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author:("iraqi, Ismail")
1.  The Chromatin Assembly Factor 1 Promotes Rad51-Dependent Template Switches at Replication Forks by Counteracting D-Loop Disassembly by the RecQ-Type Helicase Rqh1 
PLoS Biology  2014;12(10):e1001968.
A molecular switch for times of replication stress - Chromatin Assembly Factor 1 helps to protect DNA during recombination-mediated template-switching, favoring the rescue of stalled replication forks by both beneficial and detrimental homologous recombination events.
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.
Author Summary
Obstacles to the progression of DNA replication forks can result in genome rearrangements that are often observed in cancer cells and genomic disorders. Homologous recombination is a mechanism of restarting stalled replication fork that involves synthesis of the new DNA strands switching templates to a second (allelic) copy of the DNA sequence. However, the new strands can also occasionally recombine with nonallelic repeats (distinct regions of the genome that resemble the correct one) and thereby cause the inappropriate fusion of normally distant DNA segments; this is known as faulty template switching. The chromatin assembly factor 1 (CAF-1) is already known to be involved in depositing nucleosomes on DNA during DNA replication and repair. We have found that CAF-1 is also involved in the recombination-mediated template switch pathway in response to replication stress. Using both genetic and physical assays that allow the different steps of template switch to be analyzed, we reveal that CAF-1 protects recombination intermediates from disassembly by the RecQ-type helicase Rqh1, the homologue of BLM (people with mutations that affect BLM have Bloom's syndrome, an inherited predisposition to genome instability and cancer). Consequently, the likelihood of faulty template switch is controlled by the antagonistic activities of CAF-1 and Rqh1. We thus identified an evolutionarily conserved interplay between CAF-1 and RecQ-type helicases that helps to maintain genome stability in the face of replication stress.
PMCID: PMC4196752  PMID: 25313826
2.  Loss of the Thioredoxin Reductase Trr1 Suppresses the Genomic Instability of Peroxiredoxin tsa1 Mutants 
PLoS ONE  2014;9(9):e108123.
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 CanR mutation rate 5-fold lower than wild-type cells. Additional deletion of TRR1 in tsa1Δ mutant reduced substantially the CanR 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.
PMCID: PMC4172583  PMID: 25247923
3.  Redox-sensitive YFP sensors monitor dynamic nuclear and cytosolic glutathione redox changes 
Free radical biology & medicine  2012;52(0):2254-2265.
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 S. cerevisiae. Parallel analyses of the redox state of nucleus-rxYFP and cytosol-rxYFP allow us to monitor distinctively dynamic glutathione (GSH) redox changes within these two compartments in 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 system play 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.
PMCID: PMC3382975  PMID: 22561702
redox-sensitive sensor; redox; glutathione; yeast
4.  Recovery of Arrested Replication Forks by Homologous Recombination Is Error-Prone 
PLoS Genetics  2012;8(10):e1002976.
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.
Author Summary
The appropriate transmission of genetic material during successive cell divisions requires the accurate duplication and segregation of parental DNA. The semi-conservative replication of chromosomes during S-phase is highly accurate and prevents accumulation of deleterious mutations. However, during each round of duplication, there are many impediments to the replication fork machinery that may hinder faithful chromosome duplication. Homologous recombination is a universal mechanism involved in the rescue of replication forks by rebuilding a replication apparatus at the fork (by mechanisms that are not yet understood). However, recombination can jeopardize genome stability because it allows genetic exchanges between homologous repeated sequences dispersed through the genome. In this study, we employ a fission yeast-based arrest of a single replication fork to investigate the consequences of replication fork arrest for genome stability. We report that a single blocked fork favours genomic deletions, translocations, and mutations; and this instability occurs during fork recovery by recombination. We also report that a single arrested fork that resumes its progression by recombination is prone to causing replication slippage mediated by micro-homology. We propose that deletions/duplications observed in human cancer cells suffering from replication stress can be viewed as scars left by error-prone replication forks restarted by recombination.
PMCID: PMC3475662  PMID: 23093942
5.  Human peroxiredoxin PrxI is an ortholog of yeast Tsa1, capable of suppressing genome instability and protecting against cell death in Saccharomyces cerevisiae 
Cancer research  2008;68(4):1055-1063.
The peroxiredoxins (Prxs) are conserved antioxidant proteins that utilize 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 peroxiredoxins PrxI and PrxII, which share a high degree of sequence identity with Tsa1, by expressing them in S. cerevisiae cells under control of the native TSA1 promoter. We found that expression of PrxI, but not PrxII, was capable of complementing a tsa1Δ mutant for a variety of defects including genome instability, the synthetic lethality observed in rad6Δ tsa1Δ and rad51Δ tsa1Δ double mutants and mutagen sensitivity. Moreover, expression of either Tsa1 or PrxI prevented Bax-induced cell death. These data indicate that PrxI is an ortholog of Tsa1. PrxI and Tsa1 appear 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.
PMCID: PMC2761232  PMID: 18281480
peroxiredoxin; oxidative stress; genome instability; S. cerevisiae
6.  Peroxiredoxin Tsa1 Is the Key Peroxidase Suppressing Genome Instability and Protecting against Cell Death in Saccharomyces cerevisiae 
PLoS Genetics  2009;5(6):e1000524.
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 (Cys47) was essential for Tsa1 activity, while Tsa1C170S lacking the resolving Cys was partially functional. In the absence of Tsa1 activity (tsa1 or tsa1CCS 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 tsa1CCS 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.
Author Summary
Aerobically growing cells are continuously challenged by potent oxidants produced during normal cellular metabolism. These oxidants, including hydrogen peroxide and organic peroxides, are important components mediating various cell functions. However, they can also cause cell damage when present at toxic levels. Aerobic organisms possess extensive antioxidant systems to regulate oxidant levels. Among these, peroxiredoxins have received considerable attention in recent years as an expanding protein family involved in the enzymatic degradation of hydrogen peroxide and organic peroxides. To better understand the physiological role of the five peroxiredoxins in budding yeast S. cerevisiae, we performed a comparative study and found that one, Tsa1, played a key role in preventing DNA damage and assuring genome stability. Tsa1 also cooperated with other peroxiredoxins in antioxidant defense. These functions of Tsa1 required the presence of a cysteine at the catalytic site of this enzyme. Additional studies revealed that Tsa1 activity, in cooperation with appropriate DNA repair and checkpoint mechanisms, acts to protect cells against toxic levels of DNA damage that occur during aerobic growth.
PMCID: PMC2688748  PMID: 19543365

Results 1-6 (6)