Large-scale expansions of DNA repeats are implicated in numerous hereditary disorders in humans. We describe a yeast experimental system to analyze large-scale expansions of triplet GAA repeats, responsible for the human disease Friedreich’s ataxia. When GAA repeats were placed into an intron of the chimeric URA3 gene, their expansions caused gene inactivation, which was detected on the selective media. We found that the rates of expansions of GAA repeats increased exponentially with their lengths. These rates were only mildly dependent on the repeat’s orientation within the replicon, whereas the repeat-mediated replication fork stalling was exquisitely orientation-dependent. Expansion rates were significantly elevated upon inactivation of the replication fork stabilizers, Tof1 and Csm3, but decreased in the mutants of postreplication DNA repair proteins, Rad6 and Rad5, and the DNA helicase Sgs1. We propose a model for large-scale repeat expansions based on the template switching during the replication fork progression through repetitive DNA.

Friedreich's ataxia (GAA) n repeats of various lengths were cloned into a Saccharymyces cerevisiae plasmid, and their effects on DNA replication were analyzed using two-dimensional electrophoresis of replication intermediates. We found that premutation-and disease-size repeats stalled the replication fork progression in vivo, while normal-size repeats did not affect replication. Remarkably, the observed threshold repeat length for replication stalling in yeast (ϳ40 repeats) closely matched the threshold length for repeat expansion in humans. Further, replication stalling was strikingly orientation dependent, being pronounced only when the repeat's homopurine strand served as the lagging strand template. Finally, it appeared that length polymorphism of the (GAA) n ⅐ (TTC) n repeat in both expansions and contractions drastically increases in the repeat's orientation that is responsible for the replication stalling. These data represent the first direct proof of the effects of (GAA) n repeats on DNA replication in vivo. We believe that repeat-caused replication attenuation in vivo is due to triplex formation. The apparent link between the replication stalling and length polymorphism of the repeat points to a new model for the repeat expansion.

Various DNA sequences that interfere with transcription due to their unusual structural properties have been implicated in the regulation of gene expression and with genomic instability. An important example is sequences containing G-rich homopurinehomopyrimidine stretches, for which unusual transcriptional behavior is implicated in regulation of immunogenesis and in other processes such as genomic translocations and telomere function. To elucidate the mechanism of the effect of these sequences on transcription we have studied T7 RNA polymerase transcription of G-rich sequences in vitro. We have shown that these sequences produce significant transcription blockage in an orientation-, lengthand supercoiling-dependent manner. Based upon the effects of various sequence modifications, solution conditions, and ribonucleotide substitutions, we conclude that transcription blockage is due to formation of unusually stable RNA/DNA hybrids, which could be further exacerbated by triplex formation. These structures are likely responsible for transcription-dependent replication blockage by G-rich sequences in vivo.R-loops | DNA supercoiling | Hoogsteen base pairing | inosine | 7-deazaquanosine S equence-specific modulation of transcription, including transcription blockage or impediment, plays an important role in DNA transactions, for example, transcription-related mutagenesis and recombination (reviewed in refs. 1 and 2) and could also be responsible for several severe genetic diseases (reviewed in refs. 3-5).Among the DNA sequences that could affect transcription are GC-rich homopurine-homopyrimidine (hPu/hPy) stretches. These sequences could form unusual DNA structures, including triplexes and G quadruplexes (reviewed in refs. 3-5), which have been implicated in several transcription-dependent phenomena (for example, see refs. 6-9).Another important property of these sequences is a dramatic asymmetry in the stabilities of RNA/DNA duplexes: The rPu/dPy duplex is significantly more stable, whereas the rPy/dPu duplex is less stable than a DNA/DNA duplex of the same sequence (10). The increased stability of rPu/dPy duplexes is likely responsible for stable R-loop formation by these sequences (11), although alternative DNA structures might also be involved (8,12,13).The simplest example of GC-rich hPu/hPy sequences, the G n ∕C n repeats, is abundant in various genomes, including transcribed domains (14, 15).The G 32 ∕C 32 stretch was previously shown to stall DNA replication in Escherichia coli plasmids in vivo (16). Remarkably, this effect was observed only when the sequence was transcribed, which led to a model stipulating that this sequence stalled an elongating RNA polymerase, and the stalled transcription complex, in turn, blocked the replication machinery (16).To elucidate the mechanism of transcription blockage by this sequence, we have studied its effect on T7 RNA polymerase (T7 RNAP) transcription in vitro, using various sequence modifications and solution conditions that allowed us to discriminate between possible D...

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 ⅐ (CCG) n and (CAG) n ⅐ (CTG) n repeats and their propensity to expand in Saccharomyces cerevisiae. Using electrophoretic analysis of replication intermediates, we found that (CGG) n ⅐ (CCG) n repeats significantly attenuate replication fork progression. Replication inhibition for this sequence becomes evident at as few as ϳ10 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 ⅐ (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 ⅐ (CCG) 25 and (CAG) 25 ⅐ (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 ϳ50-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.Trinucleotide repeats, specifically (CGG) n ⅐ (CCG) n , (CAG) n ⅐ (CTG) n , and (GAA) n ⅐ (TTC) n , have attracted wide attention since their expansion leads to numerous hereditary neurological disorders in humans, including fragile X syndrome, Huntington's disease, myotonic dystrophy, Friedreich's ataxia, etc. (reviewed in reference 49). The inheritance of these diseases is characterized by the so-called anticipation, i.e., an increase in the probability, onset, and the severity of a disease as it passes through generations. The molecular basis for anticipation is that trinucleotide repeats are stably inherited and cause no harm unless the number of repeats exceeds a threshold of roughly 25, upon which an intergenerational transmission of expanded versions of these repeats becomes progressively more common (reviewed in reference 1).The mechanisms responsible for trinucleotide repeats expansion remain unclear. The largest volume of data supports an idea that abnormal replication of repeated stretches is responsible for their expansion. First, it is generally believed that the length dependence of expansion is linked to the ability of repeated DNAs to form unusual secondary structures, since the stability of such structures increases with repeats' lengths (reviewed in reference 27). Formation of these unusual DNA structures by trinucleotide repeats significantly compromises DNA polymerization in vitro (11,21,47). This polymerization blockage facilitates occasional misalignment between the newly synthesized and the template D...

Expanded CGG repeats cause chromosomal fragility and hereditary neurological disorders in humans. Replication forks stall at CGG repeats in a length-dependent manner in primate cells and in yeast. Yeast Tof1 and Mrc1 proteins facilitate replication fork progression through CGG repeats. Remarkably, the fork-stabilizing role of Mrc1 does not involve its checkpoint function. Thus, chromosomal fragility might occur when forks stalled at expanded CGG repeats escape the S-phase checkpoint.

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 MutSbeta and endonuclease activity of MutLalpha. We suggest that the mechanism of GAA/TTC-induced chromosomal aberrations defined in yeast can also operate in human carriers with expanded tracts.

A tandem repeat’s (TR) propensity to mutate increases with repeat number, and can become very pronounced beyond a critical boundary, transforming it into a microsatellite (MS). However, a clear understanding of the mutational behavior of different TR classes and motifs and related mechanisms is lacking, as is a consensus on the existence of a boundary separating short TRs (STRs) from MSs. This hinders our understanding of MSs’ mutational properties and their effective use as genetic markers. Using indel calls for 179 individuals from 1000 Genomes Pilot-1 Project, we determined polymorphism incidence for four major TR classes, and formalized its varying relationship with repeat number using segmented regression. We observed a biphasic regime with a transition from a faster to a slower exponential growth at 9, 5, 4, and 4 repeats for mono-, di-, tri-, and tetranucleotide TRs, respectively. We used an in vitro mutagenesis assay to evaluate the contribution of strand slippage errors to mutability. STRs and MSs differ in their absolute polymorphism levels, but more importantly in their rates of mutability growth. Although strand slippage is a major factor driving mononucleotide polymorphism incidence, dinucleotide polymorphism incidence is greater than that expected due to strand slippage alone, indicating that additional cellular factors might be driving dinucleotide mutability in the human genome. Leveraging on hundreds of human genomes, we present the first comprehensive, genome-wide analysis of TR mutational behavior, encompassing several motif sizes and compositions.

The influence of d(G) n ·d(C) n repeats on plasmid replication in Escherichia coli cells was analyzed using electrophoretic analysis of replication intermediates. These repeats impeded the replication fork in a lengthand orientation-dependent manner. Unexpectedly, the replication arrest relied primarily on the repeats' transcription. When the d(C) n sequence served as the transcriptional template, both transcription and replication were blocked. This was true for transcription driven by either bacterial or phage RNA polymerases. We hypothesize that the replication fork halts after it encounters a stalled ternary complex of the RNA polymerase, the DNA template and the r(G) n transcript. This constitutes a novel mechanism for the regulation of replication elongation. The effects of this mechanism on repeat length polymorphism and genome rearrangements are discussed.