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1.
EMBO J ; 43(6): 1043-1064, 2024 Mar.
Article in English | MEDLINE | ID: mdl-38360996

ABSTRACT

Eukaryotic cells rely on several mechanisms to ensure that the genome is duplicated precisely once in each cell division cycle, preventing DNA over-replication and genomic instability. Most of these mechanisms limit the activity of origin licensing proteins to prevent the reactivation of origins that have already been used. Here, we have investigated whether additional controls restrict the extension of re-replicated DNA in the event of origin re-activation. In a genetic screening in cells forced to re-activate origins, we found that re-replication is limited by RAD51 and enhanced by FBH1, a RAD51 antagonist. In the presence of chromatin-bound RAD51, forks stemming from re-fired origins are slowed down, leading to frequent events of fork reversal. Eventual re-initiation of DNA synthesis mediated by PRIMPOL creates ssDNA gaps that facilitate the partial elimination of re-duplicated DNA by MRE11 exonuclease. In the absence of RAD51, these controls are abrogated and re-replication forks progress much longer than in normal conditions. Our study uncovers a safeguard mechanism to protect genome stability in the event of origin reactivation.


Subject(s)
DNA-Binding Proteins , Rad51 Recombinase , DNA/genetics , DNA Replication , DNA-Binding Proteins/metabolism , MRE11 Homologue Protein/metabolism , Rad51 Recombinase/genetics , Rad51 Recombinase/metabolism , Humans
2.
Cells ; 13(2)2024 01 15.
Article in English | MEDLINE | ID: mdl-38247850

ABSTRACT

The regulated formation and resolution of R-loops is a natural process in physiological gene expression. Defects in R-loop metabolism can lead to DNA replication stress, which is associated with a variety of diseases and, ultimately, with cancer. The proteins PARP1, DIDO3, and DHX9 are important players in R-loop regulation. We previously described the interaction between DIDO3 and DHX9. Here, we show that, in mouse embryonic fibroblasts, the three proteins are physically linked and dependent on PARP1 activity. The C-terminal truncation of DIDO3 leads to the impairment of this interaction; concomitantly, the cells show increased replication stress and senescence. DIDO3 truncation also renders the cells partially resistant to in vitro oncogenic transformation, an effect that can be reversed by immortalization. We propose that PARP1, DIDO3, and DHX9 proteins form a ternary complex that regulates R-loop metabolism, preventing DNA replication stress and subsequent senescence.


Subject(s)
DNA Replication , Fibroblasts , Poly (ADP-Ribose) Polymerase-1 , Animals , Mice , Poly (ADP-Ribose) Polymerase-1/metabolism , Poly (ADP-Ribose) Polymerase-1/physiology , DEAD-box RNA Helicases/metabolism , DEAD-box RNA Helicases/physiology , Cellular Senescence/genetics , Carcinogenesis/genetics
3.
Nature ; 616(7955): 168-175, 2023 04.
Article in English | MEDLINE | ID: mdl-36949199

ABSTRACT

The resistance of cancer cells to therapy is responsible for the death of most patients with cancer1. Epithelial-to-mesenchymal transition (EMT) has been associated with resistance to therapy in different cancer cells2,3. However, the mechanisms by which EMT mediates resistance to therapy remain poorly understood. Here, using a mouse model of skin squamous cell carcinoma undergoing spontaneous EMT during tumorigenesis, we found that EMT tumour cells are highly resistant to a wide range of anti-cancer therapies both in vivo and in vitro. Using gain and loss of function studies in vitro and in vivo, we found that RHOJ-a small GTPase that is preferentially expressed in EMT cancer cells-controls resistance to therapy. Using genome-wide transcriptomic and proteomic profiling, we found that RHOJ regulates EMT-associated resistance to chemotherapy by enhancing the response to replicative stress and activating the DNA-damage response, enabling tumour cells to rapidly repair DNA lesions induced by chemotherapy. RHOJ interacts with proteins that regulate nuclear actin, and inhibition of actin polymerization sensitizes EMT tumour cells to chemotherapy-induced cell death in a RHOJ-dependent manner. Together, our study uncovers the role and the mechanisms through which RHOJ acts as a key regulator of EMT-associated resistance to chemotherapy.


Subject(s)
Carcinoma, Squamous Cell , Drug Resistance, Neoplasm , Epithelial-Mesenchymal Transition , Skin Neoplasms , rho GTP-Binding Proteins , Actins/drug effects , Actins/metabolism , Carcinoma, Squamous Cell/drug therapy , Carcinoma, Squamous Cell/metabolism , Carcinoma, Squamous Cell/pathology , Cell Line, Tumor , Drug Resistance, Neoplasm/drug effects , Drug Resistance, Neoplasm/genetics , Epithelial-Mesenchymal Transition/drug effects , Proteomics , rho GTP-Binding Proteins/genetics , rho GTP-Binding Proteins/metabolism , Animals , Mice , Skin Neoplasms/drug therapy , Skin Neoplasms/metabolism , Skin Neoplasms/pathology , Gene Expression Profiling , Genome
4.
Nucleic Acids Res ; 50(21): 12149-12165, 2022 11 28.
Article in English | MEDLINE | ID: mdl-36453993

ABSTRACT

In mammalian cells, chromosomal replication starts at thousands of origins at which replisomes are assembled. Replicative stress triggers additional initiation events from 'dormant' origins whose genomic distribution and regulation are not well understood. In this study, we have analyzed origin activity in mouse embryonic stem cells in the absence or presence of mild replicative stress induced by aphidicolin, a DNA polymerase inhibitor, or by deregulation of origin licensing factor CDC6. In both cases, we observe that the majority of stress-responsive origins are also active in a small fraction of the cell population in a normal S phase, and stress increases their frequency of activation. In a search for the molecular determinants of origin efficiency, we compared the genetic and epigenetic features of origins displaying different levels of activation, and integrated their genomic positions in three-dimensional chromatin interaction networks derived from high-depth Hi-C and promoter-capture Hi-C data. We report that origin efficiency is directly proportional to the proximity to transcriptional start sites and to the number of contacts established between origin-containing chromatin fragments, supporting the organization of origins in higher-level DNA replication factories.


Subject(s)
Chromatin , Replication Origin , Animals , Mice , Replication Origin/genetics , Chromatin/genetics , Mouse Embryonic Stem Cells/metabolism , DNA Replication/genetics , Cell Cycle Proteins/metabolism , Mammals/genetics
5.
Cell Rep ; 40(12): 111375, 2022 09 20.
Article in English | MEDLINE | ID: mdl-36130506

ABSTRACT

Stress-activated protein kinases (SAPKs) enhance survival in response to environmental changes. In yeast, the Hog1 SAPK and Mrc1, a protein required for DNA replication, define a safeguard mechanism that allows eukaryotic cells to prevent genomic instability upon stress during S-phase. Here we show that, in mammals, the p38 SAPK and Claspin-the functional homolog of Mrc1-protect cells from DNA damage upon osmostress during S-phase. We demonstrate that p38 phosphorylates Claspin and either the mutation of the p38-phosphorylation sites in Claspin or p38 inhibition suppresses the protective role of Claspin on DNA damage. In addition, wild-type Claspin but not the p38-unphosphorylatable mutant has a protective effect on cell survival in response to cisplatin treatment. These findings reveal a role of Claspin in response to chemotherapeutic drugs. Thus, this pathway protects S-phase integrity from different insults and it is conserved from yeast to mammals.


Subject(s)
Protein Serine-Threonine Kinases , Saccharomyces cerevisiae , Adaptor Proteins, Signal Transducing , Animals , Cell Cycle Proteins/metabolism , Cisplatin/pharmacology , DNA Damage , DNA Replication , Mammals/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism
6.
J Biol Chem ; 295(1): 146-157, 2020 01 03.
Article in English | MEDLINE | ID: mdl-31757807

ABSTRACT

Cohesin is a chromatin-bound complex that mediates sister chromatid cohesion and facilitates long-range interactions through DNA looping. How the transcription and replication machineries deal with the presence of cohesin on chromatin remains unclear. The dynamic association of cohesin with chromatin depends on WAPL cohesin release factor (WAPL) and on PDS5 cohesin-associated factor (PDS5), which exists in two versions in vertebrate cells, PDS5A and PDS5B. Using genetic deletion in mouse embryo fibroblasts and a combination of CRISPR-mediated gene editing and RNAi-mediated gene silencing in human cells, here we analyzed the consequences of PDS5 depletion for DNA replication. We found that either PDS5A or PDS5B is sufficient for proper cohesin dynamics and that their simultaneous removal increases cohesin's residence time on chromatin and slows down DNA replication. A similar phenotype was observed in WAPL-depleted cells. Cohesin down-regulation restored normal replication fork rates in PDS5-deficient cells, suggesting that chromatin-bound cohesin hinders the advance of the replisome. We further show that PDS5 proteins are required to recruit WRN helicase-interacting protein 1 (WRNIP1), RAD51 recombinase (RAD51), and BRCA2 DNA repair associated (BRCA2) to stalled forks and that in their absence, nascent DNA strands at unprotected forks are degraded by MRE11 homolog double-strand break repair nuclease (MRE11). These findings indicate that PDS5 proteins participate in replication fork protection and also provide insights into how cohesin and its regulators contribute to the response to replication stress, a common feature of cancer cells.


Subject(s)
Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/metabolism , DNA Replication , DNA-Binding Proteins/metabolism , Nuclear Proteins/metabolism , Transcription Factors/metabolism , ATPases Associated with Diverse Cellular Activities/metabolism , Animals , BRCA2 Protein/metabolism , Cells, Cultured , Chromatin/metabolism , DNA-Binding Proteins/genetics , HeLa Cells , Humans , MRE11 Homologue Protein/metabolism , Mice , Nuclear Proteins/genetics , Rad51 Recombinase/metabolism , Transcription Factors/genetics , Cohesins
7.
EMBO Rep ; 20(1)2019 01.
Article in English | MEDLINE | ID: mdl-30538118

ABSTRACT

The G2/M checkpoint coordinates DNA replication with mitosis and thereby prevents chromosome segregation in the presence of unreplicated or damaged DNA Here, we show that the RNA-binding protein TIAR is essential for the G2/M checkpoint and that TIAR accumulates in nuclear foci in late G2 and prophase in cells suffering from replication stress. These foci, which we named G2/M transition granules (GMGs), occur at low levels in normally cycling cells and are strongly induced by replication stress. In addition to replication stress response proteins, GMGs contain factors involved in RNA metabolism as well as CDK1. Depletion of TIAR accelerates mitotic entry and leads to chromosomal instability in response to replication stress, in a manner that can be alleviated by the concomitant depletion of Cdc25B or inhibition of CDK1. Since TIAR retains CDK1 in GMGs and attenuates CDK1 activity, we propose that the assembly of GMGs may represent a so far unrecognized mechanism that contributes to the activation of the G2/M checkpoint in mammalian cells.


Subject(s)
CDC2 Protein Kinase/genetics , G2 Phase Cell Cycle Checkpoints/genetics , RNA-Binding Proteins/genetics , cdc25 Phosphatases/genetics , Cell Cycle/genetics , Chromosome Segregation/genetics , DNA Damage/genetics , DNA Replication/genetics , HeLa Cells , Humans , Mitosis/genetics , Phosphorylation
8.
EMBO Rep ; 19(10)2018 10.
Article in English | MEDLINE | ID: mdl-30126925

ABSTRACT

The Myc family of oncogenic transcription factors regulates myriad cellular functions. Myc proteins contain a basic region/helix-loop-helix/leucine zipper domain that mediates DNA binding and heterodimerization with its partner Max. Among the Myc proteins, c-Myc is the most widely expressed and relevant in primary B lymphocytes. There is evidence suggesting that c-Myc can perform some of its functions in the absence of Max in different cellular contexts. However, the functional in vivo interplay between c-Myc and Max during B lymphocyte differentiation is not well understood. Using in vivo and ex vivo models, we show that while c-Myc requires Max in primary B lymphocytes, several key biological processes, such as cell differentiation and DNA replication, can initially progress without the formation of c-Myc/Max heterodimers. We also describe that B lymphocytes lacking Myc, Max, or both show upregulation of signaling pathways associated with the B-cell receptor. These data suggest that c-Myc/Max heterodimers are not essential for the initiation of a subset of important biological processes in B lymphocytes, but are required for fine-tuning the initial response after activation.


Subject(s)
B-Lymphocytes/chemistry , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/genetics , Cell Differentiation/genetics , Proto-Oncogene Proteins c-myc/genetics , Amino Acid Sequence/genetics , Animals , B-Lymphocytes/metabolism , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/chemistry , DNA Replication/genetics , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Dimerization , Helix-Loop-Helix Motifs/genetics , Humans , Leucine Zippers/genetics , Mice , Protein Binding/genetics , Proto-Oncogene Proteins c-myc/chemistry , Transcriptional Activation/genetics
9.
J Biol Chem ; 293(33): 12855-12861, 2018 08 17.
Article in English | MEDLINE | ID: mdl-29959228

ABSTRACT

In growing cells, DNA replication precedes mitotic cell division to transmit genetic information to the next generation. The slowing or stalling of DNA replication forks at natural or exogenous obstacles causes "replicative stress" that promotes genomic instability and affects cellular fitness. Replicative stress phenotypes can be characterized at the single-molecule level with DNA combing or stretched DNA fibers, but interpreting the results obtained with these approaches is complicated by the fact that the speed of replication forks is connected to the frequency of origin activation. Primary alterations in fork speed trigger secondary responses in origins, and, conversely, primary alterations in the number of active origins induce compensatory changes in fork speed. Here, by employing interventions that temporally restrict either fork speed or origin firing while still allowing interrogation of the other variable, we report a set of experimental conditions to separate cause and effect in any manipulation that affects DNA replication dynamics. Using HeLa cells and chemical inhibition of origin activity (through a CDC7 kinase inhibitor) and of DNA synthesis (via the DNA polymerase inhibitor aphidicolin), we found that primary effects of replicative stress on velocity of replisomes (fork rate) can be readily distinguished from primary effects on origin firing. Identifying the primary cause of replicative stress in each case as demonstrated here may facilitate the design of methods to counteract replication stress in primary cells or to enhance it in cancer cells to increase their susceptibility to therapies that target DNA repair.


Subject(s)
Aphidicolin/pharmacology , Cell Cycle Proteins/antagonists & inhibitors , Cellular Senescence/drug effects , DNA Replication/drug effects , DNA/biosynthesis , Protein Serine-Threonine Kinases/antagonists & inhibitors , Replication Origin , Cell Cycle Proteins/metabolism , DNA Repair/drug effects , HeLa Cells , Humans , Protein Serine-Threonine Kinases/metabolism
10.
Cell Rep ; 19(5): 928-938, 2017 05 02.
Article in English | MEDLINE | ID: mdl-28467906

ABSTRACT

Mammalian DNA replication origins are "licensed" by the loading of DNA helicases, a reaction that is mediated by CDC6 and CDT1 proteins. After initiation of DNA synthesis, CDC6 and CDT1 are inhibited to prevent origin reactivation and DNA overreplication before cell division. CDC6 and CDT1 are highly expressed in many types of cancer cells, but the impact of their deregulated expression had not been investigated in vivo. Here, we have generated mice strains that allow the conditional overexpression of both proteins. Adult mice were unharmed by the individual overexpression of either CDC6 or CDT1, but their combined deregulation led to DNA re-replication in progenitor cells and lethal tissue dysplasias. This study offers mechanistic insights into the necessary cooperation between CDC6 and CDT1 for facilitation of origin reactivation and describes the physiological consequences of DNA overreplication.


Subject(s)
DNA Replication , Diarrhea, Infantile/genetics , Intestinal Mucosa/metabolism , Malabsorption Syndromes/genetics , Animals , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Diarrhea, Infantile/metabolism , Female , Intestinal Mucosa/pathology , Malabsorption Syndromes/metabolism , Male , Mice , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Transgenes
11.
Mol Oncol ; 10(8): 1196-206, 2016 10.
Article in English | MEDLINE | ID: mdl-27296872

ABSTRACT

DNA replication control is a key process in maintaining genomic integrity. Monitoring DNA replication initiation is particularly important as it needs to be coordinated with other cellular events and should occur only once per cell cycle. Crucial players in the initiation of DNA replication are the ORC protein complex, marking the origin of replication, and the Cdt1 and Cdc6 proteins, that license these origins to replicate by recruiting the MCM2-7 helicase. To accurately achieve its functions, Cdt1 is tightly regulated. Cdt1 levels are high from metaphase and during G1 and low in S/G2 phases of the cell cycle. This control is achieved, among other processes, by ubiquitination and proteasomal degradation. In an overexpression screen for Cdt1 deubiquitinating enzymes, we isolated USP37, to date the first ubiquitin hydrolase controlling Cdt1. USP37 overexpression stabilizes Cdt1, most likely a phosphorylated form of the protein. In contrast, USP37 knock down destabilizes Cdt1, predominantly during G1 and G1/S phases of the cell cycle. USP37 interacts with Cdt1 and is able to de-ubiquitinate Cdt1 in vivo and, USP37 is able to regulate the loading of MCM complexes onto the chromatin. In addition, downregulation of USP37 reduces DNA replication fork speed. Taken together, here we show that the deubiquitinase USP37 plays an important role in the regulation of DNA replication. Whether this is achieved via Cdt1, a central protein in this process, which we have shown to be stabilized by USP37, or via additional factors, remains to be tested.


Subject(s)
Cell Cycle Proteins/metabolism , DNA Replication , Endopeptidases/metabolism , Ubiquitination , Cell Line , G1 Phase , Humans , Phosphorylation , Protein Binding , S Phase
12.
Nat Struct Mol Biol ; 23(4): 270-7, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26950370

ABSTRACT

Post-translational modification of proteins by ubiquitin (Ub) and Ub-like modifiers regulates DNA replication. We have previously shown that chromatin around replisomes is rich in SUMO and poor in Ub, whereas mature chromatin exhibits an opposite pattern. How this SUMO-rich, Ub-poor environment is maintained at sites of DNA replication in mammalian cells remains unexplored. Here we identify USP7 as a replisome-enriched SUMO deubiquitinase that is essential for DNA replication. By acting on SUMO and SUMOylated proteins, USP7 counteracts their ubiquitination. Inhibition or genetic deletion of USP7 leads to the accumulation of Ub on SUMOylated proteins, which are displaced away from replisomes. Our findings provide a model explaining the differential accumulation of SUMO and Ub at replication forks and identify an essential role of USP7 in DNA replication that should be considered in the development of USP7 inhibitors as anticancer agents.


Subject(s)
DNA Replication , Small Ubiquitin-Related Modifier Proteins/metabolism , Ubiquitin Thiolesterase/metabolism , Ubiquitin-Specific Proteases/metabolism , DNA Damage , DNA Repair , HCT116 Cells , HeLa Cells , Humans , Models, Molecular , Small Ubiquitin-Related Modifier Proteins/analysis , Sumoylation , Ubiquitin Thiolesterase/analysis , Ubiquitin-Specific Peptidase 7 , Ubiquitin-Specific Proteases/analysis , Ubiquitination
13.
EMBO J ; 34(21): 2604-19, 2015 Nov 03.
Article in English | MEDLINE | ID: mdl-26443207

ABSTRACT

The SMC5/6 complex is the least understood of SMC complexes. In yeast, smc5/6 mutants phenocopy mutations in sgs1, the BLM ortholog that is deficient in Bloom's syndrome (BS). We here show that NSMCE2 (Mms21, in Saccharomyces cerevisiae), an essential SUMO ligase of the SMC5/6 complex, suppresses cancer and aging in mice. Surprisingly, a mutation that compromises NSMCE2-dependent SUMOylation does not have a detectable impact on murine lifespan. In contrast, NSMCE2 deletion in adult mice leads to pathologies resembling those found in patients of BS. Moreover, and whereas NSMCE2 deletion does not have a detectable impact on DNA replication, NSMCE2-deficient cells also present the cellular hallmarks of BS such as increased recombination rates and an accumulation of micronuclei. Despite the similarities, NSMCE2 and BLM foci do not colocalize and concomitant deletion of Blm and Nsmce2 in B lymphocytes further increases recombination rates and is synthetic lethal due to severe chromosome mis-segregation. Our work reveals that SUMO- and BLM-independent activities of NSMCE2 limit recombination and facilitate segregation; functions of the SMC5/6 complex that are necessary to prevent cancer and aging in mice.


Subject(s)
Aging , Neoplasms/enzymology , Ubiquitin-Protein Ligases/physiology , Animals , B-Lymphocytes/enzymology , Base Sequence , Cells, Cultured , Chromosome Segregation , DNA Breaks, Double-Stranded , DNA Mutational Analysis , DNA Replication , Female , Haploinsufficiency , Humans , Ligases , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Protein Transport , RecQ Helicases/metabolism , Sumoylation , Tumor Suppressor Proteins/physiology
14.
Nat Commun ; 6: 8548, 2015 Oct 12.
Article in English | MEDLINE | ID: mdl-26456157

ABSTRACT

Replicative stress during embryonic development influences ageing and predisposition to disease in adults. A protective mechanism against replicative stress is provided by the licensing of thousands of origins in G1 that are not necessarily activated in the subsequent S-phase. These 'dormant' origins provide a backup in the presence of stalled forks and may confer flexibility to the replication program in specific cell types during differentiation, a role that has remained unexplored. Here we show, using a mouse strain with hypomorphic expression of the origin licensing factor mini-chromosome maintenance (MCM)3 that limiting origin licensing in vivo affects the functionality of hematopoietic stem cells and the differentiation of rapidly-dividing erythrocyte precursors. Mcm3-deficient erythroblasts display aberrant DNA replication patterns and fail to complete maturation, causing lethal anemia. Our results indicate that hematopoietic progenitors are particularly sensitive to replication stress, and full origin licensing ensures their correct differentiation and functionality.


Subject(s)
DNA Replication , Erythropoiesis , Hematopoietic Stem Cells/physiology , Minichromosome Maintenance Complex Component 3/metabolism , Animals , Checkpoint Kinase 1 , DNA Damage , Disease Susceptibility , Embryo, Mammalian/physiology , Embryonic Development , Female , Genes, Lethal , Hematologic Neoplasms , Liver/physiology , Male , Mice, Inbred C57BL , Mice, Knockout , Minichromosome Maintenance Complex Component 3/genetics , Protein Kinases/metabolism
15.
Biochim Biophys Acta ; 1851(9): 1240-53, 2015 Sep.
Article in English | MEDLINE | ID: mdl-26055626

ABSTRACT

The mevalonate pathway is tightly linked to cell division. Mevalonate derived non-sterol isoprenoids and cholesterol are essential for cell cycle progression and mitosis completion respectively. In the present work, we studied the effects of fluoromevalonate, a competitive inhibitor of mevalonate diphosphate decarboxylase, on cell proliferation and cell cycle progression in both HL-60 and MOLT-4 cells. This enzyme catalyzes the synthesis of isopentenyl diphosphate, the first isoprenoid in the cholesterol biosynthesis pathway, consuming ATP at the same time. Inhibition of mevalonate diphosphate decarboxylase was followed by a rapid accumulation of mevalonate diphosphate and the reduction of ATP concentrations, while the cell content of cholesterol was barely affected. Strikingly, mevalonate diphosphate decarboxylase inhibition also resulted in the depletion of dNTP pools, which has never been reported before. These effects were accompanied by inhibition of cell proliferation and cell cycle arrest at S phase, together with the appearance of γ-H2AX foci and Chk1 activation. Inhibition of Chk1 in cells treated with fluoromevalonate resulted in premature entry into mitosis and massive cell death, indicating that the inhibition of mevalonate diphosphate decarboxylase triggered a DNA damage response. Notably, the supply of exogenously deoxyribonucleosides abolished γ-H2AX formation and prevented the effects of mevalonate diphosphate decarboxylase inhibition on DNA replication and cell growth. The results indicate that dNTP pool depletion caused by mevalonate diphosphate decarboxylase inhibition hampered DNA replication with subsequent DNA damage, which may have important consequences for replication stress and genomic instability.


Subject(s)
Carboxy-Lyases/metabolism , Deoxyribonucleosides/metabolism , Lymphocytes/drug effects , Mevalonic Acid/pharmacology , Adenosine Triphosphate/metabolism , Carboxy-Lyases/antagonists & inhibitors , Carboxy-Lyases/genetics , Cell Cycle Checkpoints/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Checkpoint Kinase 1 , DNA Damage , DNA Replication/drug effects , Deoxyribonucleosides/pharmacology , Gene Expression Regulation , HL-60 Cells , Halogenation , Hemiterpenes/metabolism , Histones/genetics , Histones/metabolism , Humans , Lymphocytes/cytology , Lymphocytes/metabolism , Mevalonic Acid/analogs & derivatives , Mevalonic Acid/metabolism , Organophosphorus Compounds/metabolism , Protein Kinases/genetics , Protein Kinases/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Signal Transduction
16.
Dev Cell ; 32(2): 155-67, 2015 Jan 26.
Article in English | MEDLINE | ID: mdl-25625205

ABSTRACT

Polyploidization is a natural process that frequently accompanies differentiation; its deregulation is linked to genomic instability and cancer. Despite its relevance, why cells select different polyploidization mechanisms is unknown. Here we report a systematic genetic analysis of endomitosis, a process in which megakaryocytes become polyploid by entering mitosis but aborting anaphase. Whereas ablation of the APC/C cofactor Cdc20 results in mitotic arrest and severe thrombocytopenia, lack of the kinases Aurora-B, Cdk1, or Cdk2 does not affect megakaryocyte polyploidization or platelet levels. Ablation of Cdk1 forces a switch to endocycles without mitosis, whereas polyploidization in the absence of Cdk1 and Cdk2 occurs in the presence of aberrant re-replication events. Importantly, ablation of these kinases rescues the defects in Cdc20 null megakaryocytes. These findings suggest that endomitosis can be functionally replaced by alternative polyploidization mechanisms in vivo and provide the cellular basis for therapeutic approaches aimed to discriminate mitotic and polyploid cells.


Subject(s)
Megakaryocytes/cytology , Megakaryocytes/metabolism , Mitosis/physiology , Polyploidy , Anaphase/physiology , Animals , Cdc20 Proteins/metabolism , Cells, Cultured , Mice , Mitosis/genetics , Protein Serine-Threonine Kinases/metabolism
17.
J Pathol ; 233(4): 344-56, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24931331

ABSTRACT

Aberrant mitosis is a common feature of cancer, yet little is known about the altered genes causing mitotic defects. We screened human tumours for cells with morphological signatures of highly specific mitotic defects previously assigned to candidate genes in a genome-wide RNA interference screen carried out in HeLa cells (www.mitocheck.org). We discovered a striking enrichment of early mitotic configurations indicative of prophase/prometaphase delay in breast cancer. Promoter methylation analysis of MitoCheck candidate genes assigned to the corresponding 'mitotic delay' class linked this defect to epigenetic silencing of the gene encoding pregnancy-associated plasma protein-A (PAPPA), a secreted protease. PAPPA silencing was highly prevalent in precursor lesions and invasive breast cancer. Experimental manipulation of PAPPA protein levels in human mammary epithelial cells and in breast cancer cell lines demonstrates that progression through early mitosis is dependent on PAPPA function, and that breast cancer cells become more invasive after down-regulation of this protease. PAPPA regulates mitotic progression through modulating the IGF-1 signalling pathway resulting in activation of the forkhead transcription factor FoxM1, which drives a transcriptional cluster of essential mitotic genes. Our results show that PAPPA has a critical function in normal cell division and is targeted early in breast cancer development.


Subject(s)
Breast Neoplasms/genetics , Breast Neoplasms/physiopathology , Epigenomics , Gene Expression Regulation, Neoplastic/physiology , Gene Silencing/physiology , Mitosis/physiology , Pregnancy-Associated Plasma Protein-A/physiology , Aged , Biomarkers, Tumor/metabolism , Breast Neoplasms/pathology , Cell Line, Tumor , Disease Progression , Epithelial Cells/metabolism , Epithelial Cells/pathology , Female , Forkhead Box Protein M1 , Forkhead Transcription Factors/metabolism , Humans , Insulin-Like Growth Factor I/metabolism , Middle Aged , Phenotype , Pregnancy-Associated Plasma Protein-A/genetics , RNA Interference/physiology , Signal Transduction/physiology
18.
Nat Struct Mol Biol ; 20(12): 1383-9, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24240614

ABSTRACT

DNA replication forks that collapse during the process of genomic duplication lead to double-strand breaks and constitute a threat to genomic stability. The risk of fork collapse is higher in the presence of replication inhibitors or after UV irradiation, which introduces specific modifications in the structure of DNA. In these cases, fork progression may be facilitated by error-prone translesion synthesis (TLS) DNA polymerases. Alternatively, the replisome may skip the damaged DNA, leaving an unreplicated gap to be repaired after replication. This mechanism strictly requires a priming event downstream of the lesion. Here we show that PrimPol, a new human primase and TLS polymerase, uses its primase activity to mediate uninterrupted fork progression after UV irradiation and to reinitiate DNA synthesis after dNTP depletion. As an enzyme involved in tolerance to DNA damage, PrimPol might become a target for cancer therapy.


Subject(s)
DNA Primase/physiology , DNA Replication/physiology , DNA-Directed DNA Polymerase/physiology , Multifunctional Enzymes/physiology , DNA Breaks, Double-Stranded , DNA Damage , DNA Primase/chemistry , DNA Primase/metabolism , DNA-Directed DNA Polymerase/chemistry , DNA-Directed DNA Polymerase/metabolism , Genomic Instability , Humans , Multifunctional Enzymes/chemistry , Multifunctional Enzymes/metabolism , RNA, Messenger/metabolism , S Phase , Ultraviolet Rays
19.
Cell Rep ; 3(4): 1105-16, 2013 Apr 25.
Article in English | MEDLINE | ID: mdl-23545495

ABSTRACT

DNA replication is facilitated by multiple factors that concentrate in the vicinity of replication forks. Here, we developed an approach that combines the isolation of proteins on nascent DNA chains with mass spectrometry (iPOND-MS), allowing a comprehensive proteomic characterization of the human replisome and replisome-associated factors. In addition to known replisome components, we provide a broad list of proteins that reside in the vicinity of the replisome, some of which were not previously associated with replication. For instance, our data support a link between DNA replication and the Williams-Beuren syndrome and identify ZNF24 as a replication factor. In addition, we reveal that SUMOylation is widespread for factors that concentrate near replisomes, which contrasts with lower UQylation levels at these sites. This resource provides a panoramic view of the proteins that concentrate in the surroundings of the replisome, which should facilitate future investigations on DNA replication and genome maintenance.


Subject(s)
DNA/metabolism , Proteomics , Cell Line , DNA Replication , Gene Regulatory Networks , HEK293 Cells , Humans , Kruppel-Like Transcription Factors/genetics , Kruppel-Like Transcription Factors/metabolism , Mass Spectrometry , Small Ubiquitin-Related Modifier Proteins/metabolism , Ubiquitination , Williams Syndrome/genetics , Williams Syndrome/metabolism
20.
Biosci Rep ; 33(1): 57-69, 2012 Dec 06.
Article in English | MEDLINE | ID: mdl-23050906

ABSTRACT

DHCR24 (3ß-hydroxysterol Δ(24)-reductase) catalyses the reduction of the C-24 double bond of sterol intermediates during cholesterol biosynthesis. DHCR24 has also been involved in cell growth, senescence and cellular response to oncogenic and oxidative stress. Despite its important roles, little is known about the transcriptional mechanisms controlling DHCR24 gene expression. We analysed the proximal promoter region and the cholesterol-mediated regulation of DHCR24. A putative SRE (sterol-regulatory element) at -98/-90 bp of the transcription start site was identified. Other putative regulatory elements commonly found in SREBP (SRE-binding protein)-targeted genes were also identified. Sterol responsiveness was analysed by luciferase reporter assays of approximately 1 kb 5'-flanking region of the human DHCR24 gene in HepG2 and SK-N-MC cells. EMSAs (electrophoretic mobility-shift assays) and ChIP (chromatin immunoprecipitation) assays demonstrated cholesterol-dependent recruitment and binding of SREBPs to the putative SRE. Given the presence of several CACCC-boxes in the DHCR24 proximal promoter, we assessed the role of KLF5 (Krüppel-like factor 5) in androgen-regulated DHCR24 expression. DHT (dihydrotestosterone) increased DHCR24 expression synergistically with lovastatin. However, DHT was unable to activate the DHCR24 proximal promoter, whereas KLF5 did, indicating that this mechanism is not involved in the androgen-induced stimulation of DHCR24 expression. The results of the present study allow the elucidation of the mechanism of regulation of the DHCR24 gene by cholesterol availability and identification of other putative cis-acting elements which may be relevant for the regulation of DHCR24 expression.


Subject(s)
Nerve Tissue Proteins/genetics , Oxidoreductases Acting on CH-CH Group Donors/genetics , Promoter Regions, Genetic , Sterol Regulatory Element Binding Protein 1/metabolism , Transcriptional Activation , Base Sequence , Chromatin Immunoprecipitation , Cloning, Molecular , Dihydrotestosterone/pharmacology , Drug Synergism , Electrophoretic Mobility Shift Assay , Genes, Reporter , Hep G2 Cells , Humans , Kruppel-Like Transcription Factors/genetics , Kruppel-Like Transcription Factors/metabolism , Lipoproteins, LDL/metabolism , Lipoproteins, LDL/pharmacology , Lovastatin/pharmacology , Luciferases/genetics , Luciferases/metabolism , Molecular Sequence Data , Nerve Tissue Proteins/metabolism , Oxidoreductases Acting on CH-CH Group Donors/metabolism , Protein Binding , RNA, Messenger/genetics , RNA, Messenger/metabolism , Regulatory Sequences, Nucleic Acid , Sterol Regulatory Element Binding Protein 1/genetics , Transcription Initiation Site
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