Maria M. Krasilnikova

Replication Stalling at Friedreich's Ataxia (GAA) n Repeats In Vivo

ABSTRACT Friedreichs 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 (∼40 repeats) closely matched the threshold length for repeat expansion in humans. Further, replication stalling was strikingly orientation dependent, being pronounced only when the repeats homopurine strand served as the lagging strand template. Finally, it appeared that length polymorphism of the (GAA) n  · (TTC) n repeat in both expansions and contractions drastically increases in the repeats 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.

Replication and Expansion of Trinucleotide Repeats in Yeast

ABSTRACT 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  · (CCG) n and (CAG) n  · (CTG) n repeats and their propensity to expand in Saccharomyces cerevisiae. Using electrophoretic analysis of replication intermediates, we found that (CGG) n  · (CCG) n repeats significantly attenuate replication fork progression. Replication inhibition for this sequence becomes evident at as few as ∼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  · (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 · (CCG)25 and (CAG)25 · (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 ∼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.

Large-Scale Expansions of Friedreich's Ataxia GAA Repeats in Yeast

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 Friedreichs 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 repeats 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 knockouts of postreplication DNA repair proteins, Rad6 and Rad5, and the DNA helicase Sgs1. We propose a model for large-scale repeat expansions based on template switching during replication fork progression through repetitive DNA.

Replisome stalling and stabilization at CGG repeats, which are responsible for chromosomal fragility

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. Saccharomyces cerevisiae proteins Tof1 and Mrc1 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.

Mechanisms and implications of transcription blockage by guanine-rich DNA sequences

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 homopurine-homopyrimidine 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-, length- and 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.

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 Friedreichs 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 MutSβ and endonuclease activity of MutLα. We suggest that the mechanism of GAA/TTC‐induced chromosomal aberrations defined in yeast can also operate in human carriers with expanded tracts.

Distinct Mutational Behaviors Differentiate Short Tandem Repeats from Microsatellites in the Human Genome

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.

Effects of Friedreich's ataxia GAA repeats on DNA replication in mammalian cells

Friedreichs ataxia (FRDA) is a common hereditary degenerative neuro-muscular disorder caused by expansions of the (GAA)n repeat in the first intron of the frataxin gene. The expanded repeats from parents frequently undergo further significant length changes as they are passed on to progeny. Expanded repeats also show an age-dependent instability in somatic cells, albeit on a smaller scale than during intergenerational transmissions. Here we studied the effects of (GAA)n repeats of varying lengths and orientations on the episomal DNA replication in mammalian cells. We have recently shown that the very first round of the transfected DNA replication occurs in the lack of the mature chromatin, does not depend on the episomal replication origin and initiates at multiple single-stranded regions of plasmid DNA. We now found that expanded GAA repeats severely block this first replication round post plasmid transfection, while the subsequent replication cycles are only mildly affected. The fact that GAA repeats affect various replication modes in a different way might shed light on their differential expansions characteristic for FRDA.

Mature Microsatellites: Mechanisms Underlying Dinucleotide Microsatellite Mutational Biases in Human Cells

Dinucleotide microsatellites are dynamic DNA sequences that affect genome stability. Here, we focused on mature microsatellites, defined as pure repeats of lengths above the threshold and unlikely to mutate below it in a single mutational event. We investigated the prevalence and mutational behavior of these sequences by using human genome sequence data, human cells in culture, and purified DNA polymerases. Mature dinucleotides (≥10 units) are present within exonic sequences of >350 genes, resulting in vulnerability to cellular genetic integrity. Mature dinucleotide mutagenesis was examined experimentally using ex vivo and in vitro approaches. We observe an expansion bias for dinucleotide microsatellites up to 20 units in length in somatic human cells, in agreement with previous computational analyses of germ-line biases. Using purified DNA polymerases and human cell lines deficient for mismatch repair (MMR), we show that the expansion bias is caused by functional MMR and is not due to DNA polymerase error biases. Specifically, we observe that the MutSα and MutLα complexes protect against expansion mutations. Our data support a model wherein different MMR complexes shift the balance of mutations toward deletion or expansion. Finally, we show that replication fork progression is stalled within long dinucleotides, suggesting that mutational mechanisms within long repeats may be distinct from shorter lengths, depending on the biochemistry of fork resolution. Our work combines computational and experimental approaches to explain the complex mutational behavior of dinucleotide microsatellites in humans.

A distinct first replication cycle of DNA introduced in mammalian cells

Many mutation events in microsatellite DNA sequences were traced to the first embryonic divisions. It was not known what makes the first replication cycles of embryonic DNA different from subsequent replication cycles. Here we demonstrate that an unusual replication mode is involved in the first cycle of replication of DNA introduced in mammalian cells. This alternative replication starts at random positions, and occurs before the chromatin is fully assembled. It is detected in various cell lines and primary cells. The presence of single-stranded regions increases the efficiency of this alternative replication mode. The alternative replication cannot progress through the A/T-rich FRA16B fragile site, while the regular replication mode is not affected by it. A/T-rich microsatellites are associated with the majority of chromosomal breakpoints in cancer. We suggest that the alternative replication mode may be initiated at the regions with immature chromatin structure in embryonic and cancer cells resulting in increased genomic instability. This work demonstrates, for the first time, differences in the replication progression during the first and subsequent replication cycles in mammalian cells.