Search

Atg6 Beclin 1 mediates autophagy and endosomal trafficking. We investigated how Atg6 influences replication stress. Combining genetic, genomic, metabolomic, and proteomic approaches, we found that the Vps34-Vps15-Atg6 Beclin 1 -Vps38 UVRAG -phosphatydilinositol-3 phosphate (PtdIns(3)P) axis sensitizes cells to replication stress by favoring the degradation of plasma membrane amino acid (AA) transporters via endosomal trafficking and ESCRT proteins, while the PtdIns(3)P phosphatases Ymr1 and Inp53 promote survival to replication stress by reversing this process. An impaired AA uptake triggers activation of Gcn2, which attenuates protein synthesis by phosphorylating eIF2α. Mec1 Atr -Rad53 Chk1/Chk2 activation during replication stress further hinders translation efficiency by counteracting eIF2α dephosphorylation through Glc7 PP1 . AA shortage-induced hyperphosphorylation of eIF2α inhibits the synthesis of 65 stress response proteins, thus resulting in cell sensitization to replication stress, while TORC1 promotes cell survival. Our findings reveal an integrated network mediated by endosomal trafficking, translational control pathways, and checkpoint kinases linking AA availability to the response to replication stress., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The DNA damage response (DDR) coordinates DNA metabolism with nuclear and non-nuclear processes. The DDR kinase Rad53 CHK1/CHK2 controls histone degradation to assist DNA repair. However, Rad53 deficiency causes histone-dependent growth defects in the absence of DNA damage, pointing out unknown physiological functions of the Rad53-histone axis. Here we show that histone dosage control by Rad53 ensures metabolic homeostasis. Under physiological conditions, Rad53 regulates histone levels through inhibitory phosphorylation of the transcription factor Spt21 NPAT on Ser276. Rad53-Spt21 mutants display severe glucose dependence, caused by excess histones through two separable mechanisms: dampening of acetyl-coenzyme A-dependent carbon metabolism through histone hyper-acetylation, and Sirtuin-mediated silencing of starvation-induced subtelomeric domains. We further demonstrate that repression of subtelomere silencing by physiological Tel1 ATM and Rpd3 HDAC activities coveys tolerance to glucose restriction. Our findings identify DDR mutations, histone imbalances and aberrant subtelomeric chromatin as interconnected causes of glucose dependence, implying that DDR kinases coordinate metabolism and epigenetic changes.

Replication stress activates the Mec1(ATR) and Rad53 kinases. Rad53 phosphorylates nuclear pores to counteract gene gating, thus preventing aberrant transitions at forks approaching transcribed genes. Here, we show that Rrm3 and Pif1, DNA helicases assisting fork progression across pausing sites, are detrimental in rad53 mutants experiencing replication stress. Rrm3 and Pif1 ablations rescue cell lethality, chromosome fragmentation, replisome-fork dissociation, fork reversal, and processing in rad53 cells. Through phosphorylation, Rad53 regulates Rrm3 and Pif1; phospho-mimicking rrm3 mutants ameliorate rad53 phenotypes following replication stress without affecting replication across pausing elements under normal conditions. Hence, the Mec1-Rad53 axis protects fork stability by regulating nuclear pores and DNA helicases. We propose that following replication stress, forks stall in an asymmetric conformation by inhibiting Rrm3 and Pif1, thus impeding lagging strand extension and preventing fork reversal; conversely, under unperturbed conditions, the peculiar conformation of forks encountering pausing sites would depend on active Rrm3 and Pif1., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Recombination is important for DNA repair, but it can also contribute to genome rearrangements. RecQ helicases, including yeast Sgs1 and human BLM, safeguard genome integrity through their functions in DNA recombination. Sgs1 prevents the accumulation of Rad51-dependent sister chromatid junctions at damaged replication forks, and its functionality seems to be regulated by Ubc9- and Mms21-dependent sumoylation. We show that mutations in Smc5-6 and Esc2 also lead to an accumulation of recombinogenic structures at damaged replication forks. Because Smc5-6 is sumoylated in an Mms21-dependent manner, this finding suggests that Smc5-6 may be a crucial target of Mms21 implicated in this process. Our data reveal that Smc5-6 and Esc2 are required to tolerate DNA damage and that their functionality is critical in genotoxic conditions in the absence of Sgs1. As reported previously for Sgs1 and Smc5-6, we find that Esc2 physically interacts with Ubc9 and SUMO. This interaction is correlated with the ability of Esc2 to promote DNA damage tolerance. Collectively, these data suggest that Esc2 and Smc5-6 act in concert with Sgs1 to prevent the accumulation of recombinogenic structures at damaged replication forks, likely by integrating sumoylation activities to regulate the repair pathways in response to damaged DNA.

DNA replication is an essential process that occurs in all growing cells and needs to be tightly regulated in order to preserve genetic integrity. Eukaryotic cells have developed multiple mechanisms to ensure the fidelity of replication and to coordinate the progression of replication forks. Replication is often impeded by DNA damage or replication blocks, and the resulting stalled replication forks are sensed and protected by specialized surveillance mechanisms called checkpoints. The replication checkpoint plays an essential role in preventing the breakdown of stalled replication forks and the accumulation of DNA structures that enhance recombination and chromosomal rearrangements that ultimately lead to genomic instability and cancer development. In addition, the replication checkpoint is thought to assist and coordinate replication fork restart processes by controlling DNA repair pathways, regulating chromatin structure, promoting the recruitment of proteins to sites of damage, and controlling cell cycle progression. In this review we focus mainly on the results obtained in budding yeast to discuss on the multiple roles of checkpoints in maintaining fork integrity and on the enzymatic activities that cooperate with the checkpoint pathway to promote fork resumption and repair of DNA lesions thereby contributing to genome integrity.

Cells are continually exposed to genomic insults resulting from exogenous and endogenous damage as well as by challenges posed by DNA replication. In order to maintain genome integrity, the cells must monitor and coordinate different aspects of chromosome metabolism with cell cycle events that are performed in a predetermined order. Checkpoints are cellular surveillance and signaling pathways that coordinate these physiological responses, and growing evidence suggests that failure of these controls can lead to profound genome instability and genetic disorders. In this review, we focus on the different types of signals and mechanisms that contribute to the budding yeast checkpoint activation, the role of the activated replication checkpoint in stabilizing replication forks and in assisting different types of DNA repair and fork restart mechanisms, as well as on the ability of cells to recover from checkpoint arrest after repairing the lesions or adapt when faced with unrepairable DNA damage.

The replication checkpoint controls the integrity of replicating chromosomes by stabilizing stalled forks, thus preventing the accumulation of abnormal replication and recombination intermediates that contribute to genome instability. Checkpoint-defective cells are susceptible to rearrangements at chromosome fragile sites when replication pauses, and certain human cancer prone diseases suffer checkpoint abnormalities. It is unclear as to how the checkpoint stabilizes stalled forks and how cells sense replication blocks. We have analysed the checkpoint contribution in controlling replisome-fork association when replication pauses. We show that in yeast wild-type cells, stalled forks exhibit stable replisome complexes and the checkpoint sensors Ddc1 and Ddc2, thus activating Rad53 checkpoint kinase. Ddc1/Ddc2 recruitment on stalled forks and Rad53 activation are influenced by the single-strand-binding protein replication factor A (RFA). rad53 forks exhibit a defective association with DNA polymerases alpha, epsilon and delta. Further, in rad53 mutants, stalled forks progressively generate abnormal structures that turn into checkpoint signals by accumulating RFA, Ddc1 and Ddc2. We suggest that, following replication blocks, checkpoint activation mediated by RFA-ssDNA filaments stabilizes stalled forks by controlling replisome-fork association, thus preventing unscheduled recruitment of recombination enzymes that could otherwise cause the pathological processing of the forks.

Cells overcome intra-S DNA damage and replication impediments by coupling chromosome replication to sister chromatid-mediated recombination and replication-bypass processes. Further, molecular junctions between replicated molecules have been suggested to assist sister chromatid cohesion until anaphase. Using two-dimensional gel electrophoresis, we have identified, in yeast cells, replication-dependent X-shaped molecules that appear during origin activation, branch migrate, and distribute along the replicon through a mechanism influenced by the rate of fork progression. Their formation is independent of Rad51- and Rad52-mediated homologous recombination events and is not affected by DNA damage or replication blocks. Further, in hydroxyurea-treated rad53 mutants, altered in the replication checkpoint, the branched molecules progressively degenerate and likely contribute to generate pathological structures. We suggest that cells couple sister chromatid tethering with replication initiation by generating specialized joint molecules resembling hemicatenanes: this process might prime cohesion and assist sister chromatid-mediated recombination and replication events.

Saccharomyces cells with a single unrepaired double-strand break adapt after checkpoint-mediated G(2)/M arrest. We have found that both Rad51 and Rad52 recombination proteins play key roles in adaptation. Cells lacking Rad51p fail to adapt, but deleting RAD52 suppresses rad51Delta. rad52Delta also suppresses adaptation defects of srs2Delta mutants but not those of yku70Delta or tid1Delta mutants. Neither rad54Delta nor rad55Delta affects adaptation. A Rad51 mutant that fails to interact with Rad52p is adaptation defective; conversely, a C-terminal truncation mutant of Rad52p, impaired in interaction with Rad51p, is also adaptation defective. In contrast, rad51-K191A, a mutation that abolishes recombination and results in a protein that does not bind to single-stranded DNA (ssDNA), supports adaptation, as do Rad51 mutants impaired in interaction with Rad54p or Rad55p. An rfa1-t11 mutation in the ssDNA binding complex RPA partially restores adaptation in rad51Delta mutants and fully restores adaptation in yku70Delta and tid1Delta mutants. Surprisingly, although neither rfa1-t11 nor rad52Delta mutants are adaptation defective, the rad52Delta rfa1-t11 double mutant fails to adapt and exhibits the persistent hyperphosphorylation of the DNA damage checkpoint protein Rad53 after HO induction. We suggest that monitoring of the extent of DNA damage depends on independent binding of RPA and Rad52p to ssDNA, with Rad52p's activity modulated by Rad51p whereas RPA's action depends on Tid1p.

The Rad17-Mec3-Ddc1 complex is essential for the cellular response to genotoxic agents and is thought to be important for sensing DNA lesions. Deletion of any of the RAD17, MEC3 or DDC1 genes abolishes the G(1) and G(2) and impairs the intra-S DNA-damage checkpoints. We characterize a dominant-negative mec3-dn mutation that has an unexpected phenotype. It inactivates the G(1) checkpoint while it leaves the G(2) response functional, thus revealing a difference in the requirements of the DNA-damage response in different phases of the cell cycle. In an attempt to identify the molecular defect imparted by the mutation, we dissected step-by-step the signaling cascade, which is triggered by DNA lesions and requires the activity of Mec1 and Rad53 kinases. The analysis of the phosphorylation state of checkpoint factors and critical protein interactions showed that, in mec3-dn cells, the signal transduction cascade is triggered normally, and the central kinase Mec1 can be activated. In G(1) cells expressing the mutation, the signaling cannot proceed any further along the pathway, indicating that the Rad17 complex acts after the activation of Mec1, possibly recruiting targets for the kinase. We also show that the function of the G(2) checkpoint in mutant cells is maintained by an uncharacterized activity of Tel1, the yeast homologue of ATM. This work thus reports a previously undiscovered role for Tel1 in checkpoint control.

In Saccharomyces strains in which homologous recombination is delayed sufficiently to activate the DNA damage checkpoint, Rad53p checkpoint kinase activity appears 1 hr after DSB induction and disappears soon after completion of repair. Cells lacking Srs2p helicase fail to recover even though they apparently complete DNA repair; Rad53p kinase remains activated. srs2Delta cells also fail to adapt when DSB repair is prevented. The recovery defect of srs2Delta is suppressed in mec1Delta strains lacking the checkpoint or when DSB repair occurs before checkpoint activation. Permanent preanaphase arrest of srs2Delta cells is reversed by the addition of caffeine after cells have arrested. Thus, in addition to its roles in recombination, Srs2p appears to be needed to turn off the DNA damage checkpoint.

Checkpoint-mediated control of replicating chromosomes is essential for preventing cancer. In yeast, Rad53 kinase protects stalled replication forks from pathological rearrangements. To characterize the mechanisms controlling fork integrity, we analyzed replication intermediates formed in response to replication blocks using electron microscopy. At the forks, wild-type cells accumulate short single-stranded regions, which likely causes checkpoint activation, whereas rad53 mutants exhibit extensive single-stranded gaps and hemi-replicated intermediates, consistent with a lagging-strand synthesis defect. Further, rad53 cells accumulate Holliday junctions through fork reversal. We speculate that, in checkpoint mutants, abnormal replication intermediates begin to form because of uncoordinated replication and are further processed by unscheduled recombination pathways, causing genome instability.

In response to DNA damage and blocks to replication, eukaryotes activate the checkpoint pathways that prevent genomic instability and cancer by coordinating cell cycle progression with DNA repair. In budding yeast, the checkpoint response requires the Mec1-dependent activation of the Rad53 protein kinase. Active Rad53 slows DNA synthesis when DNA is damaged and prevents firing of late origins of replication. Further, rad53 mutants are unable to recover from a replication block. Mec1 and Rad53 also modulate the phosphorylation state of different DNA replication and repair enzymes. Little is known of the mechanisms by which checkpoint pathways interact with the replication apparatus when DNA is damaged or replication blocked. We used the two-dimensional gel technique to examine replication intermediates in response to hydroxyurea-induced replication blocks. Here we show that hydroxyurea-treated rad53 mutants accumulate unusual DNA structures at replication forks. The persistence of these abnormal molecules during recovery from the hydroxyurea block correlates with the inability to dephosphorylate Rad53. Further, Rad53 is required to properly maintain stable replication forks during the block. We propose that Rad53 prevents collapse of the fork when replication pauses.

The Saccharomyces cerevisiae Rad53 protein kinase is required for the execution of checkpoint arrest at multiple stages of the cell cycle. We found that Rad53 autophosphorylation activity depends on in trans phosphorylation mediated by Mec1 and does not require physical association with other proteins. Uncoupling in trans phosphorylation from autophosphorylation using a rad53 kinase-defective mutant results in a dominant-negative checkpoint defect. Activation of Rad53 in response to DNA damage in G(1) requires the Rad9, Mec3, Ddc1, Rad17 and Rad24 checkpoint factors, while this dependence is greatly reduced in S phase cells. Furthermore, during recovery from checkpoint activation, Rad53 activity decreases through a process that does not require protein synthesis. We also found that Rad53 modulates the lagging strand replication apparatus by controlling phosphorylation of the DNA polymerase alpha-primase complex in response to intra-S DNA damage.

Eukaryotic DNA replication is limited to once per cell cycle because cyclin-dependent kinases (cdks), which are required to fire origins, also prevent re-replication. Components of the replication apparatus, therefore, are 'reset' by cdk inactivation at the end of mitosis. In budding yeast, assembly of Cdc6p-dependent pre-replicative complexes (pre-RCs) at origins can only occur during G1 because it is blocked by cdk1 (Cdc28) together with B cyclins (Clbs). Here we describe a second, separate process which is also blocked by Cdc28/Clb kinase and, therefore, can only occur during G1; the recruitment of DNA polymerase alpha-primase (pol alpha) to chromatin. The recruitment of pol alpha to chromatin during G1 is independent of pre-RC formation since it can occur in the absence of Cdc6 protein. Paradoxically, overproduction of Cdc6p can drive both dephosphorylation and chromatin association of pol alpha. Overproduction of a mutant in which the N-terminus of Cdc6 has been deleted is unable to drive pol alpha chromatin binding. Since this mutant is still competent for pre-RC formation and DNA replication, we suggest that Cdc6p overproduction resets pol alpha chromatin binding by a mechanism which is independent of that used in pre-RC assembly.

The temperature-sensitive yeast DNA primase mutant pri1-M4 fails to execute an early step of DNA replication and exhibits a dominant, allele-specific sensitivity to DNA-damaging agents. pri1-M4 is defective in slowing down the rate of S phase progression and partially delaying the G1-S transition in response to DNA damage. Conversely, the G2 DNA damage response and the S-M checkpoint coupling completion of DNA replication to mitosis are unaffected. The signal transduction pathway leading to Rad53p phosphorylation induced by DNA damage is proficient in pri1-M4, and cell cycle delay caused by Rad53p overexpression is counteracted by the pri1-M4 mutation. Altogether, our results suggest that DNA primase plays an essential role in a subset of the Rad53p-dependent checkpoint pathways controlling cell cycle progression in response to DNA damage.

In this report we study the regulation of premeiotic DNA synthesis in Saccharomyces cerevisiae. DNA replication was monitored by fluorescence-activated cell sorting analysis and by analyzing the pattern of expression of the DNA polymerase alpha-primase complex. Wild-type cells and cells lacking one of the two principal regulators of meiosis, Ime1 and Ime2, were compared. We show that premeiotic DNA synthesis does not occur in ime1 delta diploids, but does occur in ime2 delta diploids with an 8-9 h delay. At late meiotic times, ime2 delta diploids exhibit an additional round of DNA synthesis. Furthermore, we show that in wild-type cells the B-subunit of DNA polymerase alpha is phosphorylated during premeiotic DNA synthesis, a phenomenon that has previously been reported for the mitotic cell cycle. Moreover, the catalytic subunit and the B-subunit of DNA polymerase alpha are specifically degraded during spore formation. Phosphorylation of the B-subunit does not occur in ime1 delta diploids, but does occur in ime2 delta diploids with an 8-9 h delay. In addition, we show that Ime2 is not absolutely required for commitment to meiotic recombination, spindle formation and nuclear division, although it is required for spore formation.

SPK1/RAD53/MEC2/SAD1 of Saccharomyces cerevisiae encodes an essential protein kinase that is required for activation of replication-sensitive and DNA damage-sensitive checkpoint arrest. We have investigated the regulation of phosphorylation and kinase activity of Spk1p during the cell cycle and by conditions that activate checkpoint pathways. Phosphorylation of Spk1p is induced by treatment of cells with agents that damage DNA or interfere with DNA synthesis. Although only S- and G2-phase cdc mutants arrest with hyperphosphorylated Spk1p, damage-induced phosphorylation of Spk1p can occur in G1 and M as well. Hydroxyurea (HU) induces phosphorylation of kinase-defective forms of Spk1p, demonstrating that this regulated phosphorylation of Spk1p occurs in trans. HU-induced phosphorylation is associated with increased catalytic activity of Spk1p. Furthermore, overexpression of wild-type SPK1, but not checkpoint-defective alleles, delays progression through the G1/S boundary. Damage-dependent phosphorylation of Spk1p requires both MEC1 and MEC3, whereas MEC1 but not MEC3, is required for replication block-induced phosphorylation. These data support the model that Spk1p is an essential intermediate component in a signal transduction pathway coupling damage and checkpoint functions to cell cycle arrest. This regulation is mediated through a protein kinase cascade that potentially includes Mec1p and Tel1p as the upstream kinases.