Cancer genome datamining and functional genetic analysis implicate mechanisms of ATM/ATR dysfunction underpinning carcinogenesis.

ATM and ATR are conserved regulators of the DNA damage response linked to cancer. Comprehensive DNA sequencing efforts identified ~4,000 cancer-associated mutations in ATM/ATR; however, their cancer implications remain largely unknown. To gain insights, we identify functionally important conserved residues in ATM, ATR and budding yeast Mec1ATR via cancer genome datamining and a functional genetic analysis, respectively. Surprisingly, only a small fraction of the critical residues is in the active site of the respective enzyme complexes, implying that loss of the intrinsic kinase activity is infrequent in carcinogenesis. A number of residues are solvent accessible, suggestive of their involvement in interacting with a protein-partner(s). The majority, buried inside the respective enzyme complexes, might play a structural or regulatory role. Together, these findings identify evolutionarily conserved ATM, ATR, and Mec1ATR residues involved in diverse aspects of the enzyme function and provide fresh insights into the elusive genotype-phenotype relationships in ATM/ATR and their cancer-associated variants.

Functional link between mitochondria and Rnr3, the minor catalytic subunit of yeast ribonucleotide reductase.

Ribonucleotide reductase (RNR) is an essential holoenzyme required for de novo synthesis of dNTPs. The Saccharomyces cerevisiae genome encodes for two catalytic subunits, Rnr1 and Rnr3. While Rnr1 is required for DNA replication and DNA damage repair, the function(s) of Rnr3 is unknown. Here, we show that carbon source, an essential nutrient, impacts Rnr1 and Rnr3 abundance: Non-fermentable carbon sources or limiting concentrations of glucose down regulate Rnr1 and induce Rnr3 expression. Oppositely, abundant glucose induces Rnr1 expression and down regulates Rnr3. The carbon source dependent regulation of Rnr3 is mediated by Mec1, the budding yeast ATM/ATR checkpoint response kinase. Unexpectedly, this regulation is independent of all currently known components of the Mec1 DNA damage response network, including Rad53, Dun1, and Tel1, implicating a novel Mec1 signalling axis. rnr3Δ leads to growth defects under respiratory conditions and rescues temperature sensitivity conferred by the absence of Tom6, a component of the mitochondrial TOM (translocase of outer membrane) complex responsible for mitochondrial protein import. Together, these results unveil involvement of Rnr3 in mitochondrial functions and Mec1 in mediating the carbon source dependent regulation of Rnr3.

Versatility of the Mec1ATM/ATR signaling network in mediating resistance to replication, genotoxic, and proteotoxic stresses.

The ataxia-telangiectasia mutated/ATM and Rad3-related (ATM/ATR) family proteins are evolutionarily conserved serine/threonine kinases best known for their roles in mediating the DNA damage response. Upon activation, ATM/ATR phosphorylate numerous targets to stabilize stalled replication forks, repair damaged DNA, and inhibit cell cycle progression to ensure survival of the cell and safeguard integrity of the genome. Intriguingly, separation of function alleles of the human ATM and MEC1, the budding yeast ATM/ATR, were shown to confer widespread protein aggregation and acute sensitivity to different types of proteotoxic agents including heavy metal, amino acid analogue, and an aggregation-prone peptide derived from the Huntington's disease protein. Further analyses unveiled that ATM and Mec1 promote resistance to perturbation in protein homeostasis via a mechanism distinct from the DNA damage response. In this minireview, we summarize the key findings and discuss ATM/ATR as a multifaceted signalling protein capable of mediating cellular response to both DNA and protein damage.

Essential Function of Mec1, the Budding Yeast ATM/ATR Checkpoint-Response Kinase, in Protein Homeostasis.

Unlike most checkpoint proteins, Mec1, an ATM/ATR kinase, is essential. We utilized mec1-4, a missense allele (E2130K) that confers diminished kinase activity, to interrogate the question. Unbiased screen for genetic interactors of mec1-4 identified numerous genes involved in proteostasis. mec1-4 confers sensitivity to heat, an amino acid analog, and Htt103Q, an aggregation-prone model peptide of Huntingtin. Oppositely, mec1-4 confers resistance to cycloheximide, a translation inhibitor. In response to heat, mec1-4 leads to widespread protein aggregation and cell death. Key components of the Mec1 signaling network, Rad53CHK1, Dun1, and Sml1, also impact survival in response to proteotoxic stress. Activation of autophagy or sml1Δ promotes aggregate resolution and rescues mec1-4 lethality. These findings show that proteostasis is a fundamental function of Mec1 and that Mec1 is likely to utilize its checkpoint response network to mediate resistance to proteotoxic stress, a role that may be conserved from yeast to mammalian cells.

S phase block following MEC1ATR inactivation occurs without severe dNTP depletion.

Inactivation of Mec1, the budding yeast ATR, results in a permanent S phase arrest followed by chromosome breakage and cell death during G2/M. The S phase arrest is proposed to stem from a defect in Mec1-mediated degradation of Sml1, a conserved inhibitor of ribonucleotide reductase (RNR), causing a severe depletion in cellular dNTP pools. Here, the casual link between the S phase arrest, Sml1, and dNTP-levels is examined using a temperature sensitive mec1 mutant. In addition to S phase arrest, thermal inactivation of Mec1 leads to constitutively high levels of Sml1 and an S phase arrest. Expression of a novel suppressor, GIS2, a conserved mRNA binding zinc finger protein, rescues the arrest without down-regulating Sml1 levels. The dNTP pool in mec1 is reduced by ∼17% and GIS2 expression restores it, but only partially, to ∼93% of a control. We infer that the permanent S phase block following Mec1 inactivation can be uncoupled from its role in Sml1 down-regulation. Furthermore, unexpectedly modest effects of mec1 and GIS2 on dNTP levels suggest that the S phase arrest is unlikely to result from a severe depletion of dNTP pool as assumed, but a heightened sensitivity to small changes in its availability.

Analysis of Yeast Sporulation Efficiency, Spore Viability, and Meiotic Recombination on Solid Medium.

Under conditions of nutrient deprivation, yeast cells initiate a differentiation program in which meiosis is induced and spores are formed. During meiosis, one round of genome duplication is followed by two rounds of chromosome segregation (meiosis I and meiosis II) to generate four haploid nuclei. Meiotic recombination occurs during prophase I. During sporogenesis, each nucleus becomes surrounded by an individual spore wall, and all four haploid spores become contained as a tetrad within an ascus. Important insights into the meiotic function(s) of a gene of interest can be gained by observing the effects of gene mutations on spore viability and viability patterns among tetrads. Moreover, recombination frequencies among viable spores can reveal potential involvement of the gene during meiotic exchange between homologous chromosomes. Here, we describe methods for inducing spore formation on solid medium, determining spore viability, and measuring, via tetrad analysis, frequencies of crossing over and gene conversion as indicators of meiotic chromosome exchange.

Analysis of Recombination and Chromosome Structure during Yeast Meiosis.

Meiosis is a diploid-specific differentiation program that consists of a single round of genome duplication followed by two rounds of chromosome segregation. These events result in halving of the genetic complement, which is a requirement for formation of haploid reproductive cells (i.e., spores in yeast and gametes in animals and plants). During meiosis I, homologous maternal and paternal chromosomes (homologs) pair and separate, whereas sister chromatids remain connected at the centromeres and separate during the second meiotic division. In most organisms, accurate homolog disjunction requires crossovers, which are formed as products of meiotic recombination. For the past two decades, studies of yeast meiosis have provided invaluable insights into evolutionarily conserved mechanisms of meiosis.

Induction and Analysis of Synchronous Meiotic Yeast Cultures.

Meiosis in Saccharomyces cerevisiae can be induced by deprivation of nutrients. Here, we present a protocol for inducing synchronous meiosis in SK1, the most efficient and synchronous yeast strain for meiosis, by exposing SK1 cells to liquid medium that contains potassium acetate as a nonfermentable carbon source and lacks nitrogen. These synchronous meiotic yeast cultures can be subjected to a range of molecular and cytological analyses, making them useful for investigating the genetic and molecular determinants of meiosis.

Analysis of Meiotic Recombination and Homolog Interaction during Yeast Meiosis.

During meiosis, one round of genome duplication is followed by two rounds of chromosome segregation, resulting in the halving of the genetic complement and the formation of haploid reproductive cells. In most organisms, intimate juxtaposition of homologous chromosomes and homologous recombination during meiotic prophase are required for meiotic success. Here we present a general protocol for visualizing chromosomal proteins and homolog interaction on surface-spread nuclei and a widely used protocol for analyzing meiotic recombination based on an engineered hotspot referred to as HIS4::LEU2.

Essential and Checkpoint Functions of Budding Yeast ATM and ATR during Meiotic Prophase Are Facilitated by Differential Phosphorylation of a Meiotic Adaptor Protein, Hop1.

A hallmark of the conserved ATM/ATR signalling is its ability to mediate a wide range of functions utilizing only a limited number of adaptors and effector kinases. During meiosis, Tel1 and Mec1, the budding yeast ATM and ATR, respectively, rely on a meiotic adaptor protein Hop1, a 53BP1/Rad9 functional analog, and its associated kinase Mek1, a CHK2/Rad53-paralog, to mediate multiple functions: control of the formation and repair of programmed meiotic DNA double strand breaks, enforcement of inter-homolog bias, regulation of meiotic progression, and implementation of checkpoint responses. Here, we present evidence that the multi-functionality of the Tel1/Mec1-to-Hop1/Mek1 signalling depends on stepwise activation of Mek1 that is mediated by Tel1/Mec1 phosphorylation of two specific residues within Hop1: phosphorylation at the threonine 318 (T318) ensures the transient basal level Mek1 activation required for viable spore formation during unperturbed meiosis. Phosphorylation at the serine 298 (S298) promotes stable Hop1-Mek1 interaction on chromosomes following the initial phospho-T318 mediated Mek1 recruitment. In the absence of Dmc1, the phospho-S298 also promotes Mek1 hyper-activation necessary for implementing meiotic checkpoint arrest. Taking these observations together, we propose that the Hop1 phospho-T318 and phospho-S298 constitute key components of the Tel1/Mec1- based meiotic recombination surveillance (MRS) network and facilitate effective coupling of meiotic recombination and progression during both unperturbed and challenged meiosis.

Recombinogenic conditions influence partner choice in spontaneous mitotic recombination.

Mammalian common fragile sites are loci of frequent chromosome breakage and putative recombination hotspots. Here, we utilized Replication Slow Zones (RSZs), a budding yeast homolog of the mammalian common fragile sites, to examine recombination activities at these loci. We found that rates of URA3 inactivation of a hisG-URA3-hisG reporter at RSZ and non-RSZ loci were comparable under all conditions tested, including those that specifically promote chromosome breakage at RSZs (hydroxyurea [HU], mec1Δ sml1Δ, and high temperature), and those that suppress it (sml1Δ and rrm3Δ). These observations indicate that RSZs are not recombination hotspots and that chromosome fragility and recombination activity can be uncoupled. Results confirmed recombinogenic effects of HU, mec1Δ sml1Δ, and rrm3Δ and identified temperature as a regulator of mitotic recombination. We also found that these conditions altered the nature of recombination outcomes, leading to a significant increase in the frequency of URA3 inactivation via loss of heterozygosity (LOH), the type of genetic alteration involved in cancer development. Further analyses revealed that the increase was likely due to down regulation of intrachromatid and intersister (IC/IS) bias in mitotic recombination, and that RSZs exhibited greater sensitivity to HU dependent loss of IC/IS bias than non RSZ loci. These observations suggest that recombinogenic conditions contribute to genome rearrangements not only by increasing the overall recombination activity, but also by altering the nature of recombination outcomes by their effects on recombination partner choice. Similarly, fragile sites may contribute to cancer more frequently than non-fragile loci due their enhanced sensitivity to certain conditions that down-regulate the IC/IS bias rather than intrinsically higher rates of recombination.

Global linkage map connects meiotic centromere function to chromosome size in budding yeast.

Synthetic genetic array (SGA) analysis automates yeast genetics, enabling high-throughput construction of ordered arrays of double mutants. Quantitative colony sizes derived from SGA analysis can be used to measure cellular fitness and score for genetic interactions, such as synthetic lethality. Here we show that SGA colony sizes also can be used to obtain global maps of meiotic recombination because recombination frequency affects double-mutant formation for gene pairs located on the same chromosome and therefore influences the size of the resultant double-mutant colony. We obtained quantitative colony size data for ~1.2 million double mutants located on the same chromosome and constructed a genome-scale genetic linkage map at ~5 kb resolution. We found that our linkage map is reproducible and consistent with previous global studies of meiotic recombination. In particular, we confirmed that the total number of crossovers per chromosome tends to follow a simple linear model that depends on chromosome size. In addition, we observed a previously unappreciated relationship between the size of linkage regions surrounding each centromere and chromosome size, suggesting that crossovers tend to occur farther away from the centromere on larger chromosomes. The pericentric regions of larger chromosomes also appeared to load larger clusters of meiotic cohesin Rec8, and acquire fewer Spo11-catalyzed DNA double-strand breaks. Given that crossovers too near or too far from centromeres are detrimental to homolog disjunction and increase the incidence of aneuploidy, our data suggest that chromosome size may have a direct role in regulating the fidelity of chromosome segregation during meiosis.

Budding yeast ATM/ATR control meiotic double-strand break (DSB) levels by down-regulating Rec114, an essential component of the DSB-machinery.

An essential feature of meiosis is Spo11 catalysis of programmed DNA double strand breaks (DSBs). Evidence suggests that the number of DSBs generated per meiosis is genetically determined and that this ability to maintain a pre-determined DSB level, or "DSB homeostasis", might be a property of the meiotic program. Here, we present direct evidence that Rec114, an evolutionarily conserved essential component of the meiotic DSB-machinery, interacts with DSB hotspot DNA, and that Tel1 and Mec1, the budding yeast ATM and ATR, respectively, down-regulate Rec114 upon meiotic DSB formation through phosphorylation. Mimicking constitutive phosphorylation reduces the interaction between Rec114 and DSB hotspot DNA, resulting in a reduction and/or delay in DSB formation. Conversely, a non-phosphorylatable rec114 allele confers a genome-wide increase in both DSB levels and in the interaction between Rec114 and the DSB hotspot DNA. These observations strongly suggest that Tel1 and/or Mec1 phosphorylation of Rec114 following Spo11 catalysis down-regulates DSB formation by limiting the interaction between Rec114 and DSB hotspots. We also present evidence that Ndt80, a meiosis specific transcription factor, contributes to Rec114 degradation, consistent with its requirement for complete cessation of DSB formation. Loss of Rec114 foci from chromatin is associated with homolog synapsis but independent of Ndt80 or Tel1/Mec1 phosphorylation. Taken together, we present evidence for three independent ways of regulating Rec114 activity, which likely contribute to meiotic DSBs-homeostasis in maintaining genetically determined levels of breaks.

Topoisomerase II- and condensin-dependent breakage of MEC1ATR-sensitive fragile sites occurs independently of spindle tension, anaphase, or cytokinesis.

Fragile sites are loci of recurrent chromosome breakage in the genome. They are found in organisms ranging from bacteria to humans and are implicated in genome instability, evolution, and cancer. In budding yeast, inactivation of Mec1, a homolog of mammalian ATR, leads to chromosome breakage at fragile sites referred to as replication slow zones (RSZs). RSZs are proposed to be homologous to mammalian common fragile sites (CFSs) whose stability is regulated by ATR. Perturbation during S phase, leading to elevated levels of stalled replication forks, is necessary but not sufficient for chromosome breakage at RSZs or CFSs. To address the nature of additional event(s) required for the break formation, we examined involvement of the currently known or implicated mechanisms of endogenous chromosome breakage, including errors in replication fork restart, premature mitotic chromosome condensation, spindle tension, anaphase, and cytokinesis. Results revealed that chromosome breakage at RSZs is independent of the RAD52 epistasis group genes and of TOP3, SGS1, SRS2, MMS4, or MUS81, indicating that homologous recombination and other recombination-related processes associated with replication fork restart are unlikely to be involved. We also found spindle force, anaphase, or cytokinesis to be dispensable. RSZ breakage, however, required genes encoding condensin subunits (YCG1, YSC4) and topoisomerase II (TOP2). We propose that chromosome break formation at RSZs following Mec1 inactivation, a model for mammalian fragile site breakage, is mediated by internal chromosomal stress generated during mitotic chromosome condensation.

Regulation of fragile sites expression in budding yeast by MEC1, RRM3 and hydroxyurea.

Fragile sites are specific loci within the genome that exhibit increased tendencies for chromosome breakage. They are conserved among mammals and are also found in lower eukaryotes including yeast and fly. Many conditions, including mutations and exogenous factors, contribute to fragile site expression, but the nature of interaction among them remains elusive. Here, we investigated this by examining the combined effects of rrm3Δ, mec1 and hydroxyurea (HU), three conditions that induce fragile sites, on expression of the replication slow zone (RSZ), a type of fragile site in budding yeast. Contrary to the expectation that each factor would contribute to fragile site expression in an independent manner, we show that rrm3Δ and high concentrations of HU suppressed RSZ expression in mec1-4ts cells. Further analyses revealed that rrm3Δ suppression occurs via promotion of Sml1 degradation, whereas HU suppresses RSZ via a premature commitment to inviability. Taken together, these observations demonstrate that: (1) the yeast genome contains different types of fragile site with regard to regulation of their expression, and (2) each fragile-site-inducing condition does not act independently, but can elicit a cellular response(s) that can paradoxically prevent the expression of a specific type(s) of fragile sites.

Phosphorylation of the axial element protein Hop1 by Mec1/Tel1 ensures meiotic interhomolog recombination.

An essential feature of meiosis is interhomolog recombination whereby a significant fraction of the programmed meiotic double-strand breaks (DSBs) is repaired using an intact homologous non-sister chromatid rather than a sister. Involvement of Mec1 and Tel1, the budding yeast homologs of the mammalian ATR and ATM kinases, in meiotic interhomlog bias has been implicated, but the mechanism remains elusive. Here, we demonstrate that Mec1 and Tel1 promote meiotic interhomolog recombination by targeting the axial element protein Hop1. Without Mec1/Tel1 phosphorylation of Hop1, meiotic DSBs are rapidly repaired via a Dmc1-independent intersister repair pathway, resulting in diminished interhomolog crossing-over leading to spore lethality. We find that Mec1/Tel1-mediated phosphorylation of Hop1 is required for activation of Mek1, a meiotic paralogue of the DNA-damage effector kinase, Rad53p/CHK2. Thus, Hop1 is a meiosis-specific adaptor protein of the Mec1/Tel1 signaling pathway that ensures interhomolog recombination by preventing Dmc1-independent repair of meiotic DSBs.

Meiotic roles of Mec1, a budding yeast homolog of mammalian ATR/ATM.

Budding yeast Mec1, a homolog of mammalian ATR/ATM, is an essential chromosome-based signal transduction protein. Mec1 is a key checkpoint regulator and plays a critical role in the maintenance of genome stability. Mec1 is also required for meiosis; loss of Mec1 functions leads to a number of meiotic defects including reduction in recombination, loss of inter-homolog bias, loss of crossover control, and failure in meiotic progression. Here we review currently available data on meiotic defects associated with loss of Mec1 functions and discuss the possibility that Mec1 may participate as a fundamentally positive player in coordinating and promoting basic meiotic chromosomal processes during normal meiosis.

Transcription of ribosomal genes can cause nondisjunction.

Mitotic disjunction of the repetitive ribosomal DNA (rDNA) involves specialized segregation mechanisms dependent on the conserved phosphatase Cdc14. The reason behind this requirement is unknown. We show that rDNA segregation requires Cdc14 partly because of its physical length but most importantly because a fraction of ribosomal RNA (rRNA) genes are transcribed at very high rates. We show that cells cannot segregate rDNA without Cdc14 unless they undergo genetic rearrangements that reduce rDNA copy number. We then demonstrate that cells with normal length rDNA arrays can segregate rDNA in the absence of Cdc14 as long as rRNA genes are not transcribed. In addition, our study uncovers an unexpected role for the replication barrier protein Fob1 in rDNA segregation that is independent of Cdc14. These findings demonstrate that highly transcribed loci can cause chromosome nondisjunction.

ATR homolog Mec1 promotes fork progression, thus averting breaks in replication slow zones.

Budding yeast Mec1, homolog of mammalian ATR, is an essential protein that mediates S-phase checkpoint responses and meiotic recombination. Elimination of Mec1 function leads to genomewide fork stalling followed by chromosome breakage. Breaks do not result from stochastic collapse of stalled forks or other incidental lesions; instead, they occur in specific regions of the genome during a G2 chromosomal transition. Break regions are found to be genetically encoded replication slow zones (RSZs), a newly discovered yeast chromosomal determinant. Thus, Mec1 has important functions in normal S phase and the genome instability of mec1 (and, analogously, ATR-/-) mutants stems from defects in these basic roles.