RAD53 is limiting in double-strand break repair and in protection against toxicity associated with ribonucleotide reductase inhibition

The yeast Chk2/Chk1 homolog Rad53 is a central component of the DNA damage checkpoint system. While it controls genotoxic stress responses such as cell cycle arrest, replication fork stabilization and increase in dNTP pools, little is known about the consequences of reduced Rad53 levels on the various cellular endpoints or about its roles in dealing with chronic vs. acute genotoxic challenges. Using a tetraploid gene dosage model in which only one copy of the yeast RAD53 is functional (simplex), we found that the simplex strain was not sensitive to acute UV radiation or chronic MMS exposure. However, the simplex strain was sensitized to chronic exposure of the ribonucleotide reductase inhibitor hydroxyurea (HU). Surprisingly, reduced RAD53 gene dosage did not affect sensitivity to HU acute exposure, indicating that immediate checkpoint responses and recovery from HU-induced stress were not compromised. Interestingly, cells of most of the colonies that arise after chronic HU exposure acquired heritable resistance to HU. We also found that short HU exposure before and after treatment of G₂ cells with ionizing radiation (IR) reduced the capability of RAD53 simplex cells to repair DSBs, in agreement with sensitivity of RAD53 simplex strain to high doses of IR. We propose that a modest reduction in Rad53 activity can impact the activation of the ribonucleotide reductase catalytic subunit Rnr1 following stress, reducing the ability to generate nucleotide pools sufficient for DNA repair and replication. At the same time, reduced Rad53 activity may lead to genome instability and to the acquisition of drug resistance before and/or during the chronic exposure to HU. These results have implications for developing drug enhancers as well as for understanding mechanisms of drug resistance in cells compromised for DNA damage checkpoint.     

Role for Sit4p-dependent mitochondrial dysfunction in mediating the shortened chronological lifespan and oxidative stress sensitivity of Isc1p-deficient cells

Saccharomyces cerevisiae cells lacking Isc1p, an orthologue of mammalian neutral sphingomyelinase 2, display a shortened lifespan and an increased sensitivity to oxidative stress. A lipidomic analysis revealed specific changes in sphingolipids that accompanied the premature ageing of Isc1p-deficient cells under severe calorie restriction conditions, including a decrease of dihydrosphingosine levels and an increase of dihydro-C(26) -ceramide and phyto-C(26) -ceramide levels, the latter raising the possibility of activation of ceramide-dependent protein phosphatases. Consequently, deletion of the SIT4 gene, which encodes for the catalytic subunit of type 2A ceramide-activated protein phosphatase in yeast, abolished the premature ageing and hydrogen peroxide sensitivity of isc1Δ cells. SIT4 deletion also abolished the respiratory defects and catalase A deficiency exhibited by isc1Δ mutants. These results are consistent with catabolic derepression associated with the loss of Sit4p. The overall results show that Isc1p is an upstream regulator of Sit4p and implicate Sit4p activation in mitochondrial dysfunction leading to the shortened chronological lifespan and oxidative stress sensitivity of isc1Δ mutants.     

Functional and physical interactions between yeast 14-3-3 proteins, acetyltransferases, and deacetylases in response to DNA replication perturbations

The highly conserved 14-3-3 proteins participate in many biological processes in different eukaryotes. The BMH1 and BMH2 genes encode the two functionally redundant Saccharomyces cerevisiae 14-3-3 isoforms. In this work we provide evidence that defective 14-3-3 functions not only impair the ability of yeast cells to sustain DNA replication in the presence of sublethal concentrations of methyl methanesulfonate (MMS) or hydroxyurea (HU) but also cause S-phase checkpoint hyperactivation. Inactivation of the catalytic subunit of the histone acetyltransferase NuA4 or of its interactor Yng2, besides leading to S-phase defects and persistent checkpoint activation in the presence of genotoxic agents, is lethal for bmh mutants. Conversely, the lack of the histone deacetylase subunit Rpd3 or Sin3 partially suppresses the hypersensitivity to HU of bmh mutants and restores their ability to complete DNA replication in the presence of MMS or HU. These data strongly suggest that reduced acetyltransferase functionality might account for the S-phase defects of bmh mutants in the presence of genotoxic agents. Consistent with a role of 14-3-3 proteins in acetyltransferase and deacetylase regulation, we find that acetylation of H3 and H4 histone tails is reduced in temperature-sensitive bmh mutants shifted to the restrictive temperature. Moreover, Bmh proteins physically interact, directly or indirectly, with the Esa1 acetyltransferase throughout the cell cycle and with the Rpd3 deacetylase specifically during unperturbed S phase and after HU treatment. Taken together, our results highlight a novel role for 14-3-3 proteins in the regulation of histone acetyltransferase and deacetylase functions in the response to replicative stress.     

The Swi2�CSnf2-like protein Uls1 is involved in replication stress response

The Saccharomyces cerevisiae Uls1 belongs to the Swi2–Snf2 family of DNA-dependent ATPases and a new protein family of SUMO-targeted ubiquitin ligases. Here, we examine a physiological role of Uls1 and report for the first time its involvement in response to replication stress. We found that deletion of ULS1 in cells lacking RAD52 caused a synthetic growth defect accompanied by prolonged S phase and aberrant cell morphology. uls1Δ also progressed slower through S phase upon MMS treatment and took longer to resolve replication intermediates during recovery. This suggests an important function for Uls1 during replication stress. Consistently, cells lacking Uls1 and endonuclease Mus81 were more sensitive to HU, MMS and CPT than single mus81Δ. Interestingly, deletion of ULS1 attenuated replication stress-related defects in sgs1Δ, such as sensitivity to HU and MMS while increasing the level of PCNA ubiquitination and Rad53 phosphorylation. Importantly, Uls1 interactions with Mus81 and Sgs1 were dependent on its helicase domain. We propose that Uls1 directs a subset of DNA structures arising during replication into the Sgs1-dependent pathway facilitating S phase progression. Thus, in the absence of Uls1 other modes of replication fork processing and repair are employed.     

Limiting amounts of budding yeast Rad53 S-phase checkpoint activity results in increased resistance to DNA alkylation damage

The Saccharomyces cerevisiae protein kinase Rad53 plays a key role in maintaining genomic integrity after DNA damage and is an essential component of the ‘intra-S-phase checkpoint’. In budding yeast, alkylating chemicals, such as methyl methanesulfonate (MMS), or depletion of nucleotides by hydroxyurea (HU) stall DNA replication forks and thus activate Rad53 during S-phase. This stabilizes stalled DNA replication forks and prevents the activation of later origins of DNA replication. Here, we report that a reduction in the level of Rad53 kinase causes cells to behave very differently in response to DNA alkylation or to nucleotide depletion. While cells lacking Rad53 are unable to activate the checkpoint response to HU or MMS, so that they rapidly lose viability, a reduction in Rad53 enhances cell survival only after DNA alkylation. This reduction in the level of Rad53 allows S-phase cells to maintain the stability of DNA replication forks upon MMS treatment, but does not prevent the collapse of forks in HU. Our results may have important implications for cancer therapies, as they suggest that partial impairment of the S-phase checkpoint Rad53/Chk2 kinase provides cells with a growth advantage in the presence of drugs that damage DNA.     

Critical role of DNA checkpoints in mediating genotoxic-stress-induced filamentous growth in Candida albicans

The polymorphic fungus Candida albicans switches from yeast to filamentous growth in response to a range of genotoxic insults, including inhibition of DNA synthesis by hydroxyurea (HU) or aphidicolin (AC), depletion of the ribonucleotide-reductase subunit Rnr2p, and DNA damage induced by methylmethane sulfonate (MMS) or UV light (UV). Deleting RAD53, which encodes a downstream effector kinase for both the DNA-replication and DNA-damage checkpoint pathways, completely abolished the filamentous growth caused by all the genotoxins tested. Deleting RAD9, which encodes a signal transducer of the DNA-damage checkpoint, specifically blocked the filamentous growth induced by MMS or UV but not that induced by HU or AC. Deleting MRC1, the counterpart of RAD9 in the DNA-replication checkpoint, impaired DNA synthesis and caused cell elongation even in the absence of external genotoxic insults. Together, the results indicate that the DNA-replication/damage checkpoints are critically required for the induction of filamentous growth by genotoxic stress. In addition, either of two mutations in the FHA1 domain of Rad53p, G65A, and N104A, nearly completely blocked the filamentous-growth response but had no significant deleterious effect on cell-cycle arrest. These results suggest that the FHA domain, known for its ability to bind phosphopeptides, has an important role in mediating genotoxic-stress-induced filamentous growth and that such growth is a specific, Rad53p-regulated cellular response in C. albicans.     

Regulation of tolerance to DNA alkylating damage by Dot1 and Rad53 in Saccharomyces cerevisiae

To maintain genomic integrity cells have to respond properly to a variety of exogenous and endogenous factors that produce genome injuries and interfere with DNA replication. DNA integrity checkpoints coordinate this response by slowing cell cycle progression to provide time for the cell to repair the damage, stabilizing replication forks and stimulating DNA repair to restore the original DNA sequence and structure. In addition, there are also mechanisms of damage tolerance, such as translesion synthesis (TLS), which are important for survival after DNA damage. TLS allows replication to continue without removing the damage, but results in a higher frequency of mutagenesis. Here, we investigate the functional contribution of the Dot1 histone methyltransferase and the Rad53 checkpoint kinase to TLS regulation in Saccharomyces cerevisiae. We demonstrate that the Dot1-dependent status of H3K79 methylation modulates the resistance to the alkylating agent MMS, which depends on PCNA ubiquitylation at lysine 164. Strikingkly, either the absence of DOT1, which prevents full activation of Rad53, or the expression of an HA-tagged version of RAD53, which produces low amounts of the kinase, confer increased MMS resistance. However, the dot1Δ rad53-HA double mutant is hypersensitive to MMS and shows barely detectable amounts of activated kinase. Furthermore, moderate overexpression of RAD53 partially suppresses the MMS resistance of dot1Δ. In addition, we show that MMS-treated dot1Δ and rad53-HA cells display increased number of chromosome-associated Rev1 foci. We propose that threshold levels of Rad53 activity exquisitely modulate the tolerance to alkylating damage at least by controlling the abundance of the key TLS factor Rev1 bound to chromatin.     

Small ubiquitin-related modifier ligase activity of Mms21 is required for maintenance of chromosome integrity during the unperturbed mitotic cell division cycle in Saccharomyces cerevisiae

The SUMO ligase activity of Mms21/Nse2, a conserved member of the Smc5/6 complex, is required for resisting extrinsically induced genotoxic stress. We report that the Mms21 SUMO ligase activity is also required during the unchallenged mitotic cell cycle in Saccharomyces cerevisiae. SUMO ligase-defective cells were slow growing and spontaneously incurred DNA damage. These cells required caffeine-sensitive Mec1 kinase-dependent checkpoint signaling for survival even in the absence of extrinsically induced genotoxic stress. SUMO ligase-defective cells were sensitive to replication stress and displayed synthetic growth defects with DNA damage checkpoint-defective mutants such as mec1, rad9, and rad24. MMS21 SUMO ligase and mediator of replication checkpoint 1 gene (MRC1) were epistatic with respect to hydroxyurea-induced replication stress or methyl methanesulfonate-induced DNA damage sensitivity. Subjecting Mms21 SUMO ligase-deficient cells to transient replication stress resulted in enhancement of cell cycle progression defects such as mitotic delay and accumulation of hyperploid cells. Consistent with the spontaneous activation of the DNA damage checkpoint pathway observed in the Mms21-mediated sumoylation-deficient cells, enhanced frequency of chromosome breakage and loss was detected in these mutant cells. A mutation in the conserved cysteine 221 that is engaged in coordination of the zinc ion in Loop 2 of the Mms21 SPL-RING E3 ligase catalytic domain resulted in strong replication stress sensitivity and also conferred slow growth and Mec1 dependence to unchallenged mitotically dividing cells. Our findings establish Mms21-mediated sumoylation as a determinant of cell cycle progression and maintenance of chromosome integrity during the unperturbed mitotic cell division cycle in budding yeast.     

Caffeine-mediated override of checkpoint controls. A requirement for rhp6 (Schizosaccharomyces pombe)

Cells exposed to inhibitors of DNA synthesis or suffering DNA damage are arrested or delayed in interphase through the action of checkpoint controls. If the arrested cell is exposed to caffeine, relatively normal cell cycle progression is resumed and, as observed in checkpoint control mutants, loss of checkpoint control activity is associated with a reduction in cell viability. To address the mechanism of caffeine's action on cell progression, fission yeast mutants that take up caffeine but are not sensitized to hydroxyurea (HU) by caffeine were selected. Mutants 788 and 1176 are point mutants of rhp6, the fission yeast homolog of the budding yeast RAD6 gene. Mutant rhp6-788 is slightly HU sensitive, radiosensitive, and exhibits normal checkpoint responses to HU, radiation, or inactivation of DNA ligase. However, the addition of caffeine does not override the associated cell cycle blocks. Both point and deletion mutations show synthetic lethality at room temperature with temperature-sensitive mutations in cyclin B (cdc13-117) or the phosphatase cdc25 (cdc25-22). These observations suggest that the rhp6 gene product, a ubiquitin-conjugating enzyme required for DNA damage repair, promotes entry to mitosis in response to caffeine treatment.     

The contribution of the S-phase checkpoint genes MEC1 and SGS1 to genome stability maintenance in Candida albicans

Genome rearrangements, a common feature of Candida albicans isolates, are often associated with the acquisition of antifungal drug resistance. In Saccharomyces cerevisiae, perturbations in the S-phase checkpoints result in the same sort of Gross Chromosomal Rearrangements (GCRs) observed in C. albicans. Several proteins are involved in the S. cerevisiae cell cycle checkpoints, including Mec1p, a protein kinase of the PIKK (phosphatidyl inositol 3-kinase-like kinase) family and the central player in the DNA damage checkpoint. Sgs1p, the ortholog of BLM, the Bloom's syndrome gene, is a RecQ-related DNA helicase; cells from BLM patients are characterized by an increase in genome instability. Yeast strains bearing deletions in MEC1 or SGS1 are viable (in contrast to the inviability seen with loss of MEC1 in S. cerevisiae) but the different deletion mutants have significantly different phenotypes. The mec1Δ/Δ colonies have a wild-type colony morphology, while the sgs1Δ/Δ mutants are slow-growing, producing wrinkled colonies with pseudohyphal-like cells. The mec1Δ/Δ mutants are only sensitive to ethylmethane sulfonate (EMS), methylmethane sulfonate (MMS), and hydroxyurea (HU) but the sgs1Δ/Δ mutants exhibit a high sensitivity to all DNA-damaging agents tested. In an assay for chromosome 1 integrity, the mec1Δ/Δ mutants exhibit an increase in genome instability; no change was observed in the sgs1Δ/Δ mutants. Finally, loss of MEC1 does not affect sensitivity to the antifungal drug fluconazole, while loss of SGS1 leads to an increased susceptibility to fluconazole. Neither deletion elevated the level of antifungal drug resistance acquisition.     

A Rad53 Independent Function of Rad9 Becomes Crucial for Genome Maintenance in the Absence of the RecQ Helicase Sgs1

The conserved family of RecQ DNA helicases consists of caretaker tumour suppressors, that defend genome integrity by acting on several pathways of DNA repair that maintain genome stability. In budding yeast, Sgs1 is the sole RecQ helicase and it has been implicated in checkpoint responses, replisome stability and dissolution of double Holliday junctions during homologous recombination. In this study we investigate a possible genetic interaction between SGS1 and RAD9 in the cellular response to methyl methane sulphonate (MMS) induced damage and compare this with the genetic interaction between SGS1 and RAD24. The Rad9 protein, an adaptor for effector kinase activation, plays well-characterized roles in the DNA damage checkpoint response, whereas Rad24 is characterized as a sensor protein also in the DNA damage checkpoint response. Here we unveil novel insights into the cellular response to MMS-induced damage. Specifically, we show a strong synergistic functionality between SGS1 and RAD9 for recovery from MMS induced damage and for suppression of gross chromosomal rearrangements, which is not the case for SGS1 and RAD24. Intriguingly, it is a Rad53 independent function of Rad9, which becomes crucial for genome maintenance in the absence of Sgs1. Despite this, our dissection of the MMS checkpoint response reveals parallel, but unequal pathways for Rad53 activation and highlights significant differences between MMS- and hydroxyurea (HU)-induced checkpoint responses with relation to the requirement of the Sgs1 interacting partner Topoisomerase III (Top3). Thus, whereas earlier studies have documented a Top3-independent role of Sgs1 for an HU-induced checkpoint response, we show here that upon MMS treatment, Sgs1 and Top3 together define a minor but parallel pathway to that of Rad9.     

Drosophila Claspin is required for the G2 arrest that is induced by DNA replication stress but not by DNA double-strand breaks

ATR and Chk1 are protein kinases that perform major roles in the DNA replication checkpoint that delays entry into mitosis in response to DNA replication stress by hydroxyurea (HU) treatment. They are also activated by ionizing radiation (IR) that induces DNA double-strand breaks. Studies in human tissue culture and Xenopus egg extracts identified Claspin as a mediator that increased the activity of ATR toward Chk1. Because the in vivo functions of Claspin are not known, we generated Drosophila lines that each contained a mutated Claspin gene. Similar to the Drosophila mei-41/ATR and grp/Chk1 mutants, embryos of the Claspin mutant showed defects in checkpoint activation, which normally occurs in early embryogenesis in response to incomplete DNA replication. Additionally, Claspin mutant larvae were defective in G2 arrest after HU treatment; however, the defects were less severe than those of the mei-41/ATR and grp/Chk1 mutants. In contrast, IR-induced G2 arrest, which was severely defective in mei-41/ATR and grp/Chk1 mutants, occurred normally in the Claspin mutant. We also found that Claspin was phosphorylated in response to HU and IR treatment and a hyperphosphorylated form of Claspin was generated only after HU treatment in mei-41/ATR-dependent and tefu/ATM-independent way. In summary, our data suggest that Drosophila Claspin is required for the G2 arrest that is induced by DNA replication stress but not by DNA double-strand breaks, and this difference is probably due to distinct phosphorylation statuses.     

The function and regulation of budding yeast Swe1 in response to interrupted DNA synthesis

Periodically regulated cyclin-dependent kinase (Cdk) is required for DNA synthesis and mitosis. Hydroxyurea (HU) inhibits DNA synthesis by depleting dNTPs, the basic unit for DNA synthesis. HU treatment triggers the S-phase checkpoint, which arrests cells at S-phase, inhibits late origin firing and stabilizes replication forks. Using budding yeast as a model system, we found that Swe1, a negative regulator of Cdk, appears at S-phase and accumulates in HU treatment cells. Interestingly, this accumulation is not dependent on S-phase checkpoint. Deltahsl1, Deltahsl7, and cdc5-2 mutants, which have defects in Swe1 degradation, show HU sensitivity because of high Swe1 protein levels. We further demonstrated that their HU sensitivity is not a result of DNA damage accumulation or incomplete DNA synthesis; instead the sensitivity is due to their dramatically delayed recovery from HU-induced S-phase arrest. Strikingly, our in vivo data indicate that Swe1 inhibits the kinase activity of Clb2-Cdk1, but not that of Clb5-Cdk1. Therefore, S-phase accumulated Swe1 prevents Clb2-Cdk1-mediated mitotic activities, but has little effects on Clb5-Cdk1-associated S-phase progression.     

The Saccharomyces cerevisiae DNA damage checkpoint is required for efficient repair of double strand breaks by non-homologous end joining

In this work we report that the Saccharomyces cerevisiae RAD9, RAD24, RAD17, MEC1, MEC3 and RAD53 checkpoint genes are required for efficient non-homologous end joining (NHEJ). RAD9 and RAD24 function additionally in this process. Defective NHEJ in rad9Delta-rad24Delta, but not yku80Delta cells, is only partially rescued by imposing G1 or G2/M delays. Thus, checkpoint functions other than transient cell cycle delays may be required for normal levels of NHEJ. Epistasis analysis also indicated that YKU80 and RAD9/RAD24 function in the same pathway for repair of lesions caused by MMS and gamma-irradiation. Unlike NHEJ, the checkpoint pathway is not required for efficient site-specific integration of plasmid DNA into the yeast genome, which is RAD52-dependent, but RAD51-independent.     

Cell Cycle Arrest of Cdc Mutants and Specificity of the Rad9 Checkpoint

In eucaryotes a cell cycle control called a checkpoint ensures that mitosis occurs only after chromosomes are completely replicated and any damage is repaired. The function of this checkpoint in budding yeast requires the RAD9 gene. Here we examine the role of the RAD9 gene in the arrest of the 12 cell division cycle (cdc) mutants, temperature-sensitive lethal mutants that arrest in specific phases of the cell cycle at a restrictive temperature. We found that in four cdc mutants the cdc rad9 cells failed to arrest after a shift to the restrictive temperature, rather they continued cell division and died rapidly, whereas the cdc RAD cells arrested and remained viable. The cell cycle and genetic phenotypes of the 12 cdc RAD mutants indicate the function of the RAD9 checkpoint is phase-specific and signal-specific. First, the four cdc RAD mutants that required RAD9 each arrested in the late S/G(2) phase after a shift to the restrictive temperature when DNA replication was complete or nearly complete, and second, each leaves DNA lesions when the CDC gene product is limiting for cell division. Three of the four CDC genes are known to encode DNA replication enzymes. We found that the RAD17 gene is also essential for the function of the RAD9 checkpoint because it is required for phase-specific arrest of the same four cdc mutants. We also show that both X- or UV-irradiated cells require the RAD9 and RAD17 genes for delay in the G(2) phase. Together, these results indicate that the RAD9 checkpoint is apparently activated only by DNA lesions and arrests cell division only in the late S/G(2) phase.     

Dual cell cycle checkpoints sensitive to chromosome replication and DNA damage in the budding yeast Saccharomyces cerevisiae.

In eucaryotic cells chromosomes must be fully replicated and repaired before mitosis begins. Genetic studies indicate that this dependence of mitosis on completion of DNA replication and DNA repair derives from a negative control called a checkpoint which somehow checks for replication and DNA damage and blocks cell entry into mitosis. Here we summarize our current understanding of the genetic components of the cell cycle checkpoint in budding yeast. Mutants were identified and their phase and signal specificity tested primarily through interactions of the arrest-defective mutants with cell division cycle mutants. The results indicate that dual checkpoint controls exist in budding yeast, one control sensitive to inhibition of DNA replication (S-phase checkpoint), and a distinct but overlapping control sensitive to DNA repair (G2 checkpoint). Six genes are required for arrest in G2 phase after DNA damage (RAD9, RAD17, RAD24, MEC1, MEC2, and MEC3), and two of these are also essential for arrest in S phase when DNA replication is blocked (MEC1 and MEC2).     

Regulation of DNA replication machinery by Mrc1 in fission yeast

Faithful replication of chromosomes is crucial to genome integrity. In yeast, the ORC binds replication origins throughout the cell cycle. However, Cdc45 binds these before S-phase, and, during replication, it moves along the DNA with MCM helicase. When replication progression is inhibited, checkpoint regulation is believed to stabilize the replication fork; the detailed mechanism, however, remains unclear. To examine the relationship between replication initiation and elongation defects and the response to replication elongation block, we used fission yeast mutants of Orc1 and Cdc45--orp1-4 and sna41-928, respectively--at their respective semipermissive temperatures with regard to BrdU incorporation. Both orp1 and sna41 cells exhibited HU hypersensitivity in the absence of Chk1, a DNA damage checkpoint kinase, and were defective in full activation of Cds1, a replication checkpoint kinase, indicating that normal replication is required for Cds1 activation. Mrc1 is required to activate Cds1 and prevent the replication machinery from uncoupling from DNA synthesis. We observed that, while either the orp1 or the sna41 mutation partially suppressed HU sensitivity of cds1 cells, sna41 specifically suppressed that of mrc1 cells. Interestingly, sna41 alleviated the defect in recovery from HU arrest without increasing Cds1 activity. In addition to sna41, specific mutations of MCM suppressed the HU sensitivity of mrc1 cells. Thus, during elongation, Mrc1 may negatively regulate Cdc45 and MCM helicase to render stalled forks capable of resuming replication.     

Deletion of ULS1 confers damage tolerance in sgs1 mutants through a Top3-dependent D-loop mediated fork restart pathway

Homologous recombination (HR)-based repair during DNA replication can apparently utilize several partially overlapping repair pathways in response to any given lesion. A key player in HR repair is the Sgs1-Top3-Rmi1 (STR) complex, which is critical for resolving X-shaped recombination intermediates formed following bypass of methyl methanesulfonate (MMS)-induced damage. STR mutants are also sensitive to the ribonucleotide reductase inhibitor, hydroxyurea (HU), but unlike MMS treatment, HU treatment is not accompanied by X-structure accumulation, and it is thus unclear how STR functions in this context. Here we provide evidence that HU-induced fork stalling enlists Top3 prior to recombination intermediate formation. The resistance of sgs1Δ mutants to HU is enhanced by the absence of the putative SUMO (Small Ubiquitin MOdifier)-targeted ubiquitin ligase, Uls1, and we demonstrate that Top3 is required for this enhanced resistance and for coordinated breaks and subsequent d-loop formation at forks stalled at the ribosomal DNA (rDNA) replication fork block (RFB). We also find that HU resistance depends on the catalytic activity of the E3 SUMO ligase, Mms21, and includes a rapid Rad51-dependent restart mechanism that is different from the slow Rad51-independent HR fork restart mechanism operative in sgs1Δ ULS1+ mutants. These data support a model in which repair of HU-induced damage in sgs1Δ mutants involves an error-prone break-induced replication pathway but, in the absence of Uls1, shifts to one that is higher-fidelity and involves the formation of Rad51-dependent d-loops.     

DNA replication checkpoint control mediated by the spindle checkpoint protein Mad2p in fission yeast

The relationship between the DNA replication and spindle checkpoints of the cell cycle is unclear, given that in most eukaryotes, spindle formation occurs only after DNA replication is complete. Fission yeast rad3 mutant cells, which are deficient in DNA replication checkpoint function, enter, progress through, and exit mitosis even when DNA replication is blocked. In contrast, the entry of cds1 mutant cells into mitosis is delayed by several hours when DNA replication is inhibited. We show here that this delay in mitotic entry in cds1 cells is due in part to activation of the spindle checkpoint protein Mad2p. In the presence of the DNA replication inhibitor hydroxyurea (HU), cds1 mad2 cells entered and progressed through mitosis earlier than did cds1 cells. Overexpression of Mad2p or inactivation of Slp1p, a regulator of the anaphase-promoting complex, also rescued the checkpoint defect of HU-treated rad3 cells. Rad3p was shown to be involved in the physical interaction between Mad2p and Slp1p in the presence of HU. These results suggested that Mad2p and Slp1p act downstream of Rad3p in the DNA replication checkpoint and that Mad2p is required for the DNA replication checkpoint when Cds1p is compromised.