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Homologous recombination is an important mechanism for genome integrity maintenance, and several homologous recombination genes are mutated in various cancers and cancer-prone syndromes. However, since in some cases homologous recombination can lead to mutagenic outcomes, this pathway must be tightly regulated, and mitotic hyper-recombination is a hallmark of genomic instability. We performed two screens inSaccharomyces cerevisiaefor genes that, when deleted, cause hyper-recombination between direct repeats. One was performed with the classical patch and replica-plating method. The other was performed with a high-throughput replica-pinning technique that was designed to detect low-frequency events. This approach allowed us to validate the high-throughput replica-pinning methodology independently of the replicative aging context in which it was developed. Furthermore, by combining the two approaches, we were able to identify and validate 35 genes whose deletion causes elevated spontaneous direct-repeat recombination. Among these are mismatch repair genes, the Sgs1-Top3-Rmi1 complex, the RNase H2 complex, genes involved in the oxidative stress response, and a number of other DNA replication, repair and recombination genes. Since several of our hits are evolutionary conserved, and repeated elements constitute a significant fraction of mammalian genomes, our work might be relevant for understanding genome integrity maintenance in humans.

The eukaryotic post-replication repair (PRR) pathway allows completion of DNA replication when replication forks encounter lesions on the DNA template and are mediated by post-translational ubiquitination of the DNA sliding clamp proliferating cell nuclear antigen (PCNA). Monoubiquitinated PCNA recruits translesion synthesis (TLS) polymerases to replicate past DNA lesions in an error-prone manner while addition of K63-linked polyubiquitin chains signals for error-free template switching to the sister chromatid. Central to both branches is the E3 ubiquitin ligase and DNA helicase Rad5/helicase-like transcription factor (HLTF). Mutations in PRR pathway components lead to genomic rearrangements, cancer predisposition, and cancer progression. Recent studies have challenged the notion that the PRR pathway is involved only in DNA lesion tolerance and have shed new light on its roles in cancer progression. Molecular details of Rad5/HLTF recruitment and function at replication forks have emerged. Mounting evidence indicates that PRR is required during lesion-less replication stress, leading to TLS polymerase activity on undamaged templates. Analysis of PRR mutation status in human cancers and PRR function in cancer models indicates that down regulation of PRR activity is a viable strategy to inhibit cancer cell growth and reduce chemoresistance. Here, we review these findings, discuss how they change our views of current PRR models, and look forward to targeting the PRR pathway in the clinic.

Genome instability is a condition characterized by the accumulation of genetic alterations and is a hallmark of cancer cells. To uncover new genes and cellular pathways affecting endogenous DNA damage and genome integrity, we exploited a Synthetic Genetic Array (SGA)-based screen in yeast. Among the positive genes, we identified VID22, reported to be involved in DNA double-strand break repair. vid22Δ cells exhibit increased levels of endogenous DNA damage, chronic DNA damage response activation and accumulate DNA aberrations in sequences displaying high probabilities of forming G-quadruplexes (G4-DNA). If not resolved, these DNA secondary structures can block the progression of both DNA and RNA polymerases and correlate with chromosome fragile sites. Vid22 binds to and protects DNA at G4-containing regions both in vitro and in vivo. Loss of VID22 causes an increase in gross chromosomal rearrangement (GCR) events dependent on G-quadruplex forming sequences. Moreover, the absence of Vid22 causes defects in the correct maintenance of G4-DNA rich elements, such as telomeres and mtDNA, and hypersensitivity to the G4-stabilizing ligand TMPyP4. We thus propose that Vid22 is directly involved in genome integrity maintenance as a novel regulator of G4 metabolism., Associazione Italiana per la Ricerca sul Cancro (AIRC) [15631, 21806 to M.M.F.]; MIUR [PRIN 2015-2015SJLMB9; PRIN 2017-2017KSZZJW to M.M.F.]; Telethon [GGP15227 to M.M.F.]; F.L. was supported by the University of Milano: ‘‘Piano di Sviluppo dell’Ateneo per la Ricerca. Linea B: Supporto per i Giovani Ricercatori’’; M.C.B. was supported by Fondazione Veronesi; Research at the laboratory of A.A. was funded by the Spanish Ministry of Economy and Competitiveness [BFU2016-75058-P]; B.G.G. was funded by the Spanish Association Against Cancer; MIUR [PRIN2017-2017Z55KC to T.B.]; M.C., D.S.H. are supported by MIUR [PRIN 2017] and CNRbiomics [PIR01_00017]; H2020 Projects ELIXIR-EXCELERATE, EOSC-Life, EOSC-Pillar and Elixir-IIB; G.W.B. was supported by the Canadian Institutes of Health Research[FDN-159913]. Funding for open access charge: Associazione Italiana per la Ricerca sul Cancro (AIRC) [21806].

Overexpression of the flap endonuclease FEN1 has been observed in a variety of cancer types and is a marker for poor prognosis. To better understand the cellular consequences of FEN1 overexpression we utilized a model of its Saccharomyces cerevisiae homolog, RAD27. In this system, we discovered that flap endonuclease overexpression impedes replication fork progression and leads to an accumulation of cells in mid-S phase. This was accompanied by increased phosphorylation of the checkpoint kinase Rad53 and histone H2A-S129. RAD27 overexpressing cells were hypersensitive to treatment with DNA damaging agents, and defective in ubiquitinating the replication clamp proliferating cell nuclear antigen (PCNA) at lysine 164. These effects were reversed when the interaction between overexpressed Rad27 and PCNA was ablated, suggesting that the observed phenotypes were linked to problems in DNA replication. RAD27 overexpressing cells also exhibited an unexpected dependence on the SUMO ligases SIZ1 and MMS21 for viability. Importantly, we found that overexpression of FEN1 in human cells also led to phosphorylation of CHK1, CHK2, RPA32 and histone H2AX, all markers of genome instability. Our data indicate that flap endonuclease overexpression is a driver of genome instability in yeast and human cells that impairs DNA replication in a manner dependent on its interaction with PCNA.

Complete and accurate DNA replication is fundamental to cellular proliferation and genome stability. Obstacles that delay, prevent, or terminate DNA replication cause the phenomena termed DNA replication stress. Cancer cells exhibit chronic replication stress due to the loss of proteins that protect or repair stressed replication forks and due to the continuous proliferative signaling, providing an exploitable therapeutic vulnerability in tumors. Here, we outline current and pending therapeutic approaches leveraging tumor-specific replication stress as a target, in addition to the challenges associated with such therapies. We discuss how replication stress modulates the cell-intrinsic innate immune response and highlight the integration of replication stress with immunotherapies. Together, exploiting replication stress for cancer treatment seems to be a promising strategy as it provides a selective means of eliminating tumors, and with continuous advances in our knowledge of the replication stress response and lessons learned from current therapies in use, we are moving toward honing the potential of targeting replication stress in the clinic.

In response to replication stress, signaling mediated by DNA damage checkpoint kinases protects genome integrity. However, following repair or bypass of DNA lesions, checkpoint signaling needs to be terminated for continued cell cycle progression and proliferation. In budding yeast, the PP4 phosphatase has been shown to play a key role in preventing hyperactivation of the checkpoint kinase Rad53. In addition, we recently uncovered a phosphatase-independent mechanism for downregulating Rad53 in which the DNA repair scaffold Slx4 decreases engagement of the checkpoint adaptor Rad9 at DNA lesions. Here we reveal that proper termination of checkpoint signaling following the bypass of replication blocks imposed by alkylated DNA adducts requires the concerted action of these two fundamentally distinct mechanisms of checkpoint downregulation. Cells lacking both SLX4 and the PP4-subunit PPH3 display a synergistic increase in Rad53 signaling and are exquisitely sensitive to the DNA alkylating agent methyl methanesulfonate, which induces replication blocks and extensive formation of chromosomal linkages due to template switching mechanisms required for fork bypass. Rad53 hypersignaling in these cells seems to converge to a strong repression of Mus81-Mms4, the endonuclease complex responsible for resolving chromosomal linkages, thus explaining the selective sensitivity of slx4Δ pph3Δ cells to alkylation damage. Our results support a model in which Slx4 acts locally to downregulate Rad53 activation following fork bypass, while PP4 acts on pools of active Rad53 that have diffused from the site of lesions. We propose that the proper spatial coordination of the Slx4 scaffold and PP4 action is crucial to allow timely activation of Mus81-Mms4 and, therefore, proper chromosome segregation.

DNA damage response pathways are crucial for protecting genome stability in all eukaryotes. Saccharomyces cerevisiaeDna2 has both helicase and nuclease activities that are essential for Okazaki fragment maturation, and Dna2 is involved in long-range DNA end resection at double-strand breaks. Dna2 forms nuclear foci in response to DNA replication stress and to double-strand breaks. We find that Dna2-GFP focus formation occurs mainly during S phase in unperturbed cells. Dna2 colocalizes in nuclear foci with 25 DNA repair proteins that define recombination repair centers in response to phleomycin-induced DNA damage. To systematically identify genes that affect Dna2 focus formation, we crossed Dna2-GFP into 4293 nonessential gene deletion mutants and assessed Dna2-GFP nuclear focus formation after phleomycin treatment. We identified 37 gene deletions that affect Dna2-GFP focus formation, 12 with fewer foci and 25 with increased foci. Together these data comprise a useful resource for understanding Dna2 regulation in response to DNA damage.

Condition-dependent genetic interactions can reveal functional relationships between genes that are not evident under standard culture conditions. State-of-the-art yeast genetic interaction mapping, which relies on robotic manipulation of arrays of double mutant strains, does not scale readily to multi-condition studies. Here we describe Barcode Fusion Genetics to map Genetic Interactions (BFG-GI), by which double mutant strains generated viaen masse‘party’ mating can also be monitoreden massefor growth and genetic interactions. By using site-specific recombination to fuse two DNA barcodes, each representing a specific gene deletion, BFG-GI enables multiplexed quantitative tracking of double mutants via next-generation sequencing. We applied BFG-GI to a matrix of DNA repair genes under nine different conditions, including methyl methanesulfonate (MMS), 4-nitroquinoline 1-oxide (4NQO), bleomycin, zeocin, and three other DNA-damaging environments. BFG-GI recapitulated known genetic interactions and yielded new condition-dependent genetic interactions. We validated and further explored a subnetwork of condition-dependent genetic interactions involvingMAG1,SLX4, and genes encoding the Shu complex, and inferred that loss of the Shu complex leads to a decrease in the activation or activity of the checkpoint protein kinase Rad53.

Hutchinson-Gilford progeria syndrome (HGPS) is caused by a mutation in LMNA that produces an aberrant lamin A protein, progerin. The accumulation of progerin in HGPS cells leads to an aberrant nuclear morphology, genetic instability, and p53-dependent premature senescence. How p53 is activated in response to progerin production is unknown. Here we show that young cycling HGPS fibroblasts exhibit chronic DNA damage, primarily in S phase, as well as delayed replication fork progression. We demonstrate that progerin binds to PCNA, altering its distribution away from replicating DNA in HGPS cells, leading to γH2AX formation, ATR activation, and RPA Ser33 phosphorylation. Unlike normal human cells that can be immortalized by enforced expression of telomerase alone, immortalization of HGPS cells requires telomerase expression and p53 repression. In addition, we show that the DNA damage response in HGPS cells does not originate from eroded telomeres. Together, these results establish that progerin interferes with the coordination of essential DNA replication factors, causing replication stress, and is the primary signal for p53 activation leading to premature senescence in HGPS. Furthermore, this damage response is shown to be independent of progerin farnesylation, implying that unprocessed lamin A alone causes replication stress.

Cancer is a multi-stage disease caused by sequential mutations; however, the molecular mechanism that generates cancer-initiating mutations is not well understood. Using the developing cerebellum as a model system, here we show the molecular mechanism of tumor initiation in Sonic hedgehog medulloblastoma (SHH-MB), a common subtype of the most frequent pediatric brain tumor. The most common initiating event of SHH-MB is Ptch1 loss of heterozygosity (LOH), which occurs in cerebellar granule cell progenitors (GCPs) and leads to preneoplasia formation. Since GCPs proliferate in response to Shh, we tested whether the normal proliferative effects of Shh can lead to genomic instability and DNA lesions responsible for Ptch1 LOH in the SHH-MB cell-of-origin. We found that Shh profoundly alters DNA replication dynamics in GCPs, leading to DNA damage, formation of DNA breaks in S-phase and hyper-recombination. Shh led to DNA helicase loading and activation, resulting in high levels of replication origin firing. Shh-dependent origin firing was required for Shh-induced DNA damage and recombination. Moreover, reducing origin firing decreased recombination and tumor initiation in a pre-clinical model of MB. These results show that reduction of Shh-dependent, DNA replication-associated DNA damage in tumor-prone Ptch1(+/-) GCPs before cancer initiation is capable of preventing MB-initiating mutations. Thus, we demonstrate that a developmental mitogen can cause cancer-initiating mutations through an increase in origin firing, and attenuating origin firing can prevent cancer initiation.

G-quadruplex DNAs form four-stranded helical structures and are proposed to play key roles in different cellular processes. Targeting G-quadruplex DNAs for cancer treatment is a very promising prospect. Here, we show that CX-5461 is a G-quadruplex stabilizer, with specific toxicity against BRCA deficiencies in cancer cells and polyclonal patient-derived xenograft models, including tumours resistant to PARP inhibition. Exposure to CX-5461, and its related drug CX-3543, blocks replication forks and induces ssDNA gaps or breaks. The BRCA and NHEJ pathways are required for the repair of CX-5461 and CX-3543-induced DNA damage and failure to do so leads to lethality. These data strengthen the concept of G4 targeting as a therapeutic approach, specifically for targeting HR and NHEJ deficient cancers and other tumours deficient for DNA damage repair. CX-5461 is now in advanced phase I clinical trial for patients with BRCA1/2 deficient tumours (Canadian trial, NCT02719977, opened May 2016)., Stabilization of DNA quadruplex structures (G4) is lethal for cells with a compromised DNA repair pathway. Here, the authors show that CX-5461, a small molecule in clinical trials as RNA polymerase inhibitor, has G4-stablization properties and can be repurposed to target DNA repair-defective cancers cells.

A significant challenge of functional genomics is to develop methods for genome-scale acquisition and analysis of cell biological data. Here, we present an integrated method that combines genome-wide genetic perturbation of Saccharomyces cerevisiae with high-content screening to facilitate the genetic description of sub-cellular structures and compartment morphology. As proof of principle, we used a Rad52-GFP marker to examine DNA damage foci in ∼20 million single cells from ∼5,000 different mutant backgrounds in the context of selected genetic or chemical perturbations. Phenotypes were classified using a machine learning-based automated image analysis pipeline. 345 mutants were identified that had elevated numbers of DNA damage foci, almost half of which were identified only in sensitized backgrounds. Subsequent analysis of Vid22, a protein implicated in the DNA damage response, revealed that it acts together with the Sgs1 helicase at sites of DNA damage and preferentially binds G-quadruplex regions of the genome. This approach is extensible to numerous other cell biological markers and experimental systems.

A mechanism is shown by which Mms1 and Mms22 promote DNA replication in the presence of replication stress: they stabilize the replisome at stalled replication forks., Mms1 and Mms22 form a Cul4Ddb1-like E3 ubiquitin ligase with the cullin Rtt101. In this complex, Rtt101 is bound to the substrate-specific adaptor Mms22 through a linker protein, Mms1. Although the Rtt101Mms1/Mms22 ubiquitin ligase is important in promoting replication through damaged templates, how it does so has yet to be determined. Here we show that mms1Δ and mms22Δ cells fail to properly regulate DNA replication fork progression when replication stress is present and are defective in recovery from replication fork stress. Consistent with a role in promoting DNA replication, we find that Mms1 is enriched at sites where replication forks have stalled and that this localization requires the known binding partners of Mms1—Rtt101 and Mms22. Mms1 and Mms22 stabilize the replisome during replication stress, as binding of the fork-pausing complex components Mrc1 and Csm3, and DNA polymerase ε, at stalled replication forks is decreased in mms1Δ and mms22Δ. Taken together, these data indicate that Mms1 and Mms22 are important for maintaining the integrity of the replisome when DNA replication forks are slowed by hydroxyurea and thereby promote efficient recovery from replication stress.

Human topoisomerase IIIalpha is a type IA DNA topoisomerase that functions with BLM and RMI1 to resolve DNA replication and recombination intermediates. BLM, human topoisomerase IIIalpha, and RMI1 catalyze the dissolution of double Holliday junctions into noncrossover products via a strand-passage mechanism. We generated single-stranded catenanes that resemble the proposed dissolution intermediate recognized by human topoisomerase IIIalpha. We demonstrate that human topoisomerase IIIalpha is a single-stranded DNA decatenase that is specifically stimulated by the BLM-RMI1 pair. In addition, RMI1 interacts with human topoisomerase IIIalpha, and the interaction is required for the stimulatory effect of RMI1 on decatenase activity. Our data provide direct evidence that human topoisomerase IIIalpha functions as a decatenase with the assistance of BLM and RMI1 to facilitate the processing of homologous recombination intermediates without crossing over as a mechanism to preserve genome integrity.

Recent findings suggest important roles for nuclear organization in gene expression. In contrast, little is known about how nuclear organization contributes to genome stability. Epistasis analysis (E-MAP) using DNA repair factors in yeast indicated a functional relationship between a nuclear pore subcomplex and Slx5/Slx8, a small ubiquitin-like modifier (SUMO)–dependent ubiquitin ligase, which we show physically interact. Real-time imaging and chromatin immunoprecipitation confirmed stable recruitment of damaged DNA to nuclear pores. Relocation required the Nup84 complex and Mec1/Tel1 kinases. Spontaneous gene conversion can be enhanced in a Slx8- and Nup84-dependent manner by tethering donor sites at the nuclear periphery. This suggests that strand breaks are shunted to nuclear pores for a repair pathway controlled by a conserved SUMO-dependent E3 ligase.

Double-strand DNA breaks occur upon exposure of cells to ionizing radiation and certain chemical agents or indirectly through replication fork collapse at DNA damage sites. If left unrepaired, double-strand breaks can cause genome instability and cell death, and their repair can result in loss of heterozygosity. In response to DNA damage, proteins involved in double-strand break repair by homologous recombination relocalize into discrete nuclear foci. We identified 29 proteins that colocalize with recombination repair protein Rad52 in response to DNA damage. Of particular interest, Ygr042w/Mte1, a protein of unknown function, showed robust colocalization with Rad52. Mte1 foci fail to form when the DNA helicase gene MPH1 is absent. Mte1 and Mph1 form a complex and are recruited to double-strand breaks in vivo in a mutually dependent manner. MTE1 is important for resolution of Rad52 foci during double-strand break repair and for suppressing break-induced replication. Together our data indicate that Mte1 functions with Mph1 in double-strand break repair.

Double-strand DNA breaks occur upon exposure of cells to agents such as ionizing radiation and ultraviolet light or indirectly through replication fork collapse at DNA damage sites. If left unrepaired double-strand breaks can cause genome instability and cell death. In response to DNA damage, proteins involved in double-strand break repair by homologous recombination re-localize into discrete nuclear foci. We identified 29 proteins that co-localize with the recombination repair protein Rad52 in response to DNA damage. Of particular interest, Ygr042w/Mte1, a protein of unknown function, showed robust colocalization with Rad52. Mte1 foci fail to form when the DNA helicase Mph1 is absent. Mte1 and Mph1 form a complex, and are recruited to double-strand breaks in vivo in a mutually dependent manner. Mte1 is important for resolution of Rad52 foci during double-strand break repair, and for suppressing break-induced replication. Together our data indicate that Mte1 functions with Mph1 in double-strand break repair.

In response to DNA damage, checkpoint signalling protects genome integrity at the cost of repressing cell cycle progression and DNA replication. Mechanisms for checkpoint down-regulation are therefore necessary for proper cellular proliferation. We recently uncovered a phosphatase-independent mechanism for dampening checkpoint signalling, where the checkpoint adaptor Rad9 is counteracted by the repair scaffolds Slx4-Rtt107. Here, we establish the molecular requirements for this new mode of checkpoint regulation. We engineered a minimal multi-BRCT-domain (MBD) module that recapitulates the action of Slx4-Rtt107 in checkpoint down-regulation. MBD mimics the damage-induced Dpb11-Slx4-Rtt107 complex by synergistically interacting with lesion-specific phospho-sites in Ddc1 and H2A. We propose that efficient recruitment of Dpb11-Slx4-Rtt107 or MBD via a cooperative ‘two-site-docking’ mechanism displaces Rad9. MBD also interacts with the Mus81 nuclease following checkpoint dampening, suggesting a spatio-temporal coordination of checkpoint signalling and DNA repair via a combinatorial mode of BRCT-domains interactions.

RTT107 (ESC4, YHR154W) encodes a BRCA1 C-terminal-domain protein that is important for recovery from DNA damage during S phase. Rtt107 is a substrate of the checkpoint protein kinase Mec1, although the mechanism by which Rtt107 is targeted by Mec1 after checkpoint activation is currently unclear. Slx4, a component of the Slx1-Slx4 structure-specific nuclease, formed a complex with Rtt107. Deletion of SLX4 conferred many of the same DNA-repair defects observed in rtt107Δ, including DNA damage sensitivity, prolonged DNA damage checkpoint activation, and increased spontaneous DNA damage. These phenotypes were not shared by the Slx4 binding partner Slx1, suggesting that the functions of the Slx4 and Slx1 proteins in the DNA damage response were not identical. Of particular interest, Slx4, but not Slx1, was required for phosphorylation of Rtt107 by Mec1 in vivo, indicating that Slx4 was a mediator of DNA damage-dependent phosphorylation of the checkpoint effector Rtt107. We propose that Slx4 has roles in the DNA damage response that are distinct from the function of Slx1-Slx4 in maintaining rDNA structure and that Slx4-dependent phosphorylation of Rtt107 by Mec1 is critical for replication restart after alkylation damage.

SGS1 encodes a DNA helicase whose homologues in human cells include the BLM, WRN, and RECQ4 genes, mutations in which lead to cancer-predisposition syndromes. Clustering of synthetic genetic interactions identified by large-scale genetic network analysis revealed that the genetic interaction profile of the gene RMI1 ( RecQ-mediated genome instability, also known as NCE4 and YPL024W) was highly similar to that of SGS1 and TOP3, suggesting a functional relationship between Rmi1 and the Sgs1/Top3 complex. We show that Rmi1 physically interacts with Sgs1 and Top3 and is a third member of this complex. Cells lacking RMI1 activate the Rad53 checkpoint kinase, undergo a mitotic delay, and display increased relocalization of the recombination repair protein Rad52, indicating the presence of spontaneous DNA damage. Consistent with a role for RMI1 in maintaining genome integrity, rmi1 Delta cells exhibit increased recombination frequency and increased frequency of gross chromosomal rearrangements. In addition, rmi1 Delta strains fail to fully activate Rad53 upon exposure to DNA-damaging agents, suggesting that Rmi1 is also an important part of the Rad53-dependent DNA damage response.

Principally characterized for its requirement in the initiation of DNA replication, compelling evidence from two yeast model organisms now points to a central role for the Dbf4/Cdc7 kinase complex in S-phase checkpoint responses. Among the key findings supporting this view are observations that orthologs Dfp1 (Schizosaccharomyces pombe) and Dbf4 (Saccharomyces cerevisiae) interact with equivalent checkpoint kinases Cds1 and Rad53, respectively, and that mutants for Dbf4 and Cdc7 in these species are sensitive to genotoxic agents. Recently, these findings have been extended through mutational analyses of conserved regions in both Dfp1 and Dbf4, leading to the identification of distinct motifs which mediate cellular responses to DNA damage and replication fork arrest. The present review is a comparative survey of data obtained from studies conducted with S. pombe and S. cerevisae, and a consideration of models for the role played by Dbf4/Cdc7 in checkpoint responses.

▪ Abstract The initiation of DNA replication in eukaryotic cells is tightly controlled to ensure that the genome is faithfully duplicated once each cell cycle. Genetic and biochemical studies in several model systems indicate that initiation is mediated by a common set of proteins, present in all eukaryotic species, and that the activities of these proteins are regulated during the cell cycle by specific protein kinases. Here we review the properties of the initiation proteins, their interactions with each other, and with origins of DNA replication. We also describe recent advances in understanding how the regulatory protein kinases control the progress of the initiation reaction. Finally, we describe the checkpoint mechanisms that function to preserve the integrity of the genome when the normal course of genome duplication is perturbed by factors that damage the DNA or inhibit DNA synthesis.

The INO80 chromatin remodeling complex functions in transcriptional regulation, DNA repair, and replication. Here we uncover a novel role for INO80 in regulating chromosome segregation. First, we show that the conserved Ies6 subunit is critical for INO80 function in vivo. Strikingly, we found that loss of either Ies6 or the Ino80 catalytic subunit results in rapid increase in ploidy. One route to polyploidy is through chromosome missegregation due to aberrant centromere structure, and we found that loss of either Ies6 or Ino80 leads to defective chromosome segregation. Importantly, we show that chromatin structure flanking centromeres is altered in cells lacking these subunits and that these alterations occur not in the Cse4-containing centromeric nucleosome, but in pericentric chromatin. We provide evidence that these effects are mediated through misincorporation of H2A.Z, and these findings indicate that H2A.Z-containing pericentric chromatin, as in higher eukaryotes with regional centromeres, is important for centromere function in budding yeast. These data reveal an important additional mechanism by which INO80 maintains genome stability.

Genetic screens of the collection of ∼4500 deletion mutants in Saccharomyces cerevisiae have identified the cohort of nonessential genes that promote maintenance of genome integrity. Here we probe the role of essential genes needed for genome stability. To this end, we screened 217 tetracycline-regulated promoter alleles of essential genes and identified 47 genes whose depletion results in spontaneous DNA damage. We further showed that 92 of these 217 essential genes have a role in suppressing chromosome rearrangements. We identified a core set of 15 genes involved in DNA replication that are critical in preventing both spontaneous DNA damage and genome rearrangements. Mapping, classification, and analysis of rearrangement breakpoints indicated that yeast fragile sites, Ty retrotransposons, tRNA genes, early origins of replication, and replication termination sites are common features at breakpoints when essential replication genes that suppress chromosome rearrangements are downregulated. We propose mechanisms by which depletion of essential replication proteins can lead to double-stranded DNA breaks near these features, which are subsequently repaired by homologous recombination at repeated elements.

Platinum-based drugs have been used to successfully treat diverse cancers for several decades. Cisplatin, the original compound of this class, cross-links DNA, resulting in cell cycle arrest and cell death via apoptosis. Cisplatin is effective against several tumor types, yet it exhibits toxic side effects and tumors often develop resistance. To mitigate these liabilities while maintaining potency, we generated a library of non-classical platinum-acridine hybrid agents and assessed their mechanisms of action using a validated genome-wide screening approach in Saccharomyces cerevisiae and in the distantly related yeast Schizosaccharomyces pombe. Chemogenomic profiles from both S. cerevisiae and S. pombe demonstrate that several of the platinum-acridines damage DNA differently than cisplatin based on their requirement for distinct modules of DNA repair.

Relocalization of proteins is a hallmark of the DNA damage response. We use high-throughput microscopic screening of the yeast GFP fusion collection to develop a systems-level view of protein reorganization following drug-induced DNA replication stress. Changes in protein localization and abundance reveal drug-specific patterns of functional enrichments. Classification of proteins by subcellular destination enables the identification of pathways that respond to replication stress. We analysed pairwise combinations of GFP fusions and gene deletion mutants to define and order two previously unknown DNA damage responses. In the first, Cmr1 forms subnuclear foci that are regulated by the histone deacetylase Hos2 and are distinct from the typical Rad52 repair foci. In a second example, we find that the checkpoint kinases Mec1/Tel1 and the translation regulator Asc1 regulate P-body formation. This method identifies response pathways that were not detected in genetic and protein interaction screens, and can be readily applied to any form of chemical or genetic stress to reveal cellular response pathways.

The integrity of the genome depends on diverse pathways that regulate DNA metabolism. Defects in these pathways result in genome instability, a hallmark of cancer. Deletion of ELG1 in budding yeast, when combined with hypomorphic alleles of PCNA results in spontaneous DNA damage during S phase that elicits upregulation of ribonucleotide reductase (RNR) activity. Increased RNR activity leads to a dramatic expansion of deoxyribonucleotide (dNTP) pools in G1 that allows cells to synthesize significant fractions of the genome in the presence of hydroxyurea in the subsequent S phase. Consistent with the recognized correlation between dNTP levels and spontaneous mutation, compromising ELG1 and PCNA results in a significant increase in mutation rates. Deletion of distinct genome stability genes RAD54, RAD55, and TSA1 also results in increased dNTP levels and mutagenesis, suggesting that this is a general phenomenon. Together, our data point to a vicious circle in which mutations in gatekeeper genes give rise to genomic instability during S phase, inducing expansion of the dNTP pool, which in turn results in high levels of spontaneous mutagenesis.

The synthesis and extensive biological study of two new tridentates ligands based on pyrazole and triazole are described. The antifungal activity against the budding yeast cells of the newly synthesized compounds was determined. These compounds were toxic to yeast cells. Cell cycle analysis suggested that treatment with these compounds impairs cell division in G1 of the cell cycle. Using yeast-based functional genomics technologies, we found that these compounds tolerance requires DNA repair pathway and SKI complex function. We have also found that the PKC1 heterozygous deletion strain was the most sensitive to these compounds using HaploInsufficiency Profiling, suggesting that the Pkc1 protein may be the target for these compounds. These results strongly suggest that these compounds induce DNA damage and thus exert a different mechanism of action compared to other azole derivatives. These two compounds might therefore represent promising lead compounds for further development of antifungal drugs for human therapy.

The faithful transmission of genetic information from a mother to daughter cells can only occur if the integrity of the genome is preserved. DNA transactions within cells are tightly regulated to prevent DNA damage. When events that challenge genome integrity do occur, a complex web of DNA damage response pathways comes into play. Studies in model organisms have contributed significantly to the understanding of these pathways. In the last decade, the development of functional genomics techniques in S. cerevisiae has ushered in systematic approaches for the study of complex cellular processes. These methods have enabled the systematic interrogation of the DNA damage response.

BLM, the helicase mutated in Bloom syndrome, associates with topoisomerase 3α, RMI1 (RecQ-mediated genome instability), and RPA, to form a complex essential for the maintenance of genome stability. Here we report a novel component of the BLM complex, RMI2, which interacts with RMI1 through two oligonucleotide-binding (OB)-fold domains similar to those in RPA. The resulting complex, named RMI, differs from RPA in that it lacks obvious DNA-binding activity. Nevertheless, RMI stimulates the dissolution of a homologous recombination intermediate in vitro and is essential for the stability, localization, and function of the BLM complex in vivo. Notably, inactivation of RMI2 in chicken DT40 cells results in an increased level of sister chromatid exchange (SCE)—the hallmark feature of Bloom syndrome cells. Epistasis analysis revealed that RMI2 and BLM suppress SCE within the same pathway. A point mutation in the OB domain of RMI2 disrupts the association between BLM and the rest of the complex, and abrogates the ability of RMI2 to suppress elevated SCE. Our data suggest that multi-OB-fold complexes mediate two modes of BLM action: via RPA-mediated protein–DNA interaction, and via RMI-mediated protein–protein interactions.

ELG1 (enhanced level of genome instability) encodes a Replication Factor C (RFC) homolog that is important for the maintenance of genome stability. Elg1 interacts with Rfc2–5, forming the third alternative RFC complex identified to date. We found that Elg1 plays a role in the suppression of spontaneous DNA damage in addition to its previously identified roles in the resistance to DNA damage. Using mutational analysis we examined the function of conserved and unique regions of Elg1 in these roles. We found that the Walker A motif in the conserved RFC region is dispensable for Elg1 function in vivo. The RFC region is important for association with chromatin although residues predicted to mediate interactions with DNA are dispensable for Elg1 function. The unique C-terminus of Elg1 mediates oligomerization with Rfc2–5, nuclear import, and chromatin association, and is critical for the function of Elg1. Finally, we demonstrated that the N-terminus of Elg1 contributes to the maintenance of genome stability, and that one function of this N-terminus is to promote the nuclear localization of Elg1. Together, these studies delineate the regions of Elg1 important for its function in damage resistance and in the suppression of spontaneous DNA damage.

DNA damage response pathways have been studied extensively in the budding yeast Saccharomyces cerevisiae, yet new genes with roles in the DNA damage response are still being identified. In this chapter we describe the use of functional genomic approaches in the identification of DNA damage response genes and pathways. These techniques take advantage of the S. cerevisiae gene deletion mutant collection, either as an ordered array or as a pool, and can be automated for high throughput.

DNA damage response pathways have been studied extensively in the budding yeast Saccharomyces cerevisiae, yet new genes with roles in the DNA damage response are still being identified. In this chapter we describe the use of functional genomic approaches in the identification of DNA damage response genes and pathways. These techniques take advantage of the S. cerevisiae gene deletion mutant collection, either as an ordered array or as a pool, and can be automated for high throughput.

Replication forks can stall spontaneously at specific sites in the genome, and upon encountering DNA lesions resulting from chemical or radiation damage. In Saccharomyces cerevisiae proteins implicated in processing of stalled replication forks include those encoded by the SGS1, TOP3, MUS81, MMS4, SLX1, SLX4, SLX5/HEX3, and SLX8 genes. We tested the roles of these genes in suppressing gross chromosomal rearrangements (GCRs), which include translocations, large interstitial deletions, and loss of a chromosome arm with de novo telomere addition. We found that mus81, mms4, slx1, slx4, slx5, and slx8 mutants all have elevated levels of spontaneous GCRs, and that SLX5 and SLX8 are particularly critical suppressors of GCRs during normal cell cycle progression. In addition to increased GCRs, deletion of SLX5 or SLX8 resulted in increased relocalization of the DNA damage checkpoint protein Ddc2 and activation of the checkpoint kinase Rad53, indicating the accumulation of spontaneous DNA damage. Surprisingly, mutants in slx5 or slx8 were not sensitive to transient replication fork stalling induced by hydroxyurea, nor were they sensitive to replication dependent double-strand breaks induced by camptothecin. This suggested that Slx8 and Slx8 played limited roles in stabilizing, restarting, or resolving transiently stalled replication forks, but were critical for preventing the accumulation of DNA damage during normal cell cycle progression.

Genome-wide synthetic genetic interaction screens with mutants in the mus81 and mms4 replication fork-processing genes identified a novel replication factor C (RFC) homolog, Elg1, which forms an alternative RFC complex with Rfc2-5. This complex is distinct from the DNA replication RFC, the DNA damage checkpoint RFC and the sister chromatid cohesion RFC. As expected from its genetic interactions, elg1 mutants are sensitive to DNA damage. Elg1 is redundant with Rad24 in the DNA damage response and contributes to activation of the checkpoint kinase Rad53. We find that elg1 mutants display DNA replication defects and genome instability, including increased recombination and mutation frequencies, and minichromosome maintenance defects. Mutants in elg1 show genetic interactions with pathways required for processing of stalled replication forks, and are defective in recovery from DNA damage during S phase. We propose that Elg1-RFC functions both in normal DNA replication and in the DNA damage response.

We performed a systematic screen of the set of ≈5,000 viable Saccharomyces cerevisiae haploid gene deletion mutants and have identified 103 genes whose deletion causes sensitivity to the DNA-damaging agent methyl methanesulfonate (MMS). In total, 40 previously uncharacterized alkylation damage response genes were identified. Comparison with the set of genes known to be transcriptionally induced in response to MMS revealed surprisingly little overlap with those required for MMS resistance, indicating that transcriptional regulation plays little, if any, role in the response to MMS damage. Clustering of the MMS response genes on the basis of their cross-sensitivities to hydroxyurea, UV radiation, and ionizing radiation revealed a DNA damage core of genes required for responses to a broad range of DNA-damaging agents. Of particular significance, we identified a subset of genes that show a specific MMS response, displaying defects in S phase progression only in the presence of MMS. These genes may promote replication fork stability or processivity during encounters between replication forks and DNA damage.

The fission yeast Dbf4 homologue Dfp1 has a well-characterized role in regulating the initiation of DNA replication. Sequence analysis of Dfp1 homologues reveals three highly conserved regions, referred to as motifs N, M, and C. To determine the roles of these conserved regions in Dfp1 function, we have generated dfp1 alleles with mutations in these regions. Mutations in motif N render cells sensitive to a broad range of DNA-damaging agents and replication inhibitors, yet these mutant proteins are efficient activators of Hsk1 kinase in vitro. In contrast, mutations in motif C confer sensitivity to the alkylating agent methyl methanesulfonate (MMS) but, surprisingly, not to UV, ionizing radiation, or hydroxyurea. Motif C mutants are poor activators of Hsk1 in vitro but can fulfill the essential function(s) of Dfp1 in vivo. Strains carrying dfp1 motif C mutants have an intact mitotic and intra-S-phase checkpoint, and epistasis analysis indicates that dfp1 motif C mutants function outside of the known MMS damage repair pathways, suggesting that the observed MMS sensitivity is due to defects in recovery from DNA damage. The motif C mutants are most sensitive to MMS during S phase and are partially suppressed by deletion of the S-phase checkpoint kinase cds1. Following treatment with MMS, dfp1 motif C mutants exhibit nuclear fragmentation, chromosome instability, precocious recombination, and persistent checkpoint activation. We propose that Dfp1 plays at least two genetically separable roles in the DNA damage response in addition to its well-characterized role in the initiation of DNA replication and that motif C plays a critical role in the response to alkylation damage, perhaps by restarting or stabilizing stalled replication forks.

Small molecules have been shown to be potent and selective probes to understand cell physiology. Here, we show that imidazo[1,2-a]pyridines and imidazo[1,2-a]pyrimidines compose a class of compounds that target essential, conserved cellular processes. Using validated chemogenomic assays in Saccharomyces cerevisiae, we discovered that two closely related compounds, an imidazo[1,2-a]pyridine and -pyrimidine that differ by a single atom, have distinctly different mechanisms of action in vivo. 2-phenyl-3-nitroso-imidazo[1,2-a]pyridine was toxic to yeast strains with defects in electron transport and mitochondrial functions and caused mitochondrial fragmentation, suggesting that compound 13 acts by disrupting mitochondria. By contrast, 2-phenyl-3-nitroso-imidazo[1,2-a]pyrimidine acted as a DNA poison, causing damage to the nuclear DNA and inducing mutagenesis. We compared compound 15 to known chemotherapeutics and found resistance required intact DNA repair pathways. Thus, subtle changes in the structure of imidazo-pyridines and -pyrimidines dramatically alter both the intracellular targeting of these compounds and their effects in vivo. Of particular interest, these different modes of action were evident in experiments on human cells, suggesting that chemical–genetic profiles obtained in yeast are recapitulated in cultured cells, indicating that our observations in yeast can: (1) be leveraged to determine mechanism of action in mammalian cells and (2) suggest novel structure–activity relationships., Author Summary We have shown that chemical–genetic screening allows structure–activity studies of chemical compounds at a very high resolution. In analyzing the effects of closely related imidazo-pyridine and -pyrimidine compounds, we found two compounds that likely act as oxidizing agents, yet target different organelles. The imidazo-pyridine affected mitochondrial functions whereas the imidazo-pyrimidine caused nuclear DNA damage. Remarkably, the only difference between these two compounds is the presence of a nitrogen atom at position 8. Thus, in addition to demonstrating the potential for high resolution in chemical–genetic studies, our work suggests that subtle changes in compound chemistry can be exploited to target different intracellular compartments with very different biological effects. Finally, we show that chemical–genetic profiling in yeast can be used to infer mode of action in mammalian cells. The specificity of compound 15 in eliciting a nuclear DNA damage response in evolutionarily diverse eukaryotes suggests that it will be of great utility in studying the cellular response to nuclear oxidative damage.

Versión postprint próximamente disponible en: http://hdl.handle.net/10261/228202, The response to DNA damage is critical for cellular homeostasis, tumor suppression, immunity and gametogenesis. In order to provide an unbiased and global view of the DNA damage response in human cells, we undertook 28 CRISPR/Cas9 screens against 25 genotoxic agents in the retinal pigment epithelium-1 (RPE1) cell line. These screens identified 840 genes whose loss causes either sensitivity or resistance to DNA damaging agents. Mining this dataset, we uncovered that ERCC6L2, which is mutated in a bone-marrow failure syndrome, codes for a canonical non-homologous end-joining pathway factor; that the RNA polymerase II component ELOF1 modulates the response to transcription-blocking agents and that the cytotoxicity of the G-quadruplex ligand pyridostatin involves trapping topoisomerase II on DNA. This map of the DNA damage response provides a rich resource to study this fundamental cellular system and has implications for the development and use of genotoxic agents in cancer therapy., AAQ, GSM and AMH are recipients of long-term EMBO fellowships (ALTF 910-2017, 795-2017 under a CC-BY-NC-ND 4.0 International license. not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available bioRxiv preprint doi: https://doi.org/10.1101/845446; this version posted November 18, 2019. The copyright holder for this preprint (which was 27 and 124-2019, respectively), NH was supported by a Human Frontier Science Program long-term Fellowship, SER is supported by a fellowship from AIRC and SA was a Banting post-doctoral fellow. ASB was supported by a PhD Studentship from AEFAT (Asociación Española Familia Ataxia Telangiectasia) and an EMBO short-term fellowship for a visit to the DD laboratory. The ICRF187 screen in FCL laboratory was funded by grants from the Spanish Government (SAF2017- 89619-R, European Regional Development Fund) and the European Research Council (ERC-CoG2014-647359). Work in the RSW laboratory was supported in part by the US National Institute of Health Intramural Program, US National Institute of Environmental Health Sciences (NIEHS, 1Z01ES102765). DD is a Canada Research Chair (Tier I) and the work was supported from grants from the CIHR (FDN143343 to DD; FRN 123518, PJT-156330 to AM) Canadian Cancer Society grant 705644 (to DD) with additional support to DD from the Krembil Foundation.

Here, we present two approaches to map DNA double-strand breaks (DSBs) and single-strand breaks (SSBs) in the genome of human cells. We named these methods respectively DSB-Seq and SSB-Seq. We tested the DSB and SSB-Seq in HCT1116, human colon cancer cells, and validated the results using the topoisomerase 2 (Top2)-poisoning agent etoposide (ETO). These methods are powerful tools for the direct detection of the physiological and pathological “breakome” of the DNA in human cells.