PP2A Controls Genome Integrity by Integrating Nutrient-Sensing and Metabolic Pathways with the DNA Damage Response.

Mec1ATR mediates the DNA damage response (DDR), integrating chromosomal signals and mechanical stimuli. We show that the PP2A phosphatases, ceramide-activated enzymes, couple cell metabolism with the DDR. Using genomic screens, metabolic analysis, and genetic and pharmacological studies, we found that PP2A attenuates the DDR and that three metabolic circuits influence the DDR by modulating PP2A activity. Irc21, a putative cytochrome b5 reductase that promotes the condensation reaction generating dihydroceramides (DHCs), and Ppm1, a PP2A methyltransferase, counteract the DDR by activating PP2A; conversely, the nutrient-sensing TORC1-Tap42 axis sustains DDR activation by inhibiting PP2A. Loss-of-function mutations in IRC21, PPM1, and PP2A and hyperactive tap42 alleles rescue mec1 mutants. Ceramides synergize with rapamycin, a TORC1 inhibitor, in counteracting the DDR. Hence, PP2A integrates nutrient-sensing and metabolic pathways to attenuate the Mec1ATR response. Our observations imply that metabolic changes affect genome integrity and may help with exploiting therapeutic options and repositioning known drugs.

The MRX complex regulates Exo1 resection activity by altering DNA end structure.

Homologous recombination is triggered by nucleolytic degradation (resection) of DNA double-strand breaks (DSBs). DSB resection requires the Mre11-Rad50-Xrs2 (MRX) complex, which promotes the activity of Exo1 nuclease through a poorly understood mechanism. Here, we describe the Mre11-R10T mutant variant that accelerates DSB resection compared to wild-type Mre11 by potentiating Exo1-mediated processing. This increased Exo1 resection activity leads to a decreased association of the Ku complex to DSBs and an enhanced DSB resection in G1, indicating that Exo1 has a direct function in preventing Ku association with DSBs. Molecular dynamics simulations show that rotation of the Mre11 capping domains is able to induce unwinding of double-strand DNA (dsDNA). The R10T substitution causes altered orientation of the Mre11 capping domain that leads to persistent melting of the dsDNA end. We propose that MRX creates a specific DNA end structure that promotes Exo1 resection activity by facilitating the persistence of this nuclease on the DSB ends, uncovering a novel MRX function in DSB resection.

The ATP-bound conformation of the Mre11-Rad50 complex is essential for Tel1/ATM activation.

Activation of the checkpoint protein Tel1 requires the Mre11-Rad50-Xrs2 (MRX) complex, which recruits Tel1 at DNA double-strand breaks (DSBs) through direct interaction between Tel1 and Xrs2. However, in vitro Tel1 activation by MRX requires ATP binding to Rad50, suggesting a role also for the MR subcomplex in Tel1 activation. Here we describe two separation-of-functions alleles, mre11-S499P and rad50-A78T, which we show to specifically affect Tel1 activation without impairing MRX functions in DSB repair. Both Mre11-S499P and Rad50-A78T reduce Tel1-MRX interaction leading to poor Tel1 association at DSBs and consequent loss of Tel1 activation. The Mre11-S499P variant reduces Mre11-Rad50 interaction, suggesting an important role for MR complex formation in Tel1 activation. Molecular dynamics simulations show that the wild type MR subcomplex bound to ATP lingers in a tightly 'closed' conformation, while ADP presence leads to the destabilization of Rad50 dimer and of Mre11-Rad50 association, both events being required for MR conformational transition to an open state. By contrast, MRA78T undertakes complex opening even if Rad50 is bound to ATP, indicating that defective Tel1 activation caused by MRA78T results from destabilization of the ATP-bound conformational state.

Structurally distinct Mre11 domains mediate MRX functions in resection, end-tethering and DNA damage resistance.

Sae2 cooperates with the Mre11-Rad50-Xrs2 (MRX) complex to initiate resection of DNA double-strand breaks (DSBs) and to maintain the DSB ends in close proximity to allow their repair. How these diverse MRX-Sae2 functions contribute to DNA damage resistance is not known. Here, we describe mre11 alleles that suppress the hypersensitivity of sae2Δ cells to genotoxic agents. By assessing the impact of these mutations at the cellular and structural levels, we found that all the mre11 alleles that restore sae2Δ resistance to both camptothecin and phleomycin affect the Mre11 N-terminus and suppress the resection defect of sae2Δ cells by lowering MRX and Tel1 association to DSBs. As a consequence, the diminished Tel1 persistence potentiates Sgs1-Dna2 resection activity by decreasing Rad9 association to DSBs. By contrast, the mre11 mutations restoring sae2Δ resistance only to phleomycin are located in Mre11 C-terminus and bypass Sae2 function in end-tethering but not in DSB resection, possibly by destabilizing the Mre11-Rad50 open conformation. These findings unmask the existence of structurally distinct Mre11 domains that support resistance to genotoxic agents by mediating different processes.

Structure-function relationships of the Mre11 protein in the control of DNA end bridging and processing.

The evolutionarily conserved Mre11-Rad50-Xrs2 (MRX) complex cooperates with the Sae2 protein in initiating resection of DNA double-strand breaks (DSBs) and in maintaining the DSB ends tethered to each other for their accurate repair. How these MRX-Sae2 functions contribute to DNA damage resistance is not understood. By taking advantage of mre11 alleles that suppress the hypersensitivity of sae2∆ cells to genotoxic agents, we have recently found that Mre11 can be divided in two structurally distinct domains that support resistance to genotoxic agents by mediating different processes. While the Mre11 N-terminal domain impacts on the resection activity of long-range resection nucleases by mediating MRX and Tel1/ATM association to DNA DSBs, the C-terminus influences the MRX-tethering activity by its virtue to interact with Rad50. Given the evolutionary conservation of the MRX complex, our results have implications for understanding the consequences of its dysfunctions in human diseases.

Tel1 and Rif2 Regulate MRX Functions in End-Tethering and Repair of DNA Double-Strand Breaks.

The cellular response to DNA double-strand breaks (DSBs) is initiated by the MRX/MRN complex (Mre11-Rad50-Xrs2 in yeast; Mre11-Rad50-Nbs1 in mammals), which recruits the checkpoint kinase Tel1/ATM to DSBs. In Saccharomyces cerevisiae, the role of Tel1 at DSBs remains enigmatic, as tel1Δ cells do not show obvious hypersensitivity to DSB-inducing agents. By performing a synthetic phenotype screen, we isolated a rad50-V1269M allele that sensitizes tel1Δ cells to genotoxic agents. The MRV1269MX complex associates poorly to DNA ends, and its retention at DSBs is further reduced by the lack of Tel1. As a consequence, tel1Δ rad50-V1269M cells are severely defective both in keeping the DSB ends tethered to each other and in repairing a DSB by either homologous recombination (HR) or nonhomologous end joining (NHEJ). These data indicate that Tel1 promotes MRX retention to DSBs and this function is important to allow proper MRX-DNA binding that is needed for end-tethering and DSB repair. The role of Tel1 in promoting MRX accumulation to DSBs is counteracted by Rif2, which is recruited to DSBs. We also found that Rif2 enhances ATP hydrolysis by MRX and attenuates MRX function in end-tethering, suggesting that Rif2 can regulate MRX activity at DSBs by modulating ATP-dependent conformational changes of Rad50.

Regulation of telomere metabolism by the RNA processing protein Xrn1.

Telomeric DNA consists of repetitive G-rich sequences that terminate with a 3΄-ended single stranded overhang (G-tail), which is important for telomere extension by telomerase. Several proteins, including the CST complex, are necessary to maintain telomere structure and length in both yeast and mammals. Emerging evidence indicates that RNA processing factors play critical, yet poorly understood, roles in telomere metabolism. Here, we show that the lack of the RNA processing proteins Xrn1 or Rrp6 partially bypasses the requirement for the CST component Cdc13 in telomere protection by attenuating the activation of the DNA damage checkpoint. Xrn1 is necessary for checkpoint activation upon telomere uncapping because it promotes the generation of single-stranded DNA. Moreover, Xrn1 maintains telomere length by promoting the association of Cdc13 to telomeres independently of ssDNA generation and exerts this function by downregulating the transcript encoding the telomerase inhibitor Rif1. These findings reveal novel roles for RNA processing proteins in the regulation of telomere metabolism with implications for genome stability in eukaryotes.

Escape of Sgs1 from Rad9 inhibition reduces the requirement for Sae2 and functional MRX in DNA end resection.

Homologous recombination requires nucleolytic degradation (resection) of DNA double-strand break (DSB) ends. In Saccharomyces cerevisiae, the MRX complex and Sae2 are involved in the onset of DSB resection, whereas extensive resection requires Exo1 and the concerted action of Dna2 and Sgs1. Here, we show that the checkpoint protein Rad9 limits the action of Sgs1/Dna2 in DSB resection by inhibiting Sgs1 binding/persistence at the DSB ends. When inhibition by Rad9 is abolished by the Sgs1-ss mutant variant or by deletion of RAD9, the requirement for Sae2 and functional MRX in DSB resection is reduced. These results provide new insights into how early and long-range resection is coordinated.

Coupling end resection with the checkpoint response at DNA double-strand breaks.

DNA double-strand breaks (DSBs) are a nasty form of damage that needs to be repaired to ensure genome stability. The DSB ends can undergo a strand-biased nucleolytic processing (resection) to generate 3'-ended single-stranded DNA (ssDNA) that channels DSB repair into homologous recombination. Generation of ssDNA also triggers the activation of the DNA damage checkpoint, which couples cell cycle progression with DSB repair. The checkpoint response is intimately linked to DSB resection, as some checkpoint proteins regulate the resection process. The present review will highlight recent works on the mechanism and regulation of DSB resection and its interplays with checkpoint activation/inactivation in budding yeast.

Budding yeast Swe1 is involved in the control of mitotic spindle elongation and is regulated by Cdc14 phosphatase during mitosis.

Cyclin-dependent kinase (Cdk1) activity is required for mitotic entry, and this event is restrained by an inhibitory phosphorylation of the catalytic subunit Cdc28 on a conserved tyrosine (Tyr(19)). This modification is brought about by the protein kinase Swe1 that inhibits Cdk1 activation thus blocking mitotic entry. Swe1 levels are regulated during the cell cycle, and they decrease during G2/M concomitantly to Cdk1 activation, which drives entry into mitosis. However, after mitotic entry, a pool of Swe1 persists, and we collected evidence that it is involved in controlling mitotic spindle elongation. We also describe that the protein phosphatase Cdc14 is implicated in Swe1 regulation; in fact, we observed that Swe1 dephosphorylation in vivo depends on Cdc14 that, in turn, is able to control its subcellular localization. In addition we show that the lack of Swe1 causes premature mitotic spindle elongation and that high levels of Swe1 block mitotic spindle elongation, indicating that Swe1 inhibits this process. Importantly, these effects are not dependent upon the role of in Cdk1 inhibition. These data fit into a model in which Cdc14 binds and inhibits Swe1 to allow timely mitotic spindle elongation.

Sae2 and Rif2 regulate MRX endonuclease activity at DNA double-strand breaks in opposite manners.

The Mre11-Rad50-Xrs2 (MRX) complex detects and processes DNA double-strand breaks (DSBs). Its DNA binding and processing activities are regulated by transitions between an ATP-bound state and a post-hydrolysis cutting state that is nucleolytically active. Mre11 endonuclease activity is stimulated by Sae2, whose lack increases MRX persistence at DSBs and checkpoint activation. Here we show that the Rif2 protein inhibits Mre11 endonuclease activity and is responsible for the increased MRX retention at DSBs in sae2Δ cells. We identify a Rad50 residue that is important for Rad50-Rif2 interaction and Rif2 inhibition of Mre11 nuclease. This residue is located near a Rad50 surface that binds Sae2 and is important in stabilizing the Mre11-Rad50 (MR) interaction in the cutting state. We propose that Sae2 stimulates Mre11 endonuclease activity by stabilizing a post-hydrolysis MR conformation that is competent for DNA cleavage, whereas Rif2 antagonizes this Sae2 function and stabilizes an endonuclease inactive MR conformation.

Functions and regulation of the MRX complex at DNA double-strand breaks.

DNA double-strand breaks (DSBs) pose a serious threat to genome stability and cell survival. Cells possess mechanisms that recognize DSBs and promote their repair through either homologous recombination (HR) or non-homologous end joining (NHEJ). The evolutionarily conserved Mre11-Rad50-Xrs2 (MRX) complex plays a central role in the cellular response to DSBs, as it is implicated in controlling end resection and in maintaining the DSB ends tethered to each other. Furthermore, it is responsible for DSB signaling by activating the checkpoint kinase Tel1 that, in turn, supports MRX function in a positive feedback loop. The present review focuses mainly on recent works in the budding yeast Saccharomyces cerevisiae to highlight structure and regulation of MRX as well as its interplays with Tel1.

Vhs2 is a novel regulator of septin dynamics in budding yeast.

In budding yeast, septins are assembled into structures that undergo dramatic changes during the cell cycle. The molecular mechanisms that drive these remodelings are not fully uncovered. In this study, we describe a characterization of Vhs2, a nonessential protein that revealed to be a new player in septin dynamics. In particular, we report that Vhs2 is important to maintain the stability of the double septin ring structure until telophase. In addition, we show that Vhs2 undergoes multiple phosphorylations during the cell cycle, being phosphorylated during S phase until nuclear division and dephosphorylated just before cell division. Importantly we report that cyclin-dependent protein kinase Cdk1 and protein phosphatase Cdc14 control these Vhs2 post-translational modifications. These results reveal that Vhs2 is a novel Cdc14 substrate that is involved in the control of septin organization.

Telomere uncapping at the crossroad between cell cycle arrest and carcinogenesis.

Telomeres are nucleoprotein complexes that protect the natural ends of chromosomes from fusion and degradation and prevent them eliciting a checkpoint response. This protective function, which is called telomere capping, is largely mediated by telomere-binding proteins that suppress checkpoint activation and DNA repair activities. Telomere dysfunction through progressive shortening or removal of capping proteins leads to a checkpoint-mediated block of cell proliferation, which acts as a cancer-suppressor mechanism. However, genetic alterations that inactivate the checkpoint can lead to further telomere erosion and increased genomic instability that, coupled with the activation of mechanisms to restabilize telomeres, can drive the oncogenic process.

Saccharomyces cerevisiae Dma proteins participate in cytokinesis by controlling two different pathways.

Cytokinesis completion in the budding yeast S. cerevisiae is driven by tightly regulated pathways, leading to actomyosin ring contraction coupled to plasma membrane constriction and to centripetal growth of the primary septum, respectively. These pathways can partially substitute for each other, but their concomitant inactivation leads to cytokinesis block and cell death. Here we show that both the lack of the functionally redundant FHA-RING ubiquitin ligases Dma1 and Dma2 and moderate Dma2 overproduction affect actomyosin ring contraction as well as primary septum deposition, although they do not apparently alter cell cycle progression of otherwise wild-type cells. In addition, overproduction of Dma2 impairs the interaction between Tem1 and Iqg1, which is thought to be required for AMR contraction, and causes asymmetric primary septum deposition as well as mislocalization of the Cyk3-positive regulator of this process. In agreement with these multiple inhibitory effects, a Dma2 excess that does not cause any apparent defect in wild-type cells leads to lethal cytokinesis block in cells lacking the Hof1 protein, which is essential for primary septum formation in the absence of Cyk3. Altogether, these findings suggest that the Dma proteins act as negative regulators of cytokinesis.

Budding yeast Dma1 and Dma2 participate in regulation of Swe1 levels and localization.

Timely down-regulation of the evolutionarily conserved protein kinase Swe1 plays an important role in cell cycle control, as Swe1 can block nuclear division through inhibitory phosphorylation of the catalytic subunit of cyclin-dependent kinase. In particular, Swe1 degradation is important for budding yeast cell survival in case of DNA replication stress, whereas it is inhibited by the morphogenesis checkpoint in response to alterations in actin cytoskeleton or septin structure. We show that the lack of the Dma1 and Dma2 ubiquitin ligases, which moderately affects Swe1 localization and degradation during an unperturbed cell cycle with no apparent phenotypic effects, is toxic for cells that are partially defective in Swe1 down-regulation. Moreover, Swe1 is stabilized, restrained at the bud neck, and hyperphosphorylated in dma1Δ dma2Δ cells subjected to DNA replication stress, indicating that the mechanism stabilizing Swe1 under these conditions is different from the one triggered by the morphogenesis checkpoint. Finally, the Dma proteins are required for proper Swe1 ubiquitylation. Taken together, the data highlight a previously unknown role of these proteins in the complex regulation of Swe1 and suggest that they might contribute to control, directly or indirectly, Swe1 ubiquitylation.