yeast can also operate in human carriers with expanded tracts

Friedreich's Ataxia (GAA)n•(TTC)n Repeats Strongly Stimulate Mitotic Crossovers in Saccharomyces cerevisae

Expansions of trinucleotide GAA•TTC tracts are associated with the human disease Friedreich's ataxia, and long GAA•TTC tracts elevate genome instability in yeast. We show that tracts of (GAA)230•(TTC)230 stimulate mitotic crossovers in yeast about 10,000-fold relative to a “normal” DNA sequence; (GAA)n•(TTC)n tracts, however, do not significantly elevate meiotic recombination. Most of the mitotic crossovers are associated with a region of non-reciprocal transfer of information (gene conversion). The major class of recombination events stimulated by (GAA)n•(TTC)n tracts is a tract-associated double-strand break (DSB) that occurs in unreplicated chromosomes, likely in G1 of the cell cycle. These findings indicate that (GAA)n•(TTC)n tracts can be a potent source of loss of heterozygosity in yeast