Inverted repeat-stimulated sister-chromatid exchange events are RAD1-independent but reduced in a msh2 mutant

Inverted repeats (IRs) and trinucleotide repeats (TNRs) that have the potential to form secondary structures in vivo are known to cause genome rearrangements. Expansions of TNRs in humans are associated with several neurological disorders. Both IRs and TNRs stimulate spontaneous unequal sister-chromatid exchange (SCE) in yeast. Secondary structure-associated SCE events occur via double-strand break repair. Here we show that the rate of spontaneous IR-stimulated unequal SCE events in yeast is significantly reduced in strains with mutations in the mismatch repair genes MSH2 or MSH3, but unaffected by a mutation in the nucleotide excision-repair gene RAD1. Non-IR-associated unequal SCE events are increased in both MMR- and rad1-mutant cells; however, SCE events for both IR- and non-IR-containing substrates occur at a higher level in the exo1 background. Our results suggest that spontaneous SCE occurs by a template switching mechanism. Like IRs, TNRs have been shown to generate double-strand breaks (DSBs) in yeast. TNR expansions in mice are MSH2-dependent. Since IR-mediated SCE events are reduced in msh2 cells, we propose that TNR expansion mutations arise when DSBs are repaired using the sister or the homolog as a template.     

Expanded CAG repeats activate the DNA damage checkpoint pathway

Trinucleotide repeats (TNRs) are sequences whose expansion causes several genetic diseases and chromosome breakage. We report a novel finding that expanded CAG repeats activate the DNA damage response. Mutations in yeast MEC1, RAD9, or RAD53 genes result in increased rates of fragility of a CAG repeat tract while single or double deletions of RAD17 or RAD24 have only a modest effect on TNR fragility, indicating that signaling down the Rad9 pathway and not the Rad17-Rad24 pathway plays a major role in sensing and repairing CAG-tract breaks. Deletion of CHK1 had no effect on CAG fragility, suggesting that a Chk1-mediated G2 arrest is not required for TNR repair. Absence of Mec1, Ddc2, Rad17, Rad24, or Rad53 also gives rise to increased frequency of CAG repeat contractions, indicating that components of the checkpoint machinery play an active role in the maintenance of both chromosomal integrity and repeat stability at expanded CAG sequences.     

Multiple recombination pathways for sister chromatid exchange in Saccharomyces cerevisiae: role of RAD1 and the RAD52 epistasis group genes

Sister chromatid exchange (SCE) can occur by several recombination mechanisms, including those directly initiated by double-strand breaks (DSBs), such as gap repair and break-induced replication (BIR), and those initiated when DNA polymerases stall, such as template switching. To elucidate SCE recombination mechanisms, we determined whether spontaneous and DNA damage-associated SCE requires specific genes within the RAD52 and RAD3 epistasis groups in Saccharomyces cerevisiae strains containing two his3 fragments, his3-Δ5′ and his3-Δ3′::HOcs. SCE frequencies were measured after cells were exposed to UV, X-rays, 4-nitroquinoline 1-oxide (4-NQO) and methyl methanesulfonate (MMS), or when an HO endonuclease-induced DSB was introduced at his3-Δ3′::HOcs. Our data indicate that genes involved in gap repair, such as RAD55, RAD57 and RAD54, are required for DNA damage-associated SCE but not for spontaneous SCE. RAD50 and RAD59, genes required for BIR, are required for X-ray-associated SCE but not for SCE stimulated by HO-induced DSBs. In comparison with wild type, rates of spontaneous SCE are 10-fold lower in rad51 rad1 but not in either rad51 rad50 or rad51 rad59 double mutants. We propose that gap repair mechanisms are important in DNA damage-associated recombination, whereas alternative pathways, including a template switch pathway, play a role in spontaneous SCE.     

Saccharomyces cerevisiae Srs2 DNA helicase selectively blocks expansions of trinucleotide repeats

Trinucleotide repeats (TNRs) undergo frequent mutations in families afflicted with certain neurodegenerative disorders and in model organisms. TNR instability is modulated both by the repeat tract itself and by cellular proteins. Here we identified the Saccharomyces cerevisiae DNA helicase Srs2 as a potent and selective inhibitor of expansions. srs2 mutants had up to 40-fold increased expansion rates of CTG, CAG, and CGG repeats. The expansion phenotype was specific, as mutation rates at dinucleotide repeats, at unique sequences, or for TNR contractions in srs2 mutants were not altered. Srs2 is known to suppress inappropriate genetic recombination; however, the TNR expansion phenotype of srs2 mutants was largely independent of RAD51 and RAD52. Instead, Srs2 mainly functioned with DNA polymerase delta to block expansions. The helicase activity of Srs2 was important, because a point mutant lacking ATPase function was defective in blocking expansions. Purified Srs2 was substantially better than bacterial UvrD helicase at in vitro unwinding of a DNA substrate that mimicked a TNR hairpin. Disruption of the related helicase gene SGS1 did not lead to excess expansions, nor did wild-type SGS1 suppress the expansion phenotype of an srs2 strain. We conclude that Srs2 selectively blocks triplet repeat expansions through its helicase activity and primarily in conjunction with polymerase delta.     

Tri-nucleotide repeats and their association with genes in rice genome

Tri-nucleotide repeats (TNRs) are extremely abundant in rice genome, of which CCG/CGG repeats have an advantage over other repeats, with approximate half of all the TNRs in the genome. Our results show that rice genome has relatively abundant TNRs with high GC content, and containing only purines or pyrimidines under the same GC content. The AAT/ATT repeats that occur predominantly in intergenic and intronic regions have a considerably higher average length than that of other repeats. The highest frequency of TNRs occurs in 5′-UTR regions, followed by in coding and 5′-flanking regions. Purines-rich TNRs prefer to the coding regions, but pyrimidines-rich TNRs exhibit a stronger bias to upstream regions, suggesting that they might be considered as the regulatory elements in gene expression. As if TNRs located predominantly near the start of coding regions do not significantly influence on the protein function.     

Tri-nucleotide repeats and their association with genes in rice genome

Tri-nucleotide repeats (TNRs) are extremely abundant in rice genome, of which CCG/CGG repeats have an advantage over other repeats, with approximate half of all the TNRs in the genome. Our results show that rice genome has relatively abundant TNRs with high GC content, and containing only purines or pyrimidines under the same GC content. The AAT/ATT repeats that occur predominantly in intergenic and intronic regions have a considerably higher average length than that of other repeats. The highest frequency of TNRs occurs in 5'-UTR regions, followed by in coding and 5'-flanking regions. Purines-rich TNRs prefer to the coding regions, but pyrimidines-rich TNRs exhibit a stronger bias to upstream regions, suggesting that they might be considered as the regulatory elements in gene expression. As if TNRs located predominantly near the start of coding regions do not significantly influence on the protein function.     

Mismatch repair blocks expansions of interrupted trinucleotide repeats in yeast

Disease-causing expansions of trinucleotide repeats (TNRs) can occur very frequently. In contrast, expansions are rare if the TNR is interrupted (imperfect). The molecular mechanism stabilizing interrupted alleles and thereby preventing disease has been elusive. We show that mismatch repair is the major stabilizing force for interrupted TNRs in Saccharomyces cerevisiae. Interrupted alleles expand much more often when mismatch repair is blocked by mutation or by poorly corrected mispairs. These results suggest that interruptions lead to mismatched expansion precursors. In normal cells, expansions are prevented in trans by mismatch repair, which coexcises the mismatches plus the aberrant, TNR-mediated secondary structure that otherwise resists removal. This study indicates a novel role for mismatch repair in mutation avoidance and, potentially, in disease prevention.     

Rad9-mediated checkpoint activation is responsible for elevated expansions of GAA repeats in CST-deficient yeast

Large-scale expansion of (GAA)_n repeats in the first intron of the FXN gene is responsible for the severe neurodegenerative disease, Friedreich’s ataxia in humans. We have previously conducted an unbiased genetic screen for GAA repeat instability in a yeast experimental system. The majority of genes that came from this screen encoded the components of DNA replication machinery, strongly implying that replication irregularities are at the heart of GAA repeat expansions. This screen, however, also produced two unexpected hits: members of the CST complex, CDC13 and TEN1 genes, which are required for telomere maintenance. To understand how the CST complex could affect intra-chromosomal GAA repeats, we studied the well-characterized temperature-sensitive cdc13-1 mutation and its effects on GAA repeat instability in yeast. We found that in-line with the screen results, this mutation leads to ∼10-fold increase in the rate of large-scale expansions of the (GAA)₁₀₀ repeat at semi-permissive temperature. Unexpectedly, the hyper-expansion phenotype of the cdc13-1 mutant largely depends on activation of the G2/M checkpoint, as deletions of individual genes RAD9, MEC1, RAD53, and EXO1 belonging to this pathway rescued the increased GAA expansions. Furthermore, the hyper-expansion phenotype of the cdc13-1 mutant depended on the subunit of DNA polymerase δ, Pol32. We hypothesize, therefore, that increased repeat expansions in the cdc13-1 mutant happen during post-replicative repair of nicks or small gaps within repetitive tracts during the G2 phase of the cell cycle upon activation of the G2/M checkpoint.     

Small interfering RNAs based on huntingtin trinucleotide repeats are highly toxic to cancer cells

Trinucleotide repeat (TNR) expansions in the genome cause a number of degenerative diseases. A prominent TNR expansion involves the triplet CAG in the huntingtin (HTT) gene responsible for Huntington's disease (HD). Pathology is caused by protein and RNA generated from the TNR regions including small siRNA‐sized repeat fragments. An inverse correlation between the length of the repeats in HTT and cancer incidence has been reported for HD patients. We now show that siRNAs based on the CAG TNR are toxic to cancer cells by targeting genes that contain long reverse complementary TNRs in their open reading frames. Of the 60 siRNAs based on the different TNRs, the six members in the CAG/CUG family of related TNRs are the most toxic to both human and mouse cancer cells. siCAG/CUG TNR‐based siRNAs induce cell death in vitro in all tested cancer cell lines and slow down tumor growth in a preclinical mouse model of ovarian cancer with no signs of toxicity to the mice. We propose to explore TNR‐based siRNAs as a novel form of anticancer reagents.     

58 Packaging trinucleotide repeats: the effect of CAG/CTG repeats on nucleosome formation

In neurological diseases such as fragile X syndrome, spinal and bulbar muscular atrophy, myotonic dystrophy, and Huntington’s disease, the molecular basis of pathogenicity is the presence of an expanded trinucleotide repeat (TNR) tract (Ashley & Warren, 1995). TNRs implicated in many of these diseases are composed of CAG/CTG repeats. For example, in healthy individuals 5–35, CAG/CTG TNR repeats are present in the huntingtin gene. However, individuals with 40 or greater repeats will develop Huntington’s disease (Andrew et al., 1993). We are particularly interested in how these TNR sequences are packaged in chromatin. Recent evaluations of CAG/CTG TNR sequences in our laboratory have demonstrated that the repeats increase the propensity for the DNA sequences to incorporate into nucleosomes, where nucleosomes represent the minimal unit of packaging in chromatin (Volle & Delaney, 2012). In this work, we are interested in determining the minimum number of CAG/CTG repeats required to confer a significant increase in nucleosome incorporation relative to sequences that lack the TNR sequence. By defining the changes imposed on these fundamental interactions by the presence of a CAG/CTG repeat tract, we will gain insight into the possible interactions that allow for the expansion of these TNR tracts.     

DNA Instability Maintains the Repeat Length of the Yeast RNA Polymerase II C-terminal Domain

The C-terminal domain (CTD) of RNA polymerase II in eukaryotes is comprised of tandemly repeating units of a conserved seven-amino acid sequence. The number of repeats is, however, quite variable across different organisms. Furthermore, previous studies have identified evidence of rearrangements within the CTD coding region, suggesting that DNA instability may play a role in regulating or maintaining CTD repeat number. The work described here establishes a clear connection between DNA instability and CTD repeat number in Saccharomyces cerevisiae. First, analysis of 36 diverse S. cerevisiae isolates revealed evidence of numerous past rearrangements within the DNA sequence that encodes the CTD. Interestingly, the total number of CTD repeats was relatively static (24–26 repeats in all strains), suggesting a balancing act between repeat expansion and contraction. In an effort to explore the genetic plasticity within this region, we measured the rates of repeat expansion and contraction using novel reporters and a doxycycline-regulated expression system for RPB1. In efforts to determine the mechanisms leading to CTD repeat variability, we identified the presence of DNA secondary structures, specifically G-quadruplex-like DNA, within the CTD coding region. Furthermore, we demonstrated that mutating PIF1, a G-quadruplex-specific helicase, results in increased CTD repeat length polymorphisms. We also determined that RAD52 is necessary for CTD repeat expansion but not contraction, identifying a role for recombination in repeat expansion. Results from these DNA rearrangements may help explain the CTD copy number variation seen across eukaryotes, as well as support a model of CTD expansion and contraction to maintain CTD integrity and overall length.     

Trinucleotide repeats are prevalent among cancer-related genes

Trinucleotide repeats (TNRs) have been primarily connected to neurologic and neuromuscular diseases, with few specific TNRs linked with various tumors. Here we conduct a genome-wide analysis and show that TNRs are five times more prevalent in cancer-related human genes. Interestingly, we also find that cancer-related genes are significantly longer than other genes. Our results suggest that genes containing TNRs are more prone to mutagenesis. The database of TNR genes can be used as a list of candidate cancer-related genes.     

Trinucleotide repeats are clustered in regulatory genes in Saccharomyces cerevisiae

The genome of Saccharomyces cerevisiae contains numerous unstable microsatellite sequences. Mononucleotide and dinucleotide repeats are rarely found in ORFs, and when present in an ORF are frequently located in an intron or at the C terminus of the protein, suggesting that their instability is deleterious to gene function. DNA trinucleotide repeats (TNRs) are found at a higher-than-expected frequency within ORFs, and the amino acids encoded by the TNRs represent a biased set. TNRs are rarely conserved between genes with related sequences, suggesting high instability or a recent origin. The genes in which TNRs are most frequently found are related to cellular regulation. The protein structural database is notably lacking in proteins containing amino acid tracts, suggesting that they are not located in structured regions of a protein but are rather located between domains. This conclusion is consistent with the location of amino acid tracts in two protein families. The preferred location of TNRs within the ORFs of genes related to cellular regulation together with their instability suggest that TNRs could have an important role in speciation. Specifically, TNRs could serve as hot spots for recombination leading to domain swapping, or mutation of TNRs could allow rapid evolution of new domains of protein structure.     

The guanine-rich fragile X chromosome repeats are reluctant to form tetraplexes

Using circular dichroism spectroscopy, UV absorption spectroscopy and polyacrylamide gel electrophoresis, we studied conformational properties of guanine-rich DNA strands of the fragile X chromosome repeats d(GGC)_n, d(GCG)_n and d(CGG)_n, with n = 2, 4, 8 and 16. These strands are generally considered in the literature to form guanine tetraplexes responsible for the repeat expansion. However, we show in this paper that the repeats are reluctant to form tetraplexes. At physiological concentrations of either Na⁺ or K⁺ ions, the hexamers and dodecamers associate to form homoduplexes and the longer repeats generate homoduplexes and hairpins. The tetraplexes are rarely observed being relatively most stable with d(GGC)_n and least stable with d(GCG)_n. The tetraplexes are exclusively formed in the presence of K⁺ ions, at salt concentrations higher than physiological, more easily at higher than physiological temperatures, and they arise with extremely long kinetics (even days). Moreover, the capability to form tetraplexes sharply diminishes with the oligonucleotide length. These facts make the concept of the tetraplex appearance in this motif in vivo very improbable. Rather, a hairpin of the fragile X repeats, whose stability increases with the repeat length, is the probable structure responsible for the repeat expansion in genomes.     

Mutations in yeast replication proteins that increase CAG/CTG expansions also increase repeat fragility

Expansion of trinucleotide repeats (TNRs) is the causative mutation in several human genetic diseases. Expanded TNR tracts are both unstable (changing in length) and fragile (displaying an increased propensity to break). We have investigated the relationship between fidelity of lagging-strand replication and both stability and fragility of TNRs. We devised a new yeast artificial chromomosme (YAC)-based assay for chromosome breakage to analyze fragility of CAG/CTG tracts in mutants deficient for proteins involved in lagging-strand replication: Fen1/Rad27, an endo/exonuclease involved in Okazaki fragment maturation, the nuclease/helicase Dna2, RNase HI, DNA ligase, polymerase delta, and primase. We found that deletion of RAD27 caused a large increase in breakage of short and long CAG/CTG tracts, and defects in DNA ligase and primase increased breakage of long tracts. We also found a correlation between mutations that increase CAG/CTG tract breakage and those that increase repeat expansion. These results suggest that processes that generate strand breaks, such as faulty Okazaki fragment processing or DNA repair, are an important source of TNR expansions.     

Accumulation of interspersed and sex-specific repeats in the non-recombining region of papaya sex chromosomes

Background

The papaya Y chromosome has undergone a degenerative expansion from its ancestral autosome, as a consequence of recombination suppression in the sex determining region of the sex chromosomes. The non-recombining feature led to the accumulation of repetitive sequences in the male- or hermaphrodite-specific regions of the Y or the Y^h chromosome (MSY or HSY). Therefore, repeat composition and distribution in the sex determining region of papaya sex chromosomes would be informative to understand how these repetitive sequences might be involved in the early stages of sex chromosome evolution.

Results

Detailed composition of interspersed, sex-specific, and tandem repeats was analyzed from 8.1 megabases (Mb) HSY and 5.3 Mb corresponding X chromosomal regions. Approximately 77% of the HSY and 64% of the corresponding X region were occupied by repetitive sequences. Ty3-gypsy retrotransposons were the most abundant interspersed repeats in both regions. Comparative analysis of repetitive sequences between the sex determining region of papaya X chromosome and orthologous autosomal sequences of Vasconcellea monoica, a close relative of papaya lacking sex chromosomes, revealed distinctive differences in the accumulation of Ty3-Gypsy, suggesting that the evolution of the papaya sex determining region may accompany Ty3-Gypsy element accumulation. In total, 21 sex-specific repeats were identified from the sex determining region; 20 from the HSY and one from the X. Interestingly, most HSY-specific repeats were detected in two regions where the HSY expansion occurred, suggesting that the HSY expansion may result in the accumulation of sex-specific repeats or that HSY-specific repeats might play an important role in the HSY expansion. The analysis of simple sequence repeats (SSRs) revealed that longer SSRs were less abundant in the papaya sex determining region than the other chromosomal regions.

Conclusion

Major repetitive elements were Ty3-gypsy retrotransposons in both the HSY and the corresponding X. Accumulation of Ty3-Gypsy retrotransposons in the sex determining region of papaya X chromosome was significantly higher than that in the corresponding region of V. monoica, suggesting that Ty3-Gypsy could be crucial for the expansion and evolution of the sex determining region in papaya. Most sex-specific repeats were located in the two HSY expansion regions.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-335) contains supplementary material, which is available to authorized users.     

Telomeric repeats act as nucleosome-disfavouring sequences in vivo

Telomeric DNAs consist of tandem repeats of G-clusters such as TTAGGG and TG_1-3, which are the human and yeast repeat sequences, respectively. In the yeast Saccharomyces cerevisiae, the telomeric repeats are non-nucleosomal, whereas in humans, they are organized in tightly packaged nucleosomes. However, previous in vitro studies revealed that the binding affinities of human and yeast telomeric repeat sequences to histone octamers in vitro were similar, which is apparently inconsistent with the differences in the human and yeast telomeric chromatin structures. To further investigate the relationship between telomeric sequences and chromatin structure, we examined the effect of telomeric repeats on the formation of positioned nucleosomes in vivo by indirect end-label mapping, primer extension mapping and nucleosome repeat analyses, using a defined minichromosome in yeast cells. We found that the human and yeast telomeric repeat sequences both disfavour nucleosome assembly and alter nucleosome positioning in the yeast minichromosome. We further demonstrated that the G-clusters in the telomeric repeats are required for the nucleosome-disfavouring properties. Thus, our results suggest that this inherent structural feature of the telomeric repeat sequences is involved in the functional dynamics of the telomeric chromatin structure.