Mutagenic mechanisms of cancer-associated DNA polymerase ϵ alleles.

A single amino acid residue change in the exonuclease domain of human DNA polymerase ϵ, P286R, is associated with the development of colorectal cancers, and has been shown to impart a mutator phenotype. The corresponding Pol ϵ allele in the yeast Saccharomyces cerevisiae (pol2-P301R), was found to drive greater mutagenesis than an entirely exonuclease-deficient Pol ϵ (pol2-4), an unexpected phenotype of ultra-mutagenesis. By studying the impact on mutation frequency, type, replication-strand bias, and sequence context, we show that ultra-mutagenesis is commonly observed in yeast cells carrying a range of cancer-associated Pol ϵ exonuclease domain alleles.

Trajectory and uniqueness of mutational signatures in yeast mutators.

The acquisition of mutations plays critical roles in adaptation, evolution, senescence, and tumorigenesis. Massive genome sequencing has allowed extraction of specific features of many mutational landscapes but it remains difficult to retrospectively determine the mechanistic origin(s), selective forces, and trajectories of transient or persistent mutations and genome rearrangements. Here, we conducted a prospective reciprocal approach to inactivate 13 single or multiple evolutionary conserved genes involved in distinct genome maintenance processes and characterize de novo mutations in 274 diploid mutation accumulation lines.

Chronic irradiation of human cells reduces histone levels and deregulates gene expression.

Over the past decades, there have been huge advances in understanding cellular responses to ionising radiation (IR) and DNA damage. These studies, however, were mostly executed with cell lines and mice using single or multiple acute doses of radiation. Hence, relatively little is known about how continuous exposure to low dose ionising radiation affects normal cells and organisms, even though our cells are constantly exposed to low levels of radiation.

Genome architecture and stability in the Saccharomyces cerevisiae knockout collection.

Despite major progress in defining the functional roles of genes, a complete understanding of their influences is far from being realized, even in relatively simple organisms. A major milestone in this direction arose via the completion of the yeast Saccharomyces cerevisiae gene-knockout collection (YKOC), which has enabled high-throughput reverse genetics, phenotypic screenings and analyses of synthetic-genetic interactions. Ensuing experimental work has also highlighted some inconsistencies and mistakes in the YKOC, or genome instability events that rebalance the effects of specific knockouts, but a complete overview of these is lacking.

ATM orchestrates the DNA-damage response to counter toxic non-homologous end-joining at broken replication forks.

Mutations in the ATM tumor suppressor gene confer hypersensitivity to DNA-damaging chemotherapeutic agents. To explore genetic resistance mechanisms, we performed genome-wide CRISPR-Cas9 screens in cells treated with the DNA topoisomerase I inhibitor topotecan. Thus, we here establish that inactivating terminal components of the non-homologous end-joining (NHEJ) machinery or of the BRCA1-A complex specifically confer topotecan resistance to ATM-deficient cells.

Shieldin complex promotes DNA end-joining and counters homologous recombination in BRCA1-null cells.

BRCA1 deficiencies cause breast, ovarian, prostate and other cancers, and render tumours hypersensitive to poly(ADP-ribose) polymerase (PARP) inhibitors. To understand the resistance mechanisms, we conducted whole-genome CRISPR-Cas9 synthetic-viability/resistance screens in BRCA1-deficient breast cancer cells treated with PARP inhibitors. We identified two previously uncharacterized proteins, C20orf196 and FAM35A, whose inactivation confers strong PARP-inhibitor resistance.

Detection of functional protein domains by unbiased genome-wide forward genetic screening.

Establishing genetic and chemo-genetic interactions has played key roles in elucidating mechanisms by which certain chemicals perturb cellular functions. In contrast to gene disruption/depletion strategies to identify mechanisms of drug resistance, searching for point-mutational genetic suppressors that can identify separation- or gain-of-function mutations has been limited. Here, by demonstrating its utility in identifying chemical-genetic suppressors of sensitivity to the DNA topoisomerase I poison camptothecin or the poly(ADP-ribose) polymerase inhibitor olaparib, we detail an approach allowing systematic, large-scale detection of spontaneous or chemically-induced suppressor mutations in yeast or haploid mammalian cells in a short timeframe, and with potential applications in other haploid systems.

Chromatin determinants impart camptothecin sensitivity.

Camptothecin-induced locking of topoisomerase 1 on DNA generates a physical barrier to replication fork progression and creates topological stress. By allowing replisome rotation, absence of the Tof1/Csm3 complex promotes the conversion of impending topological stress to DNA catenation and causes camptothecin hypersensitivity. Through synthetic viability screening, we discovered that histone H4 K16 deacetylation drives the sensitivity of yeast cells to camptothecin and that inactivation of this pathway by mutating H4 K16 or the genes suppresses much of the hypersensitivity of strains towards this agent.

Genome-wide genetic screening with chemically mutagenized haploid embryonic stem cells.

In model organisms, classical genetic screening via random mutagenesis provides key insights into the molecular bases of genetic interactions, helping to define synthetic lethality, synthetic viability and drug-resistance mechanisms. The limited genetic tractability of diploid mammalian cells, however, precludes this approach. Here, we demonstrate the feasibility of classical genetic screening in mammalian systems by using haploid cells, chemical mutagenesis and next-generation sequencing, providing a new tool to explore mammalian genetic interactions.

Global reorganization of budding yeast chromosome conformation in different physiological conditions.

The organization of the genome is nonrandom and important for correct function. Specifically, the nuclear envelope plays a critical role in gene regulation. It generally constitutes a repressive environment, but several genes, including the GAL locus in budding yeast, are recruited to the nuclear periphery on activation.

Synthetic viability genomic screening defines Sae2 function in DNA repair.

DNA double-strand break (DSB) repair by homologous recombination (HR) requires 3' single-stranded DNA (ssDNA) generation by 5' DNA-end resection. During meiosis, yeast Sae2 cooperates with the nuclease Mre11 to remove covalently bound Spo11 from DSB termini, allowing resection and HR to ensue. Mitotic roles of Sae2 and Mre11 nuclease have remained enigmatic, however, since cells lacking these display modest resection defects but marked DNA damage hypersensitivities.

IP3 signalling regulates exogenous RNAi in Caenorhabditis elegans.

RNA interference (RNAi) is a widespread and widely exploited phenomenon. Here, we show that changing inositol 1,4,5-trisphosphate (IP3) signalling alters RNAi sensitivity in Caenorhabditis elegans. Reducing IP3 signalling enhances sensitivity to RNAi in a broad range of genes and tissues.