ABSTRACT
Oncogene-induced replication stress (RS) promotes cancer development but also impedes tumor growth by activating anti-cancer barriers. To determine how cancer cells adapt to RS, we have monitored the expression of different components of the ATR-CHK1 pathway in primary tumor samples. We show that unlike upstream components of the pathway, the checkpoint mediators Claspin and Timeless are overexpressed in a coordinated manner. Remarkably, reducing the levels of Claspin and Timeless in HCT116 cells to pretumoral levels impeded fork progression without affecting checkpoint signaling. These data indicate that high level of Claspin and Timeless increase RS tolerance by protecting replication forks in cancer cells. Moreover, we report that primary fibroblasts adapt to oncogene-induced RS by spontaneously overexpressing Claspin and Timeless, independently of ATR signaling. Altogether, these data indicate that enhanced levels of Claspin and Timeless represent a gain of function that protects cancer cells from of oncogene-induced RS in a checkpoint-independent manner.
Subject(s)
Adaptor Proteins, Signal Transducing/biosynthesis , Adenocarcinoma of Lung/pathology , Breast Neoplasms/pathology , Cell Cycle Proteins/biosynthesis , Colorectal Neoplasms/pathology , Intracellular Signaling Peptides and Proteins/biosynthesis , Stress, Physiological/physiology , Adaptor Proteins, Signal Transducing/genetics , Adenocarcinoma of Lung/genetics , Ataxia Telangiectasia Mutated Proteins/metabolism , Breast Neoplasms/genetics , Cell Cycle Proteins/genetics , Cell Line, Tumor , Checkpoint Kinase 1/metabolism , Colorectal Neoplasms/genetics , DNA Damage/genetics , Genomic Instability/genetics , HCT116 Cells , HeLa Cells , Humans , Intracellular Signaling Peptides and Proteins/genetics , MCF-7 Cells , Stress, Physiological/geneticsABSTRACT
Ultraviolet (UV) induces distorting lesions to the DNA that can lead to stalling of the RNA polymerase II (RNAP II) and that are removed by transcription-coupled nucleotide excision repair (TC-NER). In humans, mutations in the TC-NER genes CSA and CSB lead to severe postnatal developmental defects in Cockayne syndrome patients. In Caenorhabditis elegans, mutations in the TC-NER genes csa-1 and csb-1, lead to developmental growth arrest upon UV treatment. We conducted a genetic suppressor screen in the nematode to identify mutations that could suppress the developmental defects in csb-1 mutants. We found that mutations in the ERK1/2 MAP kinase mpk-1 alleviate the developmental retardation in TC-NER mutants, while constitutive activation of the RAS-MAPK pathway exacerbates the DNA damage-induced growth arrest. We show that MPK-1 act via insulin/insulin-like signaling pathway and regulates the FOXO transcription factor DAF-16 to mediate the developmental DNA damage response.
Subject(s)
Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , DNA Repair , Forkhead Transcription Factors/metabolism , MAP Kinase Signaling System , Mitogen-Activated Protein Kinase 1/genetics , Animals , Caenorhabditis elegans/enzymology , Caenorhabditis elegans/genetics , Caenorhabditis elegans/growth & development , Larva/enzymology , Larva/genetics , Larva/metabolism , Mitogen-Activated Protein Kinase 1/metabolism , Mutation , Suppression, GeneticABSTRACT
DNA combing is a powerful method developed by Bensimon and colleagues to stretch DNA molecules on silanized glass coverslips. This technique provides a unique way to monitor the activation of replication origins and the progression of replication forks at the level of single DNA molecules, after incorporation of thymidine analogs, such as 5-bromo-2'-deoxyuridine (BrdU), 5-iodo-2'-deoxyuridine (IdU) and 5-chloro-2'-deoxyuridine (CldU) in newly-synthesized DNA. Unlike microarray-based approaches, this assay gives access to the variability of replication profiles in individual cells. It can also be used to monitor the effect of DNA lesions on fork progression, arrest and restart. In this review, we propose standard DNA combing methods to analyze DNA replication in budding yeast and in human cells. We also show that 5-ethynyl-2'-deoxyuridine (EdU) can be used as a good alternative to BrdU for DNA combing analysis, as unlike halogenated nucleotides, it can be detected without prior denaturation of DNA.
Subject(s)
DNA Replication , DNA, Fungal/biosynthesis , Staining and Labeling , Animals , Bromodeoxyuridine/metabolism , Click Chemistry , DNA/biosynthesis , DNA/chemistry , DNA/isolation & purification , DNA, Fungal/chemistry , DNA, Fungal/isolation & purification , DNA, Single-Stranded/chemistry , Data Interpretation, Statistical , Fluorescent Antibody Technique, Indirect , Genome, Fungal , Genome, Human , HCT116 Cells , Humans , Hydroxyurea/pharmacology , Immobilized Nucleic Acids/chemistry , In Situ Hybridization, Fluorescence , Mammals , Nucleic Acid Synthesis Inhibitors/pharmacology , Saccharomyces cerevisiae/genetics , Statistics, NonparametricABSTRACT
Cullin 4 (Cul4)-based ubiquitin ligases emerged as critical regulators of DNA replication and repair. Over 50 Cul4-specific adaptors (DNA damage-binding 1 (Ddb1)-Cul4-associated factors; DCAFs) have been identified and are thought to assemble functionally distinct Cul4 complexes. Using a live-cell imaging-based RNAi screen, we analysed the function of DCAFs and Cul4-linked proteins, and identified specific subsets required for progression through G1 and S phase. We discovered C6orf167/Mms22-like protein (Mms22L) as a putative human orthologue of budding yeast Mms22, which, together with cullin Rtt101, regulates genome stability by promoting DNA replication through natural pause sites and damaged templates. Loss of Mms22L function in human cells results in S phase-dependent genomic instability characterised by spontaneous double-strand breaks and DNA damage checkpoint activation. Unlike yeast Mms22, human Mms22L does not stably bind to Cul4, but is degraded in a Cul4-dependent manner and upon replication stress. Mms22L physically and functionally interacts with the scaffold-like protein Nfkbil2 that co-purifies with histones, several chromatin remodelling and DNA replication/repair factors. Together, our results strongly suggest that the Mms22L-Nfkbil2 complex contributes to genome stability by regulating the chromatin state at stalled replication forks.
Subject(s)
DNA Replication , DNA-Binding Proteins/metabolism , NF-kappa B/metabolism , Nuclear Proteins/metabolism , Cell Line, Tumor , DNA-Binding Proteins/antagonists & inhibitors , Gene Knockdown Techniques/methods , Genomic Instability , Humans , Mass Screening/methods , Nuclear Proteins/antagonists & inhibitors , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolismABSTRACT
The Rad52 pathway has a central function in the recombinational repair of chromosome breaks and in the recovery from replication stress. Tolerance to replication stress also depends on the Mec1 kinase, which activates the DNA replication checkpoint in an Mrc1-dependent manner in response to fork arrest. Although the Mec1 and Rad52 pathways are initiated by the same single-strand DNA (ssDNA) intermediate, their interplay at stalled forks remains largely unexplored. Here, we show that the replication checkpoint suppresses the formation of Rad52 foci in an Mrc1-dependent manner and prevents homologous recombination (HR) at chromosome breaks induced by the HO endonuclease. This repression operates at least in part by impeding resection of DNA ends, which is essential to generate 3' ssDNA tails, the primary substrate of HR. Interestingly, we also observed that the Mec1 pathway does not prevent recombination at stalled forks, presumably because they already contain ssDNA. Taken together, these data indicate that the DNA replication checkpoint suppresses genomic instability in S phase by blocking recombination at chromosome breaks and permitting helpful recombination at stalled forks.
