Aberrant DNA repair reveals a vulnerability in histone H3.3-mutant brain tumors.

Pediatric high-grade gliomas (pHGG) are devastating and incurable brain tumors with recurrent mutations in histone H3.3. These mutations promote oncogenesis by dysregulating gene expression through alterations of histone modifications. We identify aberrant DNA repair as an independent mechanism, which fosters genome instability in H3.3 mutant pHGG, and opens new therapeutic options. The two most frequent H3.3 mutations in pHGG, K27M and G34R, drive aberrant repair of replication-associated damage by non-homologous end joining (NHEJ). Aberrant NHEJ is mediated by the DNA repair enzyme polynucleotide kinase 3'-phosphatase (PNKP), which shows increased association with mutant H3.3 at damaged replication forks. PNKP sustains the proliferation of cells bearing H3.3 mutations, thus conferring a molecular vulnerability, specific to mutant cells, with potential for therapeutic targeting.

Rad5 dysregulation drives hyperactive recombination at replication forks resulting in cisplatin sensitivity and genome instability.

The postreplication repair gene, HLTF, is often amplified and overexpressed in cancer. Here we model HLTF dysregulation through the functionally conserved Saccharomyces cerevisiae ortholog, RAD5. Genetic interaction profiling and landscape enrichment analysis of RAD5 overexpression (RAD5OE) reveals requirements for genes involved in recombination, crossover resolution, and DNA replication. While RAD5OE and rad5Δ both cause cisplatin sensitivity and share many genetic interactions, RAD5OE specifically requires crossover resolving genes and drives recombination in a region of repetitive DNA. Remarkably, RAD5OE induced recombination does not require other post-replication repair pathway members, or the PCNA modification sites involved in regulation of this pathway. Instead, the RAD5OE phenotype depends on a conserved domain necessary for binding 3' DNA ends. Analysis of DNA replication intermediates supports a model in which dysregulated Rad5 causes aberrant template switching at replication forks. The direct effect of Rad5 on replication forks in vivo, increased recombination, and cisplatin sensitivity predicts similar consequences for dysregulated HLTF in cancer.

A role for the unfolded protein response stress sensor ERN1 in regulating the response to MEK inhibitors in KRAS mutant colon cancers.

BACKGROUND: Mutations in KRAS are frequent in human cancer, yet effective targeted therapeutics for these cancers are still lacking. Attempts to drug the MEK kinases downstream of KRAS have had limited success in clinical trials. Understanding the specific genomic vulnerabilities of KRAS-driven cancers may uncover novel patient-tailored treatment options. METHODS: We first searched for synthetic lethal (SL) genetic interactions with mutant RAS in yeast with the ultimate aim to identify novel cancer-specific targets for therapy. Our method used selective ploidy ablation, which enables replication of cancer-specific gene expression changes in the yeast gene disruption library. Second, we used a genome-wide CRISPR/Cas9-based genetic screen in KRAS mutant human colon cancer cells to understand the mechanistic connection between the synthetic lethal interaction discovered in yeast and downstream RAS signaling in human cells. RESULTS: We identify loss of the endoplasmic reticulum (ER) stress sensor IRE1 as synthetic lethal with activated RAS mutants in yeast. In KRAS mutant colorectal cancer cell lines, genetic ablation of the human ortholog of IRE1, ERN1, does not affect growth but sensitizes to MEK inhibition. However, an ERN1 kinase inhibitor failed to show synergy with MEK inhibition, suggesting that a non-kinase function of ERN1 confers MEK inhibitor resistance. To investigate how ERN1 modulates MEK inhibitor responses, we performed genetic screens in ERN1 knockout KRAS mutant colon cancer cells to identify genes whose inactivation confers resistance to MEK inhibition. This genetic screen identified multiple negative regulators of JUN N-terminal kinase (JNK) /JUN signaling. Consistently, compounds targeting JNK/MAPK8 or TAK1/MAP3K7, which relay signals from ERN1 to JUN, display synergy with MEK inhibition. CONCLUSIONS: We identify the ERN1-JNK-JUN pathway as a novel regulator of MEK inhibitor response in KRAS mutant colon cancer. The notion that multiple signaling pathways can activate JUN may explain why KRAS mutant tumor cells are traditionally seen as highly refractory to MEK inhibitor therapy. Our findings emphasize the need for the development of new therapeutics targeting JUN activating kinases, TAK1 and JNK, to sensitize KRAS mutant cancer cells to MEK inhibitors.

A Synthetic Dosage Lethal Genetic Interaction Between CKS1B and PLK1 Is Conserved in Yeast and Human Cancer Cells.

The CKS1B gene located on chromosome 1q21 is frequently amplified in breast, lung, and liver cancers. CKS1B codes for a conserved regulatory subunit of cyclin-CDK complexes that function at multiple stages of cell cycle progression. We used a high throughput screening protocol to mimic cancer-related overexpression in a library of Saccharomyces cerevisiae mutants to identify genes whose functions become essential only when CKS1 is overexpressed, a synthetic dosage lethal (SDL) interaction. Mutations in multiple genes affecting mitotic entry and mitotic exit are highly enriched in the set of SDL interactions. The interactions between Cks1 and the mitotic entry checkpoint genes require the inhibitory activity of Swe1 on the yeast cyclin-dependent kinase (CDK), Cdc28. In addition, the SDL interactions of overexpressed CKS1 with mutations in the mitotic exit network are suppressed by modulating expression of the CDK inhibitor Sic1. Mutation of the polo-like kinase Cdc5, which functions in both the mitotic entry and mitotic exit pathways, is lethal in combination with overexpressed CKS1 Therefore we investigated the effect of targeting the human Cdc5 ortholog, PLK1, in breast cancers with various expression levels of human CKS1B Growth inhibition by PLK1 knockdown correlates with increased CKS1B expression in published tumor cell data sets, and this correlation was confirmed using shRNAs against PLK1 in tumor cell lines. In addition, we overexpressed CKS1B in multiple cell lines and found increased sensitivity to PLK1 knockdown and PLK1 drug inhibition. Finally, combined inhibition of WEE1 and PLK1 results in less apoptosis than predicted based on an additive model of the individual inhibitors, showing an epistatic interaction and confirming a prediction of the yeast data. Thus, identification of a yeast SDL interaction uncovers conserved genetic interactions that can affect human cancer cell viability.

Physical and genetic-interaction density reveals functional organization and informs significance cutoffs in genome-wide screens.

Genome-wide experiments often measure quantitative differences between treated and untreated cells to identify affected strains. For these studies, statistical models are typically used to determine significance cutoffs. We developed a method termed "CLIK" (Cutoff Linked to Interaction Knowledge) that overlays biological knowledge from the interactome on screen results to derive a cutoff. The method takes advantage of the fact that groups of functionally related interacting genes often respond similarly to experimental conditions and, thus, cluster in a ranked list of screen results. We applied CLIK analysis to five screens of the yeast gene disruption library and found that it defined a significance cutoff that differed from traditional statistics. Importantly, verification experiments revealed that the CLIK cutoff correlated with the position in the rank order where the rate of true positives drops off significantly. In addition, the gene sets defined by CLIK analysis often provide further biological perspectives. For example, applying CLIK analysis retrospectively to a screen for cisplatin sensitivity allowed us to identify the importance of the Hrq1 helicase in DNA crosslink repair. Furthermore, we demonstrate the utility of CLIK to determine optimal treatment conditions by analyzing genome-wide screens at multiple rapamycin concentrations. We show that CLIK is an extremely useful tool for evaluating screen quality, determining screen cutoffs, and comparing results between screens. Furthermore, because CLIK uses previously annotated interaction data to determine biologically informed cutoffs, it provides additional insights into screen results, which supplement traditional statistical approaches.

Sec24p and Sec16p cooperate to regulate the GTP cycle of the COPII coat.

Vesicle budding from the endoplasmic reticulum (ER) employs a cycle of GTP binding and hydrolysis to regulate assembly of the COPII coat. We have identified a novel mutation (sec24-m11) in the cargo-binding subunit, Sec24p, that specifically impacts the GTP-dependent generation of vesicles in vitro. Using a high-throughput approach, we defined genetic interactions between sec24-m11 and a variety of trafficking components of the early secretory pathway, including the candidate COPII regulators, Sed4p and Sec16p. We defined a fragment of Sec16p that markedly inhibits the Sec23p- and Sec31p-stimulated GTPase activity of Sar1p, and demonstrated that the Sec24p-m11 mutation diminished this inhibitory activity, likely by perturbing the interaction of Sec24p with Sec16p. The consequence of the heightened GTPase activity when Sec24p-m11 is present is the generation of smaller vesicles, leading to accumulation of ER membranes and more stable ER exit sites. We propose that association of Sec24p with Sec16p creates a novel regulatory complex that retards the GTPase activity of the COPII coat to prevent premature vesicle scission, pointing to a fundamental role for GTP hydrolysis in vesicle release rather than in coat assembly/disassembly.

Srs2 overexpression reveals a helicase-independent role at replication forks that requires diverse cell functions.

Srs2 is a 3'-5' DNA helicase that regulates many aspects of DNA metabolism in Saccharomyces cerevisiae. It is best known for its ability to counteract homologous recombination by dismantling Rad51 filaments, but is also involved in checkpoint activation, adaptation and recovery, and in resolution of late recombination intermediates. To further address its biological roles and uncover new genetic interactions, we examined the consequences of overexpressing SRS2 as well as two helicase-dead mutants, srs2-K41A and srs2-K41R, in the collection of 4827 yeast haploid deletion mutants. We identified 274 genes affecting a large variety of cellular functions that are required for cell growth when SRS2 or its mutants are overexpressed. Further analysis of these interactions reveals that Srs2 acts independently of its helicase function at replication forks likely through its recruitment by the sumoylated PCNA replication clamp. This helicase-independent function is responsible for the negative interactions with DNA metabolism genes and for the toxicity of SRS2 overexpression in many of the diverse cellular pathways revealed in our screens.

The Shu complex, which contains Rad51 paralogues, promotes DNA repair through inhibition of the Srs2 anti-recombinase.

The Shu complex, which contains RAD51 paralogues, is involved in the decision between homologous recombination and error-prone repair. We discovered a link to ribosomal DNA (rDNA) recombination when we found an interaction between one member of the Shu complex, SHU1, and UAF30, a component of the upstream activating factor complex (UAF), which regulates rDNA transcription. In the absence of Uaf30, rDNA copy number increases, and this increase depends on several functional subunits of the Shu complex. Furthermore, in the absence of Uaf30, we find that Shu1 and Srs2, an anti-recombinase DNA helicase with which the Shu complex physically interacts, act in the same pathway regulating rDNA recombination. In addition, Shu1 modulates Srs2 recruitment to both induced and spontaneous foci correlating with a decrease in Rad51 foci, demonstrating that the Shu complex is an important regulator of Srs2 activity. Last, we show that Shu1 regulation of Srs2 to double-strand breaks is not restricted to the rDNA, indicating a more general function for the Shu complex in the regulation of Srs2. We propose that the Shu complex shifts the balance of repair toward Rad51 filament stabilization by inhibiting the disassembly reaction of Srs2.

Selective ploidy ablation, a high-throughput plasmid transfer protocol, identifies new genes affecting topoisomerase I-induced DNA damage.

We have streamlined the process of transferring plasmids into any yeast strain library by developing a novel mating-based, high-throughput method called selective ploidy ablation (SPA). SPA uses a universal plasmid donor strain that contains conditional centromeres on every chromosome. The plasmid-bearing donor is mated to a recipient, followed by removal of all donor-strain chromosomes, producing a haploid strain containing the transferred plasmid. As proof of principle, we used SPA to transfer plasmids containing wild-type and mutant alleles of DNA topoisomerase I (TOP1) into the haploid yeast gene-disruption library. Overexpression of Top1 identified only one sensitive mutation, rpa34, while overexpression of top1-T(722)A allele, a camptothecin mimetic, identified 190 sensitive gene-disruption strains along with rpa34. In addition to known camptothecin-sensitive strains, this set contained mutations in genes involved in the Rpd3 histone deacetylase complex, the kinetochore, and vesicle trafficking. We further show that mutations in several ESCRT vesicle trafficking components increase Top1 levels, which is dependent on SUMO modification. These findings demonstrate the utility of the SPA technique to introduce plasmids into the haploid gene-disruption library to discover new interacting pathways.

Most, but not all, yeast strains in the deletion library contain the [PIN(+)] prion.

The yeast deletion library is a collection of over 5100 single gene deletions that has been widely used by the yeast community. The presence of a non-Mendelian element, such as a prion, within this library could affect the outcome of many large-scale genomic studies. We previously showed that the deletion library parent strain contained the [PIN(+)] prion. [PIN(+)] is the misfolded infectious prion form of the Rnq1 protein that displays distinct fluorescent foci in the presence of RNQ1-GFP and exists in different physical conformations, called variants. Here, we show that over 97% of the library deletion strains are [PIN(+)]. Of the 141 remaining strains that have completely (58) or partially (83) lost [PIN(+)], 139 deletions were able to efficiently maintain three different [PIN(+)] variants despite extensive growth and storage at 4 degrees C. One strain, cue2Delta, displayed an alteration in the RNQ1-GFP fluorescent shape, but the Rnq1p prion aggregate shows no biochemical differences from the wild-type. Only strains containing a deletion of either HSP104 or RNQ1 are unable to maintain [PIN(+)], indicating that 5153 non-essential genes are not required for [PIN(+)] propagation.

Chromosome-scale genetic mapping using a set of 16 conditionally stable Saccharomyces cerevisiae chromosomes.

We have created a resource to rapidly map genetic traits to specific chromosomes in yeast. This mapping is done using a set of 16 yeast strains each containing a different chromosome with a conditionally functional centromere. Conditional centromere function is achieved by integration of a GAL1 promoter in cis to centromere sequences. We show that the 16 yeast chromosomes can be individually lost in diploid strains, which become hemizygous for the destabilized chromosome. Interestingly, most 2n - 1 strains endoduplicate and become 2n. We also demonstrate how chromosome loss in this set of strains can be used to map both recessive and dominant markers to specific chromosomes. In addition, we show that this method can be used to rapidly validate gene assignments from screens of strain libraries such as the yeast gene disruption collection.

Systematic hybrid LOH: a new method to reduce false positives and negatives during screening of yeast gene deletion libraries.

We have developed a new method, systematic hybrid loss of heterozygosity, to facilitate genomic screens utilizing the yeast gene deletion library. Screening is performed using hybrid diploid strains produced through mating the library haploids with strains from a different genetic background, to minimize the contribution of unpredicted recessive genetic factors present in the individual library strains. We utilize a set of strains where each contains a conditional centromere construct on one of the 16 yeast chromosomes that allows the destabilization and selectable loss of that chromosome. After mating a library gene deletion haploid to such a conditional centromere strain, which corresponds to the chromosome carrying the gene deletion, loss of heterozygosity (LOH) at the gene deletion locus can be generated in these otherwise hybrid diploids. The use of hybrid diploid strains permits complementation of any spurious recessive mutations in the library strain, facilitating attribution of the observed phenotype to the documented gene deletion and dramatically reducing false positive results commonly obtained in library screens. The systematic hybrid LOH method can be applied to virtually any screen utilizing the yeast non-essential gene deletion library and is particularly useful for screens requiring the introduction of a genetic assay into the library strains.

The role of DNA double-strand breaks in spontaneous homologous recombination in S. cerevisiae.

Homologous recombination (HR) is a source of genomic instability and the loss of heterozygosity in mitotic cells. Since these events pose a severe health risk, it is important to understand the molecular events that cause spontaneous HR. In eukaryotes, high levels of HR are a normal feature of meiosis and result from the induction of a large number of DNA double-strand breaks (DSBs). By analogy, it is generally believed that the rare spontaneous mitotic HR events are due to repair of DNA DSBs that accidentally occur during mitotic growth. Here we provide the first direct evidence that most spontaneous mitotic HR in Saccharomyces cerevisiae is initiated by DNA lesions other than DSBs. Specifically, we describe a class of rad52 mutants that are fully proficient in inter- and intra-chromosomal mitotic HR, yet at the same time fail to repair DNA DSBs. The conclusions are drawn from genetic analyses, evaluation of the consequences of DSB repair failure at the DNA level, and examination of the cellular re-localization of Rad51 and mutant Rad52 proteins after introduction of specific DSBs. In further support of our conclusions, we show that, as in wild-type strains, UV-irradiation induces HR in these rad52 mutants, supporting the view that DNA nicks and single-stranded gaps, rather than DSBs, are major sources of spontaneous HR in mitotic yeast cells.

Functional genomics of the yeast DNA-damage response.

High-throughput approaches are beginning to have an impact on many areas of yeast biology. Two recent studies, using different experimental platforms, provide insight into new pathways involved in the response of yeast to DNA damage.

Defects in SUMO (small ubiquitin-related modifier) conjugation and deconjugation alter cell sensitivity to DNA topoisomerase I-induced DNA damage.

Eukaryotic DNA topoisomerase I (Top1p) has important functions in DNA replication, transcription, and recombination. This enzyme also constitutes the cellular target of camptothecin (CPT), which induces S-phase-dependent cytotoxicity. To define cellular pathways that regulate cell sensitivity to Top1p-induced DNA lesions, we described a yeast genetic screen for conditional tah (top1T722A-hypersensitive) mutants with enhanced sensitivity to low levels of the CPT mimetic mutant top1T722A (Reid, R. J., Fiorani, P., Sugawara, M., and Bjornsti, M. A. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 11440-11445; Fiorani, P., Reid, R. J., Schepis, A., Jacquiau, H. R., Guo, H., Thimmaiah, P., Benedetti, P., and Bjornsti, M. A. (2004) J. Biol. Chem. 279, 21271-21281). Here we report that tah mutant ubc9-10 harbors a hypomorphic allele of UBC9, which encodes the essential SUMO (small ubiquitin-related modifier) E2-conjugating enzyme. The same conditional ubc9P123L mutant was also isolated in an independent screen for enhanced sensitivity to a distinct Top1p poison, Top1N726Hp. The ubc9-10 mutant exhibited a decrease in global protein sumoylation and increased sensitivity to a wide range of DNA-damaging agents independent of Top1p. Deletion of the Ulp2 SUMO protease failed to restore ubc9-10 cell resistance to Top1p poisons or hydroxyurea yet adversely affected wild-type TOP1 cell genetic stability and sensitivity to hydroxyurea. Moreover, although mutation of different consensus SUMO sites in the N terminus and linker region of yeast Top1p failed to recapitulate ubc9-10 mutant phenotypes, they revealed distinct and subtle effects on cell sensitivity to CPT. These results provide insights into the complexities of SUMO conjugation and the confounding effects of SUMO modification on Top1p function and cell sensitivity to genotoxic agents.

Stay close to your sister.

Yeast Sir2 protein regulates chromatin structure and suppresses recombination at the multiple tandem rDNA array. In the current issue of Cell, Kobayashi and colleagues reinvestigate rDNA dynamics in sir2 strains and find that Sir2 is necessary to recruit cohesins to the array, which influences the nature of rDNA recombination events.

The deubiquitinating enzyme Doa4p protects cells from DNA topoisomerase I poisons.

DNA topoisomerase I (Top1p) catalyzes changes in DNA topology via the formation of an enzyme-DNA covalent complex that is reversibly stabilized by the antitumor drug, camptothecin (CPT). During S-phase, collisions with replication forks convert these complexes into cytotoxic DNA lesions that trigger cell cycle arrest and cell death. To investigate cellular responses to CPT-induced DNA damage, a yeast genetic screen identified conditional tah mutants with enhanced sensitivity to self-poisoning DNA topoisomerase I mutant (Top1T722Ap), which mimics the action of CPT. Mutant alleles of three genes, DOA4, SLA1 and SLA2, were recovered. A nonsense mutation in DOA4 eliminated the catalytic residues of the Doa4p deubiquitinating enzyme, yet retained the rhodanase domain. At 36 degrees C, this doa4-10 mutant exhibited increased sensitivity to CPT, osmotic stress, and hydroxyurea, and a reversible petite phenotype. However, the accumulation of pre-vacuolar class E vesicles that was observed in doa4Delta cells was not detected in the doa4-10 mutant. Mutations in SLA1 or SLA2, which alter actin cytoskeleton architecture, induced a conditional synthetic lethal phenotype in combination with doa4-10 in the absence of DNA damage. Here actin cytoskeleton defects coincided with the enhanced fragility of large-budded cells. In contrast, the enhanced sensitivity of doa4-10 mutant cells to Top1T722Ap was unrelated to alterations in endocytosis and was selectively suppressed by increased dosage of the ribonucleotide reductase inhibitor Sml1p. Additional studies suggest a role for Doa4p in the Rad9p checkpoint response to Top1p poisons. These findings indicate a functional link between ubiquitin-mediated proteolysis and cellular resistance to CPT-induced DNA damage.

Cloning-free genome alterations in Saccharomyces cerevisiae using adaptamer-mediated PCR.

Each of the adaptamer-directed genome manipulation methods is predicated on the fact that recombination between two DNAs is enhanced by increasing the length of homology. Many of the current PCR-based genome manipulation techniques rely on very short homologies to promote recombination. In these cases homology length is dictated by the technical limits of oligonucleotide synthesis. Adaptamers circumvent this problem since long homology regions are produced in a first round of PCR, and then fused to the selectable marker in a second round of PCR via complementary sequence tags on the adaptamers. Furthermore, many of the techniques described here rely on preexisting and commercially available adaptamer sets that can be obtained inexpensively rather than designing new primers for every experiment. Although a cost is incurred when performing multiple PCR amplifications, the increase in recombination efficiency is dramatic. Finally, the adaptamer-mediated PCR fusion methodology is versatile and can be applied to varied genome manipulations.

Efficient PCR-based gene disruption in Saccharomyces strains using intergenic primers.

Gene disruptions are a vital tool for understanding Saccharomyces cerevisiae gene function. An arrayed library of gene disruption strains has been produced by a consortium of yeast laboratories; however their use is limited to a single genetic background. Since the yeast research community works with several different strain backgrounds, disruption libraries in other common laboratory strains are desirable. We have developed simple PCR-based methods that allow transfer of gene disruptions from the S288C-derived strain library into any Saccharomyces strain. One method transfers the unique sequence tags that flank each of the disrupted genes and replaces the kanamycin resistance marker with a recyclable URA3 gene from Kluyveromyces lactis. All gene-specific PCR amplifications for this method are performed using a pre-existing set of primers that are commercially available. We have also extended this PCR technique to develop a second general gene disruption method suitable for any transformable strain of Saccharomyces.