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).

Chromosome instability (CIN) is observed in most solid tumors and is linked to somatic mutations in genome integrity maintenance genes. The spectrum of mutations that cause CIN is only partly known and it is not possible to predict a priori all pathways whose disruption might lead to CIN. To address this issue, we generated a catalogue of CIN genes and pathways by screening ∼2,000 reduction-of-function alleles for 90% of essential genes in Saccharomyces cerevisiae. Integrating this with published CIN phenotypes for other yeast genes generated a systematic CIN gene dataset comprised of 692 genes. Enriched gene ontology terms defined cellular CIN pathways that, together with sequence orthologs, created a list of human CIN candidate genes, which we cross-referenced to published somatic mutation databases revealing hundreds of mutated CIN candidate genes. Characterization of some poorly characterized CIN genes revealed short telomeres in mutants of the ASTRA/TTT components TTI1 and ASA1. High-throughput phenotypic profiling links ASA1 to TTT (Tel2-Tti1-Tti2) complex function and to TORC1 signaling via Tor1p stability, consistent with the role of TTT in PI3-kinase related kinase biogenesis. The comprehensive CIN gene list presented here in principle comprises all conserved eukaryotic genome integrity pathways. Deriving human CIN candidate genes from the list allows direct cross-referencing with tumor mutational data and thus candidate mutations potentially driving CIN in tumors. Overall, the CIN gene spectrum reveals new chromosome biology and will help us to understand CIN phenotypes in human disease.

DNA:RNA hybrid formation is emerging as a significant cause of genome instability in biological systems ranging from bacteria to mammals. Here we describe the genome-wide distribution of DNA:RNA hybrid prone loci in Saccharomyces cerevisiae by DNA:RNA immunoprecipitation (DRIP) followed by hybridization on tiling microarray. These profiles show that DNA:RNA hybrids preferentially accumulated at rDNA, Ty1 and Ty2 transposons, telomeric repeat regions and a subset of open reading frames (ORFs). The latter are generally highly transcribed and have high GC content. Interestingly, significant DNA:RNA hybrid enrichment was also detected at genes associated with antisense transcripts. The expression of antisense-associated genes was also significantly altered upon overexpression of RNase H, which degrades the RNA in hybrids. Finally, we uncover mutant-specific differences in the DRIP profiles of a Sen1 helicase mutant, RNase H deletion mutant and Hpr1 THO complex mutant compared to wild type, suggesting different roles for these proteins in DNA:RNA hybrid biology. Our profiles of DNA:RNA hybrid prone loci provide a resource for understanding the properties of hybrid-forming regions in vivo, extend our knowledge of hybrid-mitigating enzymes, and contribute to models of antisense-mediated gene regulation. A summary of this paper was presented at the 26th International Conference on Yeast Genetics and Molecular Biology, August 2013.

Genome instability via RNA:DNA hybrid-mediated R loops has been observed in mutants involved in various aspects of transcription and RNA processing. The prevalence of this mechanism among essential chromosome instability (CIN) genes remains unclear. In a secondary screen for increased Rad52 foci in CIN mutants, representing~25% of essential genes, we identified seven essential subunits of the mRNA cleavage and polyadenylation (mCP) machinery. Genome-wide analysis of fragile sites by chromatin immunoprecipitation (ChIP) and microarray (ChIP-chip) of phosphorylated H2A in these mutants supported a transcription-dependent mechanism of DNA damage characteristic of R loops. In parallel, we directly detected increased RNA:DNA hybrid formation in mCP mutants and demonstrated that CIN is suppressed by expression of the R-loop-degrading enzyme RNaseH. To investigate the conservation of CIN in mCP mutants, we focused on FIP1L1, the human ortholog of yeast FIP1, a conserved mCP component that is part of an oncogenic fusion in eosinophilic leukemia. We found that truncation fusions of yeast FIP1 analogous to those in cancer cause loss of function and that siRNA knockdown of FIP1L1 in human cells increases DNA damage and chromosome breakage. Our findings illuminate how mCP maintains genome integrity by suppressing R-loop formation and suggest that this function may be relevant to certain human cancers.

The eukaryotic cytosolic chaperonin containing TCP-1 (CCT) has an important function in maintaining cellular homoeostasis by assisting the folding of many proteins, including the cytoskeletal components actin and tubulin. Yet the nature of the proteins and cellular pathways dependent on CCT function has not been established globally. Here, we use proteomic and genomic approaches to define CCT interaction networks involving 136 proteins/genes that include links to the nuclear pore complex, chromatin remodelling, and protein degradation. Our study also identifies a third eukaryotic cytoskeletal system connected with CCT: the septin ring complex, which is essential for cytokinesis. CCT interactions with septins are ATP dependent, and disrupting the function of the chaperonin in yeast leads to loss of CCT-septin interaction and aberrant septin ring assembly. Our results therefore provide a rich framework for understanding the function of CCT in several essential cellular processes, including epigenetics and cell division.

Summary Like Ricin, Shiga, and Cholera toxins, yeast K28 is an A/B toxin that depends on endocytosis and retrograde trafficking for toxicity. Knowledge of the specific proteins, lipids, and mechanisms required for trafficking and killing by these toxins remains incomplete. Since K28 is a model for clinically relevant toxins, we screened over 5000 yeast mutants, identifying 365 that affect K28 sensitivity. Hypersensitive mutants revealed cytoprotective pathways, including stress-activated signaling and protein degradation. Resistant mutants clustered to endocytic, lipid organization and cell wall biogenesis pathways. Furthermore, GPI anchors and transcriptional regulation are important for K28-cell binding. Strikingly, the AP2 complex, which in metazoans links endocytic cargo to the clathrin coat, but had no assigned function in yeast, was critical for K28 toxicity. Yeast AP2 localizes to endocytic sites and has a cargo-specific function in K28 uptake. This comprehensive genetic analysis identified conserved processes important for A/B toxin trafficking and killing.

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Many ATP-dependent molecular chaperones, including Hsp70, Hsp90, and the chaperonins GroEL/Hsp60, require cofactor proteins to regulate their ATPase activities and thus folding functions in vivo. One conspicuous exception has been the eukaryotic chaperonin CCT, for which no regulator of its ATPase activity, other than non-native substrate proteins, is known. We identify the evolutionarily conserved PhLP3 (phosducin-like protein 3) as a modulator of CCT function in vitro and in vivo. PhLP3 binds CCT, spanning the cylindrical chaperonin cavity and contacting at least two subunits. When present in a ternary complex with CCT and an actin or tubulin substrate, PhLP3 significantly diminishes the chaperonin ATPase activity, and accordingly, excess PhLP3 perturbs actin or tubulin folding in vitro. Most interestingly, however, the Saccharomyces cerevisiae PhLP3 homologue is required for proper actin and tubulin function. This cellular role of PhLP3 is most apparent in a strain that also lacks prefoldin, a chaperone that facilitates CCT-mediated actin and tubulin folding. We propose that the antagonistic actions of PhLP3 and prefoldin serve to modulate CCT activity and play a key role in establishing a functional cytoskeleton in vivo.