ABSTRACT
All organisms possess molecular mechanisms that govern DNA repair and associated DNA damage response (DDR) processes. Owing to their relevance to human disease, most notably cancer, these mechanisms have been studied extensively, yet new DNA repair and/or DDR factors and functional interactions between them are still being uncovered. The emergence of CRISPR technologies and CRISPR-based genetic screens has enabled genome-scale analyses of gene-gene and gene-drug interactions, thereby providing new insights into cellular processes in distinct DDR-deficiency genetic backgrounds and conditions. In this Review, we discuss the mechanistic basis of CRISPR-Cas genetic screening approaches and describe how they have contributed to our understanding of DNA repair and DDR pathways. We discuss how DNA repair pathways are regulated, and identify and characterize crosstalk between them. We also highlight the impacts of CRISPR-based studies in identifying novel strategies for cancer therapy, and in understanding, overcoming and even exploiting cancer-drug resistance, for example in the contexts of PARP inhibition, homologous recombination deficiencies and/or replication stress. Lastly, we present the DDR CRISPR screen (DDRcs) portal , in which we have collected and reanalysed data from CRISPR screen studies and provide a tool for systematically exploring them.
Subject(s)
CRISPR-Cas Systems , Neoplasms , Humans , CRISPR-Cas Systems/genetics , DNA Repair/genetics , Neoplasms/therapy , Neoplasms/drug therapy , Genome , DNA Damage/geneticsABSTRACT
Whole-genome-sequencing (WGS) of human tumors has revealed distinct mutation patterns that hint at the causative origins of cancer. We examined mutational signatures in 324 WGS human-induced pluripotent stem cells exposed to 79 known or suspected environmental carcinogens. Forty-one yielded characteristic substitution mutational signatures. Some were similar to signatures found in human tumors. Additionally, six agents produced double-substitution signatures and eight produced indel signatures. Investigating mutation asymmetries across genome topography revealed fully functional mismatch and transcription-coupled repair pathways. DNA damage induced by environmental mutagens can be resolved by disparate repair and/or replicative pathways, resulting in an assortment of signature outcomes even for a single agent. This compendium of experimentally induced mutational signatures permits further exploration of roles of environmental agents in cancer etiology and underscores how human stem cell DNA is directly vulnerable to environmental agents. VIDEO ABSTRACT.
Subject(s)
Carcinogens, Environmental/classification , Neoplasms/genetics , Carcinogens, Environmental/adverse effects , DNA Damage/genetics , DNA Mutational Analysis/methods , DNA Repair/genetics , DNA Replication , Genetic Profile , Genome, Human/genetics , Humans , INDEL Mutation/genetics , Mutagenesis , Mutation/genetics , Pluripotent Stem Cells/metabolism , Whole Genome Sequencing/methodsABSTRACT
The DNA damage response (DDR) fulfils essential roles to preserve genome integrity. Targeting the DDR in tumors has had remarkable success over the last decade, exemplified by the licensing of PARP inhibitors for cancer therapy. Recent studies suggest that the application of DDR inhibitors impacts on cellular innate and adaptive immune responses, wherein key DNA repair factors have roles in limiting chronic inflammatory signaling. Antitumor immunity plays an emerging part in cancer therapy, and extensive efforts have led to the development of immune checkpoint inhibitors overcoming immune suppressive signals in tumors. Here, we review the current understanding of the molecular mechanisms underlying DNA damage-triggered immune responses, including cytosolic DNA sensing via the cGAS/STING pathway. We highlight the implications of DDR components for therapeutic outcomes of immune checkpoint inhibitors or their use as biomarkers. Finally, we discuss the rationale for novel combinations of DDR inhibitors with antagonists of immune checkpoints and current hindrances limiting their broader therapeutic applications.
Subject(s)
DNA Repair/physiology , Immunity, Cellular/genetics , Immunotherapy , Neoplasms/therapy , Adaptive Immunity/genetics , DNA Damage/immunology , Discoidin Domain Receptors/antagonists & inhibitors , Humans , Immune Checkpoint Inhibitors/therapeutic use , Neoplasms/drug therapy , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Poly(ADP-ribose) Polymerase Inhibitors/therapeutic use , Protein Kinase Inhibitors/therapeutic useABSTRACT
The nucleoside analogue decitabine (or 5-aza-dC) is used to treat several haematological cancers. Upon its triphosphorylation and incorporation into DNA, 5-aza-dC induces covalent DNA methyltransferase 1 DNA-protein crosslinks (DNMT1-DPCs), leading to DNA hypomethylation. However, 5-aza-dC's clinical outcomes vary, and relapse is common. Using genome-scale CRISPR/Cas9 screens, we map factors determining 5-aza-dC sensitivity. Unexpectedly, we find that loss of the dCMP deaminase DCTD causes 5-aza-dC resistance, suggesting that 5-aza-dUMP generation is cytotoxic. Combining results from a subsequent genetic screen in DCTD-deficient cells with the identification of the DNMT1-DPC-proximal proteome, we uncover the ubiquitin and SUMO1 E3 ligase, TOPORS, as a new DPC repair factor. TOPORS is recruited to SUMOylated DNMT1-DPCs and promotes their degradation. Our study suggests that 5-aza-dC-induced DPCs cause cytotoxicity when DPC repair is compromised, while cytotoxicity in wild-type cells arises from perturbed nucleotide metabolism, potentially laying the foundations for future identification of predictive biomarkers for decitabine treatment.
Subject(s)
DNA (Cytosine-5-)-Methyltransferase 1 , Decitabine , Ubiquitin-Protein Ligases , Decitabine/pharmacology , Humans , DNA (Cytosine-5-)-Methyltransferase 1/metabolism , DNA (Cytosine-5-)-Methyltransferase 1/genetics , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/genetics , DNA Methylation/drug effects , Antimetabolites, Antineoplastic/pharmacology , Animals , Sumoylation/drug effectsABSTRACT
Trimethylation of histone H3 on Lys36 (H3K36me3) by SETD2 is linked to actively transcribed regions. Li et al. identify a novel role for H3K36me3 that facilitates DNA mismatch repair (MMR) in cells by targeting the MMR machinery to chromatin during the cell cycle, thereby explaining certain cases of MMR-defective cancers.
ABSTRACT
DNA single-strand breaks (SSBs) disrupt DNA replication and induce chromosome breakage. However, whether SSBs induce chromosome breakage when present behind replication forks or ahead of replication forks is unclear. To address this question, we exploited an exquisite sensitivity of SSB repair-defective human cells lacking PARP activity or XRCC1 to the thymidine analogue 5-chloro-2'-deoxyuridine (CldU). We show that incubation with CldU in these cells results in chromosome breakage, sister chromatid exchange, and cytotoxicity by a mechanism that depends on the S phase activity of uracil DNA glycosylase (UNG). Importantly, we show that CldU incorporation in one cell cycle is cytotoxic only during the following cell cycle, when it is present in template DNA. In agreement with this, while UNG induces SSBs both in nascent strands behind replication forks and in template strands ahead of replication forks, only the latter trigger fork collapse and chromosome breakage. Finally, we show that BRCA-defective cells are hypersensitive to CldU, either alone and/or in combination with PARP inhibitor, suggesting that CldU may have clinical utility.
Subject(s)
Antineoplastic Agents , Poly(ADP-ribose) Polymerase Inhibitors , Humans , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Chromosome Breakage , DNA Repair , DNA Replication , DNA , X-ray Repair Cross Complementing Protein 1/metabolismABSTRACT
The balance between self-renewal and differentiation in human foetal lung epithelial progenitors controls the size and function of the adult organ. Moreover, progenitor cell gene regulation networks are employed by both regenerating and malignant lung cells, where modulators of their effects could potentially be of therapeutic value. Details of the molecular networks controlling human lung progenitor self-renewal remain unknown. We performed the first CRISPRi screen in primary human lung organoids to identify transcription factors controlling progenitor self-renewal. We show that SOX9 promotes proliferation of lung progenitors and inhibits precocious airway differentiation. Moreover, by identifying direct transcriptional targets using Targeted DamID, we place SOX9 at the centre of a transcriptional network, which amplifies WNT and RTK signalling to stabilise the progenitor cell state. In addition, the proof-of-principle CRISPRi screen and Targeted DamID tools establish a new workflow for using primary human organoids to elucidate detailed functional mechanisms underlying normal development and disease.
Subject(s)
Lung , SOX9 Transcription Factor , Stem Cells , Humans , Cell Differentiation/physiology , Lung/embryology , Signal Transduction , SOX9 Transcription Factor/metabolism , Stem Cells/metabolismABSTRACT
In response to genotoxic stress, cells activate a signaling cascade known as the DNA damage checkpoint (DDC) that leads to a temporary cell cycle arrest and activation of DNA repair mechanisms. Because persistent DDC activation compromises cell viability, this process must be tightly regulated. However, despite its importance, the mechanisms regulating DDC recovery are not completely understood. Here, we identify a DNA-damage-regulated histone modification in Saccharomyces cerevisiae, phosphorylation of H4 threonine 80 (H4T80ph), and show that it triggers checkpoint inactivation. H4T80ph is critical for cell survival to DNA damage, and its absence causes impaired DDC recovery and persistent cell cycle arrest. We show that, in response to genotoxic stress, p21-activated kinase Cla4 phosphorylates H4T80 to recruit Rtt107 to sites of DNA damage. Rtt107 displaces the checkpoint adaptor Rad9, thereby interrupting the checkpoint-signaling cascade. Collectively, our results indicate that H4T80ph regulates DDC recovery.
Subject(s)
DNA Damage , DNA Repair , Histones/genetics , Histones/metabolism , Cell Cycle Checkpoints/genetics , Cell Cycle Proteins , Checkpoint Kinase 2/genetics , Checkpoint Kinase 2/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Nuclear Proteins/metabolism , Phosphorylation , Protein Binding , Protein Serine-Threonine Kinases/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Signal TransductionABSTRACT
This Article has been retracted; see accompanying Retraction.
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 SequencingABSTRACT
In vertebrate cells, the DNA damage response is controlled by three related kinases: ATM, ATR, and DNA-PK. It has been 20 years since the cloning of ATR, the last of the three to be identified. During this time, our understanding of how these kinases regulate DNA repair and associated events has grown profoundly, although major questions remain unanswered. Here, we provide a historical perspective of their discovery and discuss their established functions in sensing and responding to genotoxic stress. We also highlight what is known regarding their structural similarities and common mechanisms of regulation, as well as emerging non-canonical roles and how our knowledge of ATM, ATR, and DNA-PK is being translated to benefit human health.
Subject(s)
Ataxia Telangiectasia Mutated Proteins/metabolism , Calcium-Binding Proteins/metabolism , Cell Nucleus/enzymology , DNA Damage , DNA Repair , Animals , Antineoplastic Agents/therapeutic use , Ataxia Telangiectasia Mutated Proteins/antagonists & inhibitors , Ataxia Telangiectasia Mutated Proteins/chemistry , Ataxia Telangiectasia Mutated Proteins/history , Calcium-Binding Proteins/antagonists & inhibitors , Calcium-Binding Proteins/chemistry , Calcium-Binding Proteins/history , DNA Repair/drug effects , Enzyme Activation , History, 20th Century , History, 21st Century , Humans , Models, Molecular , Neoplasms/drug therapy , Neoplasms/enzymology , Neoplasms/genetics , Neoplasms/pathology , Phosphorylation , Protein Conformation , Protein Kinase Inhibitors/therapeutic use , Signal Transduction , Structure-Activity Relationship , Tumor Suppressor Protein p53/metabolismABSTRACT
The Mre11/Rad50/Nbs1 protein complex plays central enzymatic and signaling roles in the DNA-damage response. Nuclease (Mre11) and scaffolding (Rad50) components of MRN have been extensively characterized, but the molecular basis of Nbs1 function has remained elusive. Here, we present a 2.3A crystal structure of the N-terminal region of fission yeast Nbs1, revealing an unusual but conserved architecture in which the FHA- and BRCT-repeat domains structurally coalesce. We demonstrate that diphosphorylated pSer-Asp-pThr-Asp motifs, recently identified as multicopy docking sites within Mdc1, are evolutionarily conserved Nbs1 binding targets. Furthermore, we show that similar phosphomotifs within Ctp1, the fission yeast ortholog of human CtIP, promote interactions with the Nbs1 FHA domain that are necessary for Ctp1-dependent resistance to DNA damage. Finally, we establish that human Nbs1 interactions with Mdc1 occur through both its FHA- and BRCT-repeat domains, suggesting how their structural and functional interdependence underpins Nbs1 adaptor functions in the DNA-damage response.
Subject(s)
Cell Cycle Proteins/chemistry , Chromosomal Proteins, Non-Histone/chemistry , DNA Repair , Nuclear Proteins/chemistry , Schizosaccharomyces pombe Proteins/chemistry , Schizosaccharomyces/chemistry , Amino Acid Sequence , Crystallography, X-Ray , DNA Damage , DNA-Binding Proteins/metabolism , Humans , Models, Molecular , Molecular Sequence Data , Mutation , Phosphorylation , Protein Structure, Tertiary , Schizosaccharomyces/metabolism , Schizosaccharomyces pombe Proteins/metabolism , Sequence AlignmentABSTRACT
G-quadruplex (G4)-forming genomic sequences, including telomeres, represent natural replication fork barriers. Stalled replication forks can be stabilized and restarted by homologous recombination (HR), which also repairs DNA double-strand breaks (DSBs) arising at collapsed forks. We have previously shown that HR facilitates telomere replication. Here, we demonstrate that the replication efficiency of guanine-rich (G-rich) telomeric repeats is decreased significantly in cells lacking HR. Treatment with the G4-stabilizing compound pyridostatin (PDS) increases telomere fragility in BRCA2-deficient cells, suggesting that G4 formation drives telomere instability. Remarkably, PDS reduces proliferation of HR-defective cells by inducing DSB accumulation, checkpoint activation, and deregulated G2/M progression and by enhancing the replication defect intrinsic to HR deficiency. PDS toxicity extends to HR-defective cells that have acquired olaparib resistance through loss of 53BP1 or REV7. Altogether, these results highlight the therapeutic potential of G4-stabilizing drugs to selectively eliminate HR-compromised cells and tumors, including those resistant to PARP inhibition.
Subject(s)
Aminoquinolines/pharmacology , Antineoplastic Agents/pharmacology , BRCA1 Protein/deficiency , BRCA2 Protein/deficiency , Biomarkers, Tumor/deficiency , G-Quadruplexes/drug effects , Neoplasms/drug therapy , Picolinic Acids/pharmacology , Animals , BRCA1 Protein/genetics , BRCA2 Protein/genetics , Biomarkers, Tumor/genetics , Cell Proliferation/drug effects , DNA Breaks, Double-Stranded , Dose-Response Relationship, Drug , Drug Resistance, Neoplasm , G2 Phase Cell Cycle Checkpoints/drug effects , HEK293 Cells , Humans , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Mad2 Proteins/genetics , Mad2 Proteins/metabolism , Male , Mice, Nude , Molecular Targeted Therapy , Neoplasms/genetics , Neoplasms/metabolism , Neoplasms/pathology , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , RNA Interference , Telomere/drug effects , Telomere/genetics , Telomere/metabolism , Time Factors , Transfection , Tumor Burden/drug effects , Tumor Suppressor p53-Binding Protein 1 , Xenograft Model Antitumor AssaysABSTRACT
SUMOylation is critical for numerous cellular signalling pathways, including the maintenance of genome integrity via the repair of DNA double-strand breaks (DSBs). If misrepaired, DSBs can lead to cancer, neurodegeneration, immunodeficiency and premature ageing. Using systematic human proteome microarray screening combined with widely applicable carbene footprinting, genetic code expansion and high-resolution structural profiling, we define two non-conventional and topology-selective SUMO2-binding regions on XRCC4, a DNA repair protein important for DSB repair by non-homologous end-joining (NHEJ). Mechanistically, the interaction of SUMO2 and XRCC4 is incompatible with XRCC4 binding to three other proteins important for NHEJ-mediated DSB repair. These findings are consistent with SUMO2 forming a redundant NHEJ layer with the potential to regulate different NHEJ complexes at distinct levels including, but not limited to, XRCC4 interactions with XLF, LIG4 and IFFO1. Regulation of NHEJ is not only relevant for carcinogenesis, but also for the design of precision anti-cancer medicines and the optimisation of CRISPR/Cas9-based gene editing. In addition to providing molecular insights into NHEJ, this work uncovers a conserved SUMO-binding module and provides a rich resource on direct SUMO binders exploitable towards uncovering SUMOylation pathways in a wide array of cellular processes.
Subject(s)
DNA End-Joining Repair , DNA Repair , DNA Breaks, Double-Stranded , DNA Repair Enzymes/metabolism , Humans , Microarray Analysis , Protein Binding , Small Ubiquitin-Related Modifier Proteins , SumoylationABSTRACT
PAXX was identified recently as a novel nonhomologous end-joining DNA repair factor in human cells. To characterize its physiological roles, we generated Paxx-deficient mice. Like Xlf-/- mice, Paxx-/- mice are viable, grow normally, and are fertile but show mild radiosensitivity. Strikingly, while Paxx loss is epistatic with Ku80, Lig4, and Atm deficiency, Paxx/Xlf double-knockout mice display embryonic lethality associated with genomic instability, cell death in the central nervous system, and an almost complete block in lymphogenesis, phenotypes that closely resemble those of Xrcc4-/- and Lig4-/- mice. Thus, combined loss of Paxx and Xlf is synthetic-lethal in mammals.
Subject(s)
DNA-Binding Proteins/genetics , Embryonic Development/genetics , Synthetic Lethal Mutations/genetics , Trisaccharides/genetics , Animals , Apoptosis/genetics , DNA-Binding Proteins/metabolism , Epistasis, Genetic , Genomic Instability/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Protein Kinases/genetics , Protein Kinases/metabolism , Radiation Tolerance/genetics , Trisaccharides/metabolismABSTRACT
Repair of DNA double-strand breaks is crucial for maintaining genome integrity and is governed by post-translational modifications such as protein ubiquitylation. Here, we establish that the deubiquitylating enzyme USP4 promotes DNA-end resection and DNA repair by homologous recombination. We also report that USP4 interacts with CtIP and the MRE11-RAD50-NBS1 (MRN) complex and is required for CtIP recruitment to DNA damage sites. Furthermore, we show that USP4 is ubiquitylated on multiple sites including those on cysteine residues and that deubiquitylation of these sites requires USP4 catalytic activity and is required for USP4 to interact with CtIP/MRN and to promote CtIP recruitment and DNA repair. Lastly, we establish that regulation of interactor binding by ubiquitylation occurs more generally among USP-family enzymes. Our findings thus identify USP4 as a novel DNA repair regulator and invoke a model in which ubiquitin adducts regulate USP enzyme interactions and functions.
Subject(s)
DNA Breaks, Double-Stranded , Models, Biological , Recombinational DNA Repair , Ubiquitin Thiolesterase/metabolism , Ubiquitination/physiology , Acid Anhydride Hydrolases , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Line, Tumor , DNA Repair Enzymes/genetics , DNA Repair Enzymes/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Endodeoxyribonucleases , Humans , MRE11 Homologue Protein , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Ubiquitin Thiolesterase/genetics , Ubiquitin-Specific ProteasesABSTRACT
The Bloom syndrome helicase BLM and topoisomerase-IIß-binding protein 1 (TopBP1) are key regulators of genome stability. It was recently proposed that BLM phosphorylation on Ser338 mediates its interaction with TopBP1, to protect BLM from ubiquitylation and degradation (Wang et al., 2013). Here, we show that the BLM-TopBP1 interaction does not involve Ser338 but instead requires BLM phosphorylation on Ser304. Furthermore, we establish that disrupting this interaction does not markedly affect BLM stability. However, BLM-TopBP1 binding is important for maintaining genome integrity, because in its absence cells display increased sister chromatid exchanges, replication origin firing and chromosomal aberrations. Therefore, the BLM-TopBP1 interaction maintains genome stability not by controlling BLM protein levels, but via another as-yet undetermined mechanism. Finally, we identify critical residues that mediate interactions between TopBP1 and MDC1, and between BLM and TOP3A/RMI1/RMI2. Taken together, our findings provide molecular insights into a key tumor suppressor and genome stability network.
Subject(s)
Carrier Proteins/metabolism , DNA-Binding Proteins/metabolism , Genomic Instability , Nuclear Proteins/metabolism , RecQ Helicases/metabolism , Adaptor Proteins, Signal Transducing , Amino Acid Sequence , Carrier Proteins/genetics , Cell Cycle Proteins , DNA Topoisomerases, Type I/genetics , DNA Topoisomerases, Type I/metabolism , DNA-Binding Proteins/genetics , HeLa Cells , Humans , Molecular Sequence Data , Mutation , Nuclear Proteins/genetics , Phosphorylation , RecQ Helicases/genetics , Serine/metabolism , Trans-Activators/genetics , Trans-Activators/metabolismABSTRACT
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/metabolismABSTRACT
The protein kinase ATR plays pivotal roles in DNA repair, cell cycle checkpoint engagement and DNA replication. Consequently, ATR inhibitors (ATRi) are in clinical development for the treatment of cancers, including tumours harbouring mutations in the related kinase ATM. However, it still remains unclear which functions and pathways dominate long-term ATRi efficacy, and how these vary between clinically relevant genetic backgrounds. Elucidating common and genetic-background specific mechanisms of ATRi efficacy could therefore assist in patient stratification and pre-empting drug resistance. Here, we use CRISPR-Cas9 genome-wide screening in ATM-deficient and proficient mouse embryonic stem cells to interrogate cell fitness following treatment with the ATRi, ceralasertib. We identify factors that enhance or suppress ATRi efficacy, with a subset of these requiring intact ATM signalling. Strikingly, two of the strongest resistance-gene hits in both ATM-proficient and ATM-deficient cells encode Cyclin C and CDK8: members of the CDK8 kinase module for the RNA polymerase II mediator complex. We show that Cyclin C/CDK8 loss reduces S-phase DNA:RNA hybrid formation, transcription-replication stress, and ultimately micronuclei formation induced by ATRi. Overall, our work identifies novel biomarkers of ATRi efficacy in ATM-proficient and ATM-deficient cells, and highlights transcription-associated replication stress as a predominant driver of ATRi-induced cell death.
Subject(s)
Ataxia Telangiectasia Mutated Proteins/genetics , Cyclin C/genetics , Cyclin-Dependent Kinase 8/genetics , Transcription, Genetic , Animals , Ataxia Telangiectasia Mutated Proteins/antagonists & inhibitors , Cell Line, Tumor , DNA Damage/drug effects , DNA Repair/drug effects , DNA Replication/drug effects , Humans , Mice , Mouse Embryonic Stem Cells/drug effects , Protein Kinase Inhibitors/pharmacology , Signal Transduction/drug effectsABSTRACT
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.