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1.
Nucleic Acids Res ; 49(7): 3919-3931, 2021 04 19.
Article in English | MEDLINE | ID: mdl-33764464

ABSTRACT

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. Similarities between mutations generated by these alleles and those generated in pol2-4 cells indicate a shared mechanism of mutagenesis that yields a mutation pattern similar to cancer Signature 14. Comparison of POL2 ultra-mutator with pol2-M644G, a mutant in the polymerase domain decreasing Pol ϵ fidelity, revealed unexpected analogies in the sequence context and strand bias of mutations. Analysis of mutational patterns unique to exonuclease domain mutant cells suggests that backtracking of the polymerase, when the mismatched primer end cannot be accommodated in the proofreading domain, results in the observed insertions and T>A mutations in specific sequence contexts.


Subject(s)
Colorectal Neoplasms , DNA Polymerase II , Mutation Rate , Poly-ADP-Ribose Binding Proteins , Saccharomyces cerevisiae Proteins , Colorectal Neoplasms/enzymology , Colorectal Neoplasms/genetics , DNA Polymerase II/genetics , DNA Polymerase II/metabolism , DNA Replication , Humans , Mutagenesis , Mutation , Poly-ADP-Ribose Binding Proteins/genetics , Poly-ADP-Ribose Binding Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism
2.
Proc Natl Acad Sci U S A ; 117(40): 24947-24956, 2020 10 06.
Article in English | MEDLINE | ID: mdl-32968016

ABSTRACT

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 Saccharomyces cerevisiae mutation accumulation lines. This approach revealed the diversity, complexity, and ultimate uniqueness of mutational landscapes, differently composed of base substitutions, small insertions/deletions (InDels), structural variants, and/or ploidy variations. Several landscapes parallel the repertoire of mutational signatures in human cancers while others are either novel or composites of subsignatures resulting from distinct DNA damage lesions. Notably, the increase of base substitutions in the homologous recombination-deficient Rad51 mutant, specifically dependent on the Polζ translesion polymerase, yields COSMIC signature 3 observed in BRCA1/BRCA2-mutant breast cancer tumors. Furthermore, "mutome" analyses in highly polymorphic diploids and single-cell bottleneck lineages revealed a diverse spectrum of loss-of-heterozygosity (LOH) signatures characterized by interstitial and terminal chromosomal events resulting from interhomolog mitotic cross-overs. Following the appearance of heterozygous mutations, the strong stimulation of LOHs in the rad27/FEN1 and tsa1/PRDX1 backgrounds leads to fixation of homozygous mutations or their loss along the lineage. Overall, these mutomes and their trajectories provide a mechanistic framework to understand the origin and dynamics of genome variations that accumulate during clonal evolution.


Subject(s)
Breast Neoplasms/genetics , Carcinogenesis/genetics , Mutation/genetics , Saccharomyces cerevisiae/genetics , Acetyltransferases/genetics , BRCA1 Protein/genetics , BRCA2 Protein/genetics , Breast Neoplasms/pathology , DNA Damage/genetics , DNA-Directed DNA Polymerase , Diploidy , Female , Flap Endonucleases/genetics , Genome, Fungal/genetics , Humans , Loss of Heterozygosity/genetics , Membrane Proteins/genetics , Peroxiredoxins/genetics , Rad51 Recombinase/genetics , Saccharomyces cerevisiae Proteins/genetics , Whole Genome Sequencing
3.
Sci Rep ; 10(1): 2200, 2020 02 10.
Article in English | MEDLINE | ID: mdl-32042076

ABSTRACT

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. We addressed this issue by examining the consequences of exposing human primary cells to continuous ionising γ-radiation delivered at 6-20 mGy/h. Although these dose rates are estimated to inflict fewer than a single DNA double-strand break (DSB) per hour per cell, they still caused dose-dependent reductions in cell proliferation and increased cellular senescence. We concomitantly observed histone protein levels to reduce by up to 40%, which in contrast to previous observations, was not mainly due to protein degradation but instead correlated with reduced histone gene expression. Histone reductions were accompanied by enlarged nuclear size paralleled by an increase in global transcription, including that of pro-inflammatory genes. Thus, chronic irradiation, even at low dose-rates, can induce cell senescence and alter gene expression via a hitherto uncharacterised epigenetic route. These features of chronic radiation represent a new aspect of radiation biology.


Subject(s)
Chromatin/radiation effects , Gene Expression/radiation effects , Histones/radiation effects , Animals , Cell Line , Cell Proliferation/radiation effects , Cellular Senescence/radiation effects , DNA/radiation effects , DNA Breaks, Double-Stranded/radiation effects , DNA Damage/radiation effects , DNA Repair/physiology , DNA Repair/radiation effects , Dose-Response Relationship, Radiation , Gamma Rays , Histones/genetics , Humans , Male , Mice , Primary Cell Culture
4.
Nature ; 573(7774): 416-420, 2019 09.
Article in English | MEDLINE | ID: mdl-31511699

ABSTRACT

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 interactions1-3. Ensuing experimental work has also highlighted some inconsistencies and mistakes in the YKOC, or genome instability events that rebalance the effects of specific knockouts4-6, but a complete overview of these is lacking. The identification and analysis of genes that are required for maintaining genomic stability have traditionally relied on reporter assays and on the study of deletions of individual genes, but whole-genome-sequencing technologies now enable-in principle-the direct observation of genome instability globally and at scale. To exploit this opportunity, we sequenced the whole genomes of nearly all of the 4,732 strains comprising the homozygous diploid YKOC. Here, by extracting information on copy-number variation of tandem and interspersed repetitive DNA elements, we describe-for almost every single non-essential gene-the genomic alterations that are induced by its loss. Analysis of this dataset reveals genes that affect the maintenance of various genomic elements, highlights cross-talks between nuclear and mitochondrial genome stability, and shows how strains have genetically adapted to life in the absence of individual non-essential genes.


Subject(s)
Genome, Fungal/genetics , Genomic Instability , Saccharomyces cerevisiae/genetics , Adaptation, Biological/genetics , Gene Knockout Techniques , Genome, Mitochondrial/genetics , Whole Genome Sequencing
5.
Nat Commun ; 10(1): 87, 2019 01 08.
Article in English | MEDLINE | ID: mdl-30622252

ABSTRACT

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. We show that hypersensitivity of ATM-mutant cells to topotecan or the poly-(ADP-ribose) polymerase (PARP) inhibitor olaparib reflects delayed engagement of homologous recombination at DNA-replication-fork associated single-ended double-strand breaks (DSBs), allowing some to be subject to toxic NHEJ. Preventing DSB ligation by NHEJ, or enhancing homologous recombination by BRCA1-A complex disruption, suppresses this toxicity, highlighting a crucial role for ATM in preventing toxic LIG4-mediated chromosome fusions. Notably, suppressor mutations in ATM-mutant backgrounds are different to those in BRCA1-mutant scenarios, suggesting new opportunities for patient stratification and additional therapeutic vulnerabilities for clinical exploitation.


Subject(s)
Antineoplastic Agents/pharmacology , Ataxia Telangiectasia Mutated Proteins/genetics , DNA End-Joining Repair/genetics , Drug Resistance, Neoplasm/genetics , Animals , Antineoplastic Agents/therapeutic use , Ataxia Telangiectasia Mutated Proteins/metabolism , BRCA1 Protein/metabolism , CRISPR-Cas Systems/genetics , Cell Line, Tumor , Cell Survival/genetics , DNA Breaks, Double-Stranded/drug effects , DNA Ligase ATP/metabolism , DNA Replication/drug effects , DNA Replication/genetics , Female , Humans , Mice , Mice, Inbred NOD , Mice, Knockout , Mouse Embryonic Stem Cells , Mutation , Neoplasms, Experimental/drug therapy , Neoplasms, Experimental/genetics , Neoplasms, Experimental/pathology , Phthalazines/pharmacology , Phthalazines/therapeutic use , Piperazines/pharmacology , Piperazines/therapeutic use , Topotecan/pharmacology , Topotecan/therapeutic use
6.
Nat Cell Biol ; 20(8): 954-965, 2018 08.
Article in English | MEDLINE | ID: mdl-30022119

ABSTRACT

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. Mechanistically, we show that C20orf196 and FAM35A form a complex, 'Shieldin' (SHLD1/2), with FAM35A interacting with single-stranded DNA through its C-terminal oligonucleotide/oligosaccharide-binding fold region. We establish that Shieldin acts as the downstream effector of 53BP1/RIF1/MAD2L2 to promote DNA double-strand break (DSB) end-joining by restricting DSB resection and to counteract homologous recombination by antagonizing BRCA2/RAD51 loading in BRCA1-deficient cells. Notably, Shieldin inactivation further sensitizes BRCA1-deficient cells to cisplatin, suggesting how defining the SHLD1/2 status of BRCA1-deficient tumours might aid patient stratification and yield new treatment opportunities. Highlighting this potential, we document reduced SHLD1/2 expression in human breast cancers displaying intrinsic or acquired PARP-inhibitor resistance.


Subject(s)
BRCA1 Protein/genetics , Bone Neoplasms/drug therapy , Breast Neoplasms/drug therapy , DNA End-Joining Repair , Drug Resistance, Neoplasm , Osteosarcoma/drug therapy , Ovarian Neoplasms/drug therapy , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Proteins/metabolism , Recombinational DNA Repair , Animals , BRCA1 Protein/deficiency , Bone Neoplasms/genetics , Bone Neoplasms/metabolism , Bone Neoplasms/pathology , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Cycle Proteins , Cell Line, Tumor , Cisplatin/pharmacology , DNA Breaks, Double-Stranded , DNA-Binding Proteins , Dose-Response Relationship, Drug , Drug Resistance, Neoplasm/genetics , Female , HEK293 Cells , Humans , Mad2 Proteins/genetics , Mad2 Proteins/metabolism , Mice , Multiprotein Complexes , Osteosarcoma/genetics , Osteosarcoma/metabolism , Osteosarcoma/pathology , Ovarian Neoplasms/genetics , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , Proteins/genetics , Telomere-Binding Proteins/genetics , Telomere-Binding Proteins/metabolism , Tumor Suppressor p53-Binding Protein 1/genetics , Tumor Suppressor p53-Binding Protein 1/metabolism , Xenograft Model Antitumor Assays
7.
Sci Rep ; 8(1): 6161, 2018 04 18.
Article in English | MEDLINE | ID: mdl-29670134

ABSTRACT

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. In addition to applications in molecular biology research, this protocol can be used to identify drug targets and predict drug-resistance mechanisms. Mapping suppressor mutations on the primary or tertiary structures of protein suppressor hits provides insights into functionally relevant protein domains. Importantly, we show that olaparib resistance is linked to missense mutations in the DNA binding regions of PARP1, but not in its catalytic domain. This provides experimental support to the concept of PARP1 trapping on DNA as the prime source of toxicity to PARP inhibitors, and points to a novel olaparib resistance mechanism with potential therapeutic implications.


Subject(s)
Camptothecin/pharmacology , DNA Topoisomerases, Type I/genetics , Genetic Testing , Genome-Wide Association Study , Protein Domains/genetics , Protein Interaction Domains and Motifs , Amino Acid Sequence , Animals , Cell Line, Tumor , Embryonic Stem Cells , Humans , Mice , Models, Molecular , Mutation , Poly (ADP-Ribose) Polymerase-1/chemistry , Poly (ADP-Ribose) Polymerase-1/genetics , Poly (ADP-Ribose) Polymerase-1/metabolism , Protein Conformation
8.
EMBO Rep ; 18(6): 1000-1012, 2017 06.
Article in English | MEDLINE | ID: mdl-28389464

ABSTRACT

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 SIR1-4 suppresses much of the hypersensitivity of tof1∆ strains towards this agent. We show that disruption of rDNA or telomeric silencing does not mediate camptothecin resistance but that disruption of Sir1-dependent chromatin domains is sufficient to suppress camptothecin sensitivity in wild-type and tof1∆ cells. We suggest that topoisomerase 1 inhibition in proximity of these domains causes topological stress that leads to DNA hypercatenation, especially in the absence of the Tof1/Csm3 complex. Finally, we provide evidence of the evolutionarily conservation of this mechanism.


Subject(s)
Camptothecin/pharmacology , Chromatin , Saccharomyces cerevisiae Proteins/metabolism , Benzamides/pharmacology , Camptothecin/metabolism , Cell Cycle Proteins , DNA Damage , DNA Replication , DNA Topoisomerases, Type I/genetics , DNA Topoisomerases, Type I/metabolism , DNA, Fungal/genetics , DNA, Ribosomal/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Humans , Naphthols/pharmacology , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae Proteins/genetics , Silent Information Regulator Proteins, Saccharomyces cerevisiae/genetics , Silent Information Regulator Proteins, Saccharomyces cerevisiae/metabolism
9.
Nat Chem Biol ; 13(1): 12-14, 2017 Jan.
Article in English | MEDLINE | ID: mdl-27820796

ABSTRACT

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.


Subject(s)
Genetic Testing , Genome/drug effects , Genome/genetics , Mouse Embryonic Stem Cells/drug effects , Mouse Embryonic Stem Cells/metabolism , Mutagenesis/drug effects , Animals , Cell Line , Mice
10.
J Cell Biol ; 212(3): 321-34, 2016 Feb 01.
Article in English | MEDLINE | ID: mdl-26811423

ABSTRACT

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. Here, we combine imaging and computational modeling to ask how the association of a single gene locus with the nuclear envelope influences the surrounding chromosome architecture. Systematic analysis of an entire yeast chromosome establishes that peripheral recruitment of the GAL locus is part of a large-scale rearrangement that shifts many chromosomal regions closer to the nuclear envelope. This process is likely caused by the presence of several independent anchoring points. To identify novel factors required for peripheral anchoring, we performed a genome-wide screen and demonstrated that the histone acetyltransferase SAGA and the activity of histone deacetylases are needed for this extensive gene recruitment to the nuclear periphery.


Subject(s)
Chromatin Assembly and Disassembly , Chromatin/genetics , Chromosomes, Fungal/ultrastructure , Gene Expression Regulation, Fungal , Genes, Fungal , Genetic Loci , Nuclear Envelope/ultrastructure , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/genetics , Chromatin/metabolism , Computer Simulation , Galactokinase/genetics , Galactokinase/metabolism , Galactose/metabolism , Gene Library , Glucose/metabolism , Histone Deacetylases/metabolism , Models, Genetic , Nuclear Envelope/metabolism , Nucleic Acid Conformation , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Trans-Activators/genetics , Trans-Activators/metabolism
11.
EMBO J ; 34(11): 1509-22, 2015 Jun 03.
Article in English | MEDLINE | ID: mdl-25899817

ABSTRACT

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. By combining classic genetic suppressor screening with high-throughput DNA sequencing, we identify Mre11 mutations that strongly suppress DNA damage sensitivities of sae2∆ cells. By assessing the impacts of these mutations at the cellular, biochemical and structural levels, we propose that, in addition to promoting resection, a crucial role for Sae2 and Mre11 nuclease activity in mitotic DSB repair is to facilitate the removal of Mre11 from ssDNA associated with DSB ends. Thus, without Sae2 or Mre11 nuclease activity, Mre11 bound to partly processed DSBs impairs strand invasion and HR.


Subject(s)
DNA Repair/physiology , DNA, Fungal/metabolism , DNA, Single-Stranded/metabolism , Endonucleases/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , DNA, Fungal/genetics , DNA, Single-Stranded/genetics , Endodeoxyribonucleases/genetics , Endodeoxyribonucleases/metabolism , Endonucleases/genetics , Exodeoxyribonucleases/genetics , Exodeoxyribonucleases/metabolism , High-Throughput Nucleotide Sequencing , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics
12.
EMBO Rep ; 16(3): 341-50, 2015 Mar.
Article in English | MEDLINE | ID: mdl-25608529

ABSTRACT

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. Conversely up-regulating IP3 signalling decreases sensitivity. Tissue-specific rescue experiments suggest IP3 functions in the intestine. We also exploit IP3 signalling mutants to further enhance the sensitivity of RNAi hypersensitive strains. These results demonstrate that conserved cell signalling pathways can modify RNAi responses, implying that RNAi responses may be influenced by an animal's physiology or environment.


Subject(s)
Caenorhabditis elegans/physiology , Inositol 1,4,5-Trisphosphate/metabolism , RNA Interference/physiology , Signal Transduction/physiology , Animals , Caenorhabditis elegans/genetics , Image Processing, Computer-Assisted , Intestinal Mucosa/metabolism , Microscopy, Fluorescence , Models, Biological , RNA, Double-Stranded , Signal Transduction/genetics
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