Restarted replication forks are error-prone and cause CAG repeat expansions and contractions.

Disease-associated trinucleotide repeats form secondary DNA structures that interfere with replication and repair. Replication has been implicated as a mechanism that can cause repeat expansions and contractions. However, because structure-forming repeats are also replication barriers, it has been unclear whether the instability occurs due to slippage during normal replication progression through the repeat, slippage or misalignment at a replication stall caused by the repeat, or during subsequent replication of the repeat by a restarted fork that has altered properties. In this study, we have specifically addressed the fidelity of a restarted fork as it replicates through a CAG/CTG repeat tract and its effect on repeat instability. To do this, we used a well-characterized site-specific replication fork barrier (RFB) system in fission yeast that creates an inducible and highly efficient stall that is known to restart by recombination-dependent replication (RDR), in combination with long CAG repeat tracts inserted at various distances and orientations with respect to the RFB. We find that replication by the restarted fork exhibits low fidelity through repeat sequences placed 2-7 kb from the RFB, exhibiting elevated levels of Rad52- and Rad8ScRad5/HsHLTF-dependent instability. CAG expansions and contractions are not elevated to the same degree when the tract is just in front or behind the barrier, suggesting that the long-traveling Polδ-Polδ restarted fork, rather than fork reversal or initial D-loop synthesis through the repeat during stalling and restart, is the greatest source of repeat instability. The switch in replication direction that occurs due to replication from a converging fork while the stalled fork is held at the barrier is also a significant contributor to the repeat instability profile. Our results shed light on a long-standing question of how fork stalling and RDR contribute to expansions and contractions of structure-forming trinucleotide repeats, and reveal that tolerance to replication stress by fork restart comes at the cost of increased instability of repetitive sequences.

The end-joining factor Ku acts in the end-resection of double strand break-free arrested replication forks.

Replication requires homologous recombination (HR) to stabilize and restart terminally arrested forks. HR-mediated fork processing requires single stranded DNA (ssDNA) gaps and not necessarily double strand breaks. We used genetic and molecular assays to investigate fork-resection and restart at dysfunctional, unbroken forks in Schizosaccharomyces pombe. Here, we report that fork-resection is a two-step process regulated by the non-homologous end joining factor Ku. An initial resection mediated by MRN-Ctp1 removes Ku from terminally arrested forks, generating ~110 bp sized gaps obligatory for subsequent Exo1-mediated long-range resection and replication restart. The mere lack of Ku impacts the processing of arrested forks, leading to an extensive resection, a reduced recruitment of RPA and Rad51 and a slower fork-restart process. We propose that terminally arrested forks undergo fork reversal, providing a single DNA end for Ku binding. We uncover a role for Ku in regulating end-resection of unbroken forks and in fine-tuning HR-mediated replication restart.

Unprotected Replication Forks Are Converted into Mitotic Sister Chromatid Bridges.

Replication stress and mitotic abnormalities are key features of cancer cells. Temporarily paused forks are stabilized by the intra-S phase checkpoint and protected by the association of Rad51, which prevents Mre11-dependent resection. However, if a fork becomes dysfunctional and cannot resume, this terminally arrested fork is rescued by a converging fork to avoid unreplicated parental DNA during mitosis. Alternatively, dysfunctional forks are restarted by homologous recombination. Using fission yeast, we report that Rad52 and the DNA binding activity of Rad51, but not its strand-exchange activity, act to protect terminally arrested forks from unrestrained Exo1-nucleolytic activity. In the absence of recombination proteins, large ssDNA gaps, up to 3 kb long, occur behind terminally arrested forks, preventing efficient fork merging and leading to mitotic sister chromatid bridging. Thus, Rad52 and Rad51 prevent temporarily and terminally arrested forks from degrading and, despite the availability of converging forks, converting to anaphase bridges causing aneuploidy and cell death.

The chromatin assembly factor 1 promotes Rad51-dependent template switches at replication forks by counteracting D-loop disassembly by the RecQ-type helicase Rqh1.

At blocked replication forks, homologous recombination mediates the nascent strands to switch template in order to ensure replication restart, but faulty template switches underlie genome rearrangements in cancer cells and genomic disorders. Recombination occurs within DNA packaged into chromatin that must first be relaxed and then restored when recombination is completed. The chromatin assembly factor 1, CAF-1, is a histone H3-H4 chaperone involved in DNA synthesis-coupled chromatin assembly during DNA replication and DNA repair. We reveal a novel chromatin factor-dependent step during replication-coupled DNA repair: Fission yeast CAF-1 promotes Rad51-dependent template switches at replication forks, independently of the postreplication repair pathway. We used a physical assay that allows the analysis of the individual steps of template switch, from the recruitment of recombination factors to the formation of joint molecules, combined with a quantitative measure of the resulting rearrangements. We reveal functional and physical interplays between CAF-1 and the RecQ-helicase Rqh1, the BLM homologue, mutations in which cause Bloom's syndrome, a human disease associating genome instability with cancer predisposition. We establish that CAF-1 promotes template switch by counteracting D-loop disassembly by Rqh1. Consequently, the likelihood of faulty template switches is controlled by antagonistic activities of CAF-1 and Rqh1 in the stability of the D-loop. D-loop stabilization requires the ability of CAF-1 to interact with PCNA and is thus linked to the DNA synthesis step. We propose that CAF-1 plays a regulatory role during template switch by assembling chromatin on the D-loop and thereby impacting the resolution of the D-loop.

Loss of the thioredoxin reductase Trr1 suppresses the genomic instability of peroxiredoxin tsa1 mutants.

The absence of Tsa1, a key peroxiredoxin that scavenges H2O2 in Saccharomyces cerevisiae, causes the accumulation of a broad spectrum of mutations. Deletion of TSA1 also causes synthetic lethality in combination with mutations in RAD51 or several key genes involved in DNA double-strand break repair. In the present study, we propose that the accumulation of reactive oxygen species (ROS) is the primary cause of genome instability of tsa1Δ cells. In searching for spontaneous suppressors of synthetic lethality of tsa1Δ rad51Δ double mutants, we identified that the loss of thioredoxin reductase Trr1 rescues their viability. The trr1Δ mutant displayed a Can(R) mutation rate 5-fold lower than wild-type cells. Additional deletion of TRR1 in tsa1Δ mutant reduced substantially the Can(R) mutation rate of tsa1Δ strain (33-fold), and to a lesser extent, of rad51Δ strain (4-fold). Loss of Trr1 induced Yap1 nuclear accumulation and over-expression of a set of Yap1-regulated oxido-reductases with antioxidant properties that ultimately re-equilibrate intracellular redox environment, reducing substantially ROS-associated DNA damages. This trr1Δ -induced effect was largely thioredoxin-dependent, probably mediated by oxidized forms of thioredoxins, the primary substrates of Trr1. Thioredoxin Trx1 and Trx2 were constitutively and strongly oxidized in the absence of Trr1. In trx1Δ trx2Δ cells, Yap1 was only moderately activated; consistently, the trx1Δ trx2Δ double deletion failed to efficiently rescue the viability of tsa1Δ rad51Δ. Finally, we showed that modulation of the dNTP pool size also influences the formation of spontaneous mutation in trr1Δ and trx1Δ trx2Δ strains. We present a tentative model that helps to estimate the respective impact of ROS level and dNTP concentration in the generation of spontaneous mutations.

The extent of error-prone replication restart by homologous recombination is controlled by Exo1 and checkpoint proteins.

Genetic instability, a hallmark of cancer, can occur when the replication machinery encounters a barrier. The intra-S-phase checkpoint maintains stalled replication forks in a replication-competent configuration by phosphorylating replisome components and DNA repair proteins to prevent forks from catastrophically collapsing. Here, we report a novel function of the core Schizosaccharomyces pombe checkpoint sensor kinase, Rad3 (an ATR orthologue), that is independent of Chk1 and Cds1 (a CHK2 orthologue); Rad3(ATR) regulates the association of recombination factors with collapsed forks, thus limiting their genetic instability. We further reveal antagonistic roles for Rad3(ATR) and the 9-1-1 clamp - Rad3(ATR) restrains MRN- and Exo1-dependent resection, whereas the 9-1-1 complex promotes Exo1 activity. Interestingly, the MRN complex, but not its nuclease activity, promotes resection and the subsequent association of recombination factors at collapsed forks. The biological significance of this regulation is revealed by the observation that Rad3(ATR) prevents Exo1-dependent genome instability upstream of a collapsed fork without affecting the efficiency of recombination-mediated replication restart. We propose that the interplay between Rad3(ATR) and the 9-1-1 clamp functions to fine-tune the balance between the need for the recovery of replication through recombination and the risk of increased genome instability.

Recovery of arrested replication forks by homologous recombination is error-prone.

Homologous recombination is a universal mechanism that allows repair of DNA and provides support for DNA replication. Homologous recombination is therefore a major pathway that suppresses non-homology-mediated genome instability. Here, we report that recovery of impeded replication forks by homologous recombination is error-prone. Using a fork-arrest-based assay in fission yeast, we demonstrate that a single collapsed fork can cause mutations and large-scale genomic changes, including deletions and translocations. Fork-arrest-induced gross chromosomal rearrangements are mediated by inappropriate ectopic recombination events at the site of collapsed forks. Inverted repeats near the site of fork collapse stimulate large-scale genomic changes up to 1,500 times over spontaneous events. We also show that the high accuracy of DNA replication during S-phase is impaired by impediments to fork progression, since fork-arrest-induced mutation is due to erroneous DNA synthesis during recovery of replication forks. The mutations caused are small insertions/duplications between short tandem repeats (micro-homology) indicative of replication slippage. Our data establish that collapsed forks, but not stalled forks, recovered by homologous recombination are prone to replication slippage. The inaccuracy of DNA synthesis does not rely on PCNA ubiquitination or trans-lesion-synthesis DNA polymerases, and it is not counteracted by mismatch repair. We propose that deletions/insertions, mediated by micro-homology, leading to copy number variations during replication stress may arise by progression of error-prone replication forks restarted by homologous recombination.

Redox-sensitive YFP sensors monitor dynamic nuclear and cytosolic glutathione redox changes.

Intracellular redox homeostasis is crucial for many cellular functions but accurate measurements of cellular compartment-specific redox states remain technically challenging. To better characterize redox control in the nucleus, we targeted a yellow fluorescent protein-based redox sensor (rxYFP) to the nucleus of the yeast Saccharomyces cerevisiae. Parallel analyses of the redox state of nucleus-rxYFP and cytosol-rxYFP allowed us to monitor distinctively dynamic glutathione (GSH) redox changes within these two compartments under a given condition. We observed that the nuclear GSH redox environment is highly reducing and similar to the cytosol under steady-state conditions. Furthermore, these sensors are able to detect redox variations specific for their respective compartments in glutathione reductase (Glr1) and thioredoxin pathway (Trr1, Trx1, Trx2) mutants that have altered subcellular redox environments. Our mutant redox data provide in vivo evidence that glutathione and the thioredoxin redox systems have distinct but overlapping functions in controlling subcellular redox environments. We also monitored the dynamic response of nucleus-rxYFP and cytosol-rxYFP to GSH depletion and to exogenous low and high doses of H2O2 bursts. These observations indicate a rapid and almost simultaneous oxidation of both nucleus-rxYFP and cytosol-rxYFP, highlighting the robustness of the rxYFP sensors in measuring real-time compartmental redox changes. Taken together, our data suggest that the highly reduced yeast nuclear and cytosolic redox states are maintained independently to some extent and under distinct but subtle redox regulation. Nucleus- and cytosol-rxYFP register compartment-specific localized redox fluctuations that may involve exchange of reduced and/or oxidized glutathione between these two compartments. Finally, we confirmed that GSH depletion has profound effects on mitochondrial genome stability but little effect on nuclear genome stability, thereby emphasizing that the critical requirement for GSH during growth is linked to a mitochondria-dependent process.

Selective cytotoxicity of Aniba rosaeodora essential oil towards epidermoid cancer cells through induction of apoptosis.

Essential oils are complex mixtures of odorous and volatile compounds derived from secondary plant metabolism. They can be isolated from many plants by mechanical pressing or hydro- and steam-distillation and are known to induce a wide range of biological effects through their antibacterial, antifungal, cytotoxic, antioxidant and antimutagenic activities. In order to explore their beneficial properties on human skin cells, we investigated the effects of an essential oil from rosewood Aniba rosaeodora (REO) on the human epidermoid carcinoma cell line A431, on immortal HaCaT cells thought to represent an early stage of skin carcinogenesis, on transformed normal HEK001 keratinocytes and on primary normal NHEK keratinocytes. In a defined range of concentrations, REO selectively killed A431 and HaCaT cells. The same treatments had only a minor cytotoxic effect on HEK001 and NHEK cells. Preferentially in A431 and HaCaT cells, REO triggered the production of reactive oxygen species, induced depolarization of the mitochondrial membrane and caused caspase-dependent cell death characterized by phosphatidylserine externalization, an early marker of apoptosis. Both intrinsic and extrinsic apoptotic pathways were implicated in REO-induced cell death. The identification of selective induction of apoptosis in precancerous and cancerous skin cells by REO highlights the potential anticancer activity of this essential oil.

Peroxiredoxin Tsa1 is the key peroxidase suppressing genome instability and protecting against cell death in Saccharomyces cerevisiae.

Peroxiredoxins (Prxs) constitute a family of thiol-specific peroxidases that utilize cysteine (Cys) as the primary site of oxidation during the reduction of peroxides. To gain more insight into the physiological role of the five Prxs in budding yeast Saccharomyces cerevisiae, we performed a comparative study and found that Tsa1 was distinguished from the other Prxs in that by itself it played a key role in maintaining genome stability and in sustaining aerobic viability of rad51 mutants that are deficient in recombinational repair. Tsa2 and Dot5 played minor but distinct roles in suppressing the accumulation of mutations in cooperation with Tsa1. Tsa2 was capable of largely complementing the absence of Tsa1 when expressed under the control of the Tsa1 promoter. The presence of peroxidatic cysteine (Cys(47)) was essential for Tsa1 activity, while Tsa1(C170S) lacking the resolving Cys was partially functional. In the absence of Tsa1 activity (tsa1 or tsa1(CCS) lacking the peroxidatic and resolving Cys) and recombinational repair (rad51), dying cells displayed irregular cell size/shape, abnormal cell cycle progression, and significant increase of phosphatidylserine externalization, an early marker of apoptosis-like cell death. The tsa1(CCS) rad51- or tsa1 rad51-induced cell death did not depend on the caspase Yca1 and Ste20 kinase, while the absence of the checkpoint protein Rad9 accelerated the cell death processes. These results indicate that the peroxiredoxin Tsa1, in cooperation with appropriate DNA repair and checkpoint mechanisms, acts to protect S. cerevisiae cells against toxic levels of DNA damage that occur during aerobic growth.

Human peroxiredoxin PrxI is an orthologue of yeast Tsa1, capable of suppressing genome instability in Saccharomyces cerevisiae.

The peroxiredoxins (Prx) are conserved antioxidant proteins that use cysteine as the primary site of oxidation during the reduction of peroxides. Many organisms have more than one isoform of Prx. Deletion of TSA1, one of five Prxs in yeast Saccharomyces cerevisiae, results in accumulation of a broad spectrum of mutations including gross chromosomal rearrangements. Deletion of TSA1 is synthetically lethal with mutations in RAD6 and several key genes involved in DNA double-strand break repair. Here, we have examined the function of human PrxI and PrxII, which share a high degree of sequence identity with Tsa1, by expressing them in S. cerevisiae cells under the control of the native TSA1 promoter. We found that expression of PrxI, but not PrxII, was capable of complementing a tsa1Delta mutant for a variety of defects including genome instability, the synthetic lethality observed in rad6 Delta tsa1Delta and rad51 Delta tsa1Delta double mutants, and mutagen sensitivity. Moreover, expression of either Tsa1 or PrxI prevented Bax-induced cell death. These data indicate that PrxI is an orthologue of Tsa1. PrxI and Tsa1 seem to act on the same substrates in vivo and share similar mechanisms of function. The observation that PrxI is involved in suppressing genome instability and protecting against cell death potentially provides a better understanding of the consequences of PrxI dysfunction in human cells. The S. cerevisiae system described here could provide a sensitive tool to uncover the mechanisms that underlie the function of human Prxs.

The SUN41 and SUN42 genes are essential for cell separation in Candida albicans.

Completion of the yeast cell cycle involves extensive remodelling of the cell wall upon separation of mother and daughter cells. We have studied two members of the ascomycete-specific SUN gene family in Candida albicans. Inactivation of SUN41 yields defects in cell separation and hyphal elongation while inactivation of SUN42 results in minor phenotypic alterations. Simultaneous inactivation of SUN41 and SUN42 is synthetically lethal due to lysis of mother cells after septation. Electronic microscopy reveals cell wall defects mainly localized in the region surrounding the septa. This phenotype is osmoremediable and the conditional double mutants show increased sensitivity to cell wall or cell membrane perturbing agents. The essential function shared by Sun41p and Sun42p is conserved among yeasts because UTH1, a Saccharomyces cerevisiae SUN gene, suppresses the lethality of SUN41 and SUN42 conditional mutants. Investigation of functional genomic data obtained in S. cerevisiae reveals links between members of the SUN gene family and the RAM pathway regulating cell wall-degrading enzymes specifically involved during cell separation. Thus, the main function of ascomycetous Sun proteins appears linked to cell wall remodelling, with a probable role in counter-balancing cell wall degradation to avoid cell lysis upon cell separation.

Oxygen metabolism and reactive oxygen species cause chromosomal rearrangements and cell death.

The absence of Tsa1, a key peroxiredoxin that functions to scavenge H(2)O(2) in Saccharomyces cerevisiae, causes the accumulation of a broad spectrum of mutations including gross chromosomal rearrangements (GCRs). Deletion of TSA1 also causes synthetic lethality in combination with mutations in RAD6 and several key genes involved in DNA double-strand break repair. In the present study we investigated the causes of GCRs and cell death in these mutants. tsa1-associated GCRs were independent of the activity of the translesion DNA polymerases zeta, eta, and Rev1. Anaerobic growth reduced substantially GCR rates of WT and tsa1 mutants and restored the viability of tsa1 rad6, tsa1 rad51, and tsa1 mre11 double mutants. Anaerobic growth also reduced the GCR rate of rad27, pif1, and rad52 mutants, indicating a role of reactive oxygen species in GCR formation in these mutants. In addition, deletion of TSA1 or H(2)O(2) treatment of WT cells resulted in increased formation of Rad52 foci, sites of repair of multiple DNA lesions. H(2)O(2) treatment also induced the GCRs. Our results provide in vivo evidence that oxygen metabolism and reactive oxygen species are important sources of DNA damages that can lead to GCRs and lethal effects in S. cerevisiae.

Podospora anserina target of rapamycin.

We have isolated the Podospora anserina TOR gene. The PaTOR protein displayed strong identities with TOR proteins from other eukaryotes especially in the FRB domain and the kinase domain. Genome analysis suggests that a single TOR gene exists in Podospora. The serine residue known to be one site of missense mutations conferring rapamycin resistance in other organisms is conserved in the PaTOR protein (S1895). A PaTOR-S1895R mutated allele has been constructed and introduced in the wild-type strain, as expected strains expressing the PaTOR-S1895R gene become resistant to rapamycin. The dominance of the PaTOR-S1895R allele indicates that apparently the mutation does not impair the kinase activity. We confirm that all cytological modifications associated with rapamycin treatment in Podospora are indeed mediated by PaTOR. We conclude that the PaTOR gene is likely to be essential and that rapamycin treatment might be useful to further investigate rapamycin-sensitive TOR functions in Podospora and especially newly identified rapamycin-sensitive functions such as the autophagy-independent control of vacuole remodeling and septation.

The Yak1p kinase controls expression of adhesins and biofilm formation in Candida glabrata in a Sir4p-dependent pathway.

Biofilm is the predominant type of microbial development in natural environments, and potentially represents a major form of resistance or source of recurrence during host infection. Although a large number of studies have focussed on the genetics of bacterial biofilm formation, very little is known about the genes involved in this type of growth in fungi. A genetic screen for Candida glabrata Biofilm mutants was performed using a 96-well plate model of biofilm formation. Study of the isolated mutant strains allowed the identification of four genes involved in biofilm formation (RIF1, SIR4, EPA6 and YAK1). Epa6p is a newly identified adhesin required for biofilm formation in this pathogenic yeast. EPA6 and its close paralogue EPA7 are located in subtelomeric regions and their transcription is regulated by Sir4p and Rif1p, two proteins involved in subtelomeric silencing. Biofilm growth conditions induce the transcription of EPA6 and EPA7: this is dependent on the presence of an intact subtelomeric silencing machinery and is independent of the Mpk1p signalling pathway. Finally, the kinase Yak1p is required for expression of both adhesin genes and acts through a subtelomeric silencing machinery-dependent pathway.

The external amino acid signaling pathway promotes activation of Stp1 and Uga35/Dal81 transcription factors for induction of the AGP1 gene in Saccharomyces cerevisiae.

Yeast cells respond to the presence of amino acids in their environment by inducing transcription of several amino acid permease genes including AGP1, BAP2, and BAP3. The signaling pathway responsible for this induction involves Ssy1, a permease-like sensor of external amino acids, and culminates with proteolytic cleavage and translocation to the nucleus of the zinc-finger proteins Stp1 and Stp2, the lack of which abolishes induction of BAP2 and BAP3. Here we show that Stp1-but not Stp2-plays an important role in AGP1 induction, although significant induction of AGP1 by amino acids persists in stp1 and stp1 stp2 mutants. This residual induction depends on the Uga35/Dal81 transcription factor, indicating that the external amino acid signaling pathway activates not only Stp1 and Stp2, but also another Uga35/Dal81-dependent transcriptional circuit. Analysis of the AGP1 gene's upstream region revealed that Stp1 and Uga35/Dal81 act synergistically through a 21-bp cis-acting sequence similar to the UAS(AA) element previously found in the BAP2 and BAP3 upstream regions. Although cells growing under poor nitrogen-supply conditions display much higher induction of AGP1 expression than cells growing under good nitrogen-supply conditions, the UAS(AA) itself is totally insensitive to nitrogen availability. Nitrogen-source control of AGP1 induction is mediated by the GATA factor Gln3, likely acting through adjacent 5'-GATA-3' sequences, to amplify the positive effect of UAS(AA). Our data indicate that Stp1 may act in combination with distinct sets of transcription factors, according to the gene context, to promote induction of transcription in response to external amino acids. The data also suggest that Uga35/Dal81 is yet another transcription factor under the control of the external amino acid sensing pathway. Finally, the data show that the TOR pathway mediating global nitrogen control of transcription does not interfere with the external amino acid signaling pathway.

Candida albicans biofilms: a developmental state associated with specific and stable gene expression patterns.

Like many bacteria, yeast species can form biofilms on several surfaces. Candida albicans colonizes the surfaces of catheters, prostheses, and epithelia, forming biofilms that are extremely resistant to antifungal drugs. We have used transcript profiling to investigate the specific properties of C. albicans biofilms. Biofilm and planktonic cultures produced under different conditions of nutrient flow, aerobiosis, or glucose concentration were compared by overall gene expression correlation. Correlation was much higher between biofilms than planktonic populations irrespective of the growth conditions, indicating that biofilm populations formed in different environments display very similar and specific transcript profiles. A first cluster of 325 differentially expressed genes was identified. In agreement with the overrepresentation of amino acid biosynthesis genes in this cluster, Gcn4p, a regulator of amino acid metabolism, was shown to be required for normal biofilm growth. To identify biofilm-related genes that are independent of mycelial development, we studied the transcriptome of biofilms produced by a wild-type, hypha-producing strain and a cph1/cph1 efg1/efg1 strain defective for hypha production. This analysis identified a cluster of 317 genes expressed independently of hypha formation, whereas 86 genes were dependent on mycelial development. Both sets revealed the activation of the sulfur-amino acid biosynthesis pathway as a feature of C. albicans biofilms.