116 results on '"Longhese, M"'
Search Results
2. DNA damage checkpoints and telomere protection
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DI DOMENICO, Enea Gino, Auriche, Cristina, Longhese, M. P., Gilson, E., and Ascenzioni, Fiorentina
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- 2006
3. Humanized yeast telomeres: checkpoints activation, telomere protection and genome instability
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DI DOMENICO, Enea Gino, Auriche, Cristina, Longhese, M. P., Gilson, E., and Ascenzioni, Fiorentina
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- 2006
4. Chronic chromosome instability in humanized yeast
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DI DOMENICO, Enea Gino, Fanella, E., Pirone, L., Auriche, Cristina, Longhese, M. P., and Ascenzioni, Fiorentina
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- 2005
5. Saccharomyces cerevisiae Rif1 cooperates with MRX-Sae2 in promoting DNA-end resection
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Martina, M., primary, Bonetti, D., additional, Villa, M., additional, Lucchini, G., additional, and Longhese, M. P., additional
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- 2014
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6. The S-phase checkpoint and its regulation in Saccharomyces cerevisiae
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Longhese, M, primary
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- 2003
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7. DNA damage checkpoint in budding yeast
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Longhese, M. P., primary
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- 1998
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8. The novel DNA damage checkpoint protein Ddc1p is phosphorylated periodically during the cell cycle and in response to DNA damage in budding yeast
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Longhese, M. P., primary
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- 1997
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9. The 70 kDa subunit of replication protein A is required for the G1/S and intra-S DNA damage checkpoints in budding yeast
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Longhese, M., primary
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- 1996
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10. Replication factor A is required in vivo for DNA replication, repair, and recombination
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Longhese, M P, primary, Plevani, P, additional, and Lucchini, G, additional
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- 1994
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11. Purification and characterization of a new DNA polymerase from budding yeast Saccharomyces cerevisiae. A probable homolog of mammalian DNA polymerase beta.
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Shimizu, K, primary, Santocanale, C, additional, Ropp, P A, additional, Longhese, M P, additional, Plevani, P, additional, Lucchini, G, additional, and Sugino, A, additional
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- 1993
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12. Conditional mutations in the yeast DNA primase genes affect different aspects of DNA metabolism and interactions in the DNA polymerase alpha-primase complex.
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Longhese, M P, primary, Jovine, L, additional, Plevani, P, additional, and Lucchini, G, additional
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- 1993
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13. Mutations in conserved yeast DNA primase domains impair DNA replication in vivo.
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Francesconi, S, primary, Longhese, M P, additional, Piseri, A, additional, Santocanale, C, additional, Lucchini, G, additional, and Plevani, P, additional
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- 1991
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14. The set1Delta mutation unveils a novel signaling pathway relayed by the Rad53-dependent hyperphosphorylation of replication protein A that leads to transcriptional activation of repair genes.
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Schramke, V, Neecke, H, Brevet, V, Corda, Y, Lucchini, G, Longhese, M P, Gilson, E, and Géli, V
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SET domain proteins are present in chromosomal proteins involved in epigenetic control of transcription. The yeast SET domain protein Set1p regulates chromatin structure, DNA repair, and telomeric functions. We investigated the mechanism by which the absence of Set1p increases DNA repair capacities of checkpoint mutants. We show that deletion of SET1 induces a response relayed by the signaling kinase Rad53p that leads to the MEC1/TEL1-independent hyperphosphorylation of replication protein A middle subunit (Rfa2p). Consequently, the binding of Rfa2p to upstream repressing sequences (URS) of repair genes is decreased, thereby leading to their derepression. Our results correlate the set1Delta-dependent phosphorylation of Rfa2p with the transcriptional induction of repair genes. Moreover, we show that the deletion of the amino-terminal region of Rfa2p suppresses the sensitivity to ultraviolet radiation of a mec3Delta checkpoint mutant, abolishes the URS-mediated repression, and increases the expression of repair genes. This work provides an additional link for the role of Rfa2p in the regulation of the repair capacity of the cell and reveals a role for the phosphorylation of Rfa2p and unveils unsuspected connections between chromatin, signaling pathways, telomeres, and DNA repair.
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- 2001
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15. The checkpoint protein Ddc2, functionally related to S. pombe Rad26, interacts with Mec1 and is regulated by Mec1-dependent phosphorylation in budding yeast.
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Paciotti, V, Clerici, M, Lucchini, G, and Longhese, M P
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DDC2 is a novel component of the DNA integrity checkpoint pathway, which is required for proper checkpoint response to DNA damage and to incomplete DNA replication. Moreover, Ddc2 overproduction causes sensitivity to DNA-damaging agents and checkpoint defects. Ddc2 physically interacts with Mec1 and undergoes Mec1-dependent phosphorylation both in vitro and in vivo. The phosphorylation of Ddc2 takes place in late S phase and in G(2) phase during an unperturbed cell cycle and is further increased in response to DNA damage. Because Ddc2 phosphorylation does not require any other known tested checkpoint factors but Mec1, the Ddc2-Mec1 complex might respond to the presence of some DNA structures independently of the other known checkpoint proteins. Our findings suggest that Ddc2 may be the functional homolog of Schizosaccharomyces pombe Rad26, strengthening the hypothesis that the mechanisms leading to checkpoint activation are conserved throughout evolution.
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- 2000
16. Yeast pip3/mec3 mutants fail to delay entry into S phase and to slow DNA replication in response to DNA damage, and they define a functional link between Mec3 and DNA primase
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Longhese, M P, Fraschini, R, Plevani, P, and Lucchini, G
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The catalytic DNA primase subunit of the DNA polymerase alpha-primase complex is encoded by the essential PRI1 gene in Saccharomyces cerevisiae. To identify factors that functionally interact with yeast DNA primase in living cells, we developed a genetic screen for mutants that are lethal at the permissive temperature in a cold-sensitive pril-2 genetic background. Twenty-four recessive mutations belonging to seven complementation groups were identified. Some mutants showed additional phenotypes, such as increased sensitivity to UV irradiation, methyl methanesulfonate, and hydroxyurea, that were suggestive of defects in DNA repair and/or checkpoint mechanisms. We have cloned and characterized the gene of one complementation group, PIP3, whose product is necessary both for delaying entry into S phase or mitosis when cells are UV irradiated in G1 or G2 phase and for lowering the rate of ongoing DNA synthesis in the presence of methyl methanesulfonate. PIP3 turned out to be the MEC3 gene, previously identified as a component of the G2 DNA damage checkpoint. The finding that Mec3 is also required for the G1- and S-phase DNA damage checkpoints, together with the analysis of genetic interactions between a mec3 null allele and several conditional DNA replication mutations at the permissive temperature, suggests that Mec3 could be part of a mechanism coupling DNA replication with repair of DNA damage, and DNA primase might be involved in this process.
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- 1996
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17. The Ku complex promotes DNA end-bridging and this function is antagonized by Tel1/ATM kinase
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Carlo Rinaldi, Paolo Pizzul, Erika Casari, Marco Mangiagalli, Renata Tisi, Maria Pia Longhese, Rinaldi, C, Pizzul, P, Casari, E, Mangiagalli, M, Tisi, R, and Longhese, M
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Genetics ,NHEJ, HR, end-tethering, DSBs - Abstract
DNA double-strand breaks (DSBs) can be repaired by either homologous recombination (HR) or non-homologous end-joining (NHEJ). NHEJ is induced by the binding to DSBs of the Ku70–Ku80 heterodimer, which acts as a hub for the recruitment of downstream NHEJ components. An important issue in DSB repair is the maintenance of the DSB ends in close proximity, a function that in yeast involves the MRX complex and Sae2. Here, we provide evidence that Ku contributes to keep the DNA ends tethered to each other. The ku70-C85Y mutation, which increases Ku affinity for DNA and its persistence very close to the DSB ends, enhances DSB end-tethering and suppresses the end-tethering defect of sae2Δ cells. Impairing histone removal around DSBs either by eliminating Tel1 kinase activity or nucleosome remodelers enhances Ku persistence at DSBs and DSB bridging, suggesting that Tel1 antagonizes the Ku function in supporting end-tethering by promoting nucleosome removal and possibly Ku sliding inwards. As Ku provides a block to DSB resection, this Tel1 function can be important to regulate the mode by which DSBs are repaired.
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- 2023
18. The regulation of the DNA damage response at telomeres: focus on kinases
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Chiara Frigerio, Michela Clerici, Maria Pia Longhese, Michela Galli, Galli, M, Frigerio, C, Longhese, M, and Clerici, M
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Genome instability ,Senescence ,Telomerase ,Cell cycle checkpoint ,DNA Repair ,DNA repair ,DNA damage ,Telomere-Binding Proteins ,Saccharomyces cerevisiae ,Ataxia Telangiectasia Mutated Proteins ,Biology ,Biochemistry ,replicative senescence ,03 medical and health sciences ,checkpoint ,0302 clinical medicine ,Telomere Homeostasis ,Animals ,Humans ,DNA Breaks, Double-Stranded ,030304 developmental biology ,0303 health sciences ,Models, Genetic ,Proto-Oncogene Proteins c-ets ,Mec1/ATR ,DNA ,Telomere ,Cell biology ,Repressor Proteins ,Tel1/ATM ,biological phenomena, cell phenomena, and immunity ,Protein Kinases ,030217 neurology & neurosurgery ,DNA Damage - Abstract
The natural ends of linear chromosomes resemble those of accidental double-strand breaks (DSBs). DSBs induce a multifaceted cellular response that promotes the repair of lesions and slows down cell cycle progression. This response is not elicited at chromosome ends, which are organized in nucleoprotein structures called telomeres. Besides counteracting DSB response through specialized telomere-binding proteins, telomeres also prevent chromosome shortening. Despite of the different fate of telomeres and DSBs, many proteins involved in the DSB response also localize at telomeres and participate in telomere homeostasis. In particular, the DSB master regulators Tel1/ATM and Mec1/ATR contribute to telomere length maintenance and arrest cell cycle progression when chromosome ends shorten, thus promoting a tumor-suppressive process known as replicative senescence. During senescence, the actions of both these apical kinases and telomere-binding proteins allow checkpoint activation while bulk DNA repair activities at telomeres are still inhibited. Checkpoint-mediated cell cycle arrest also prevents further telomere erosion and deprotection that would favor chromosome rearrangements, which are known to increase cancer-associated genome instability. This review summarizes recent insights into functions and regulation of Tel1/ATM and Mec1/ATR at telomeres both in the presence and in the absence of telomerase, focusing mainly on discoveries in budding yeast.
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- 2021
19. The DNA damage checkpoint: A tale from budding yeast
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Paolo Pizzul, Erika Casari, Marco Gnugnoli, Carlo Rinaldi, Flavio Corallo, Maria Pia Longhese, Pizzul, P, Casari, E, Gnugnoli, M, Rinaldi, C, Corallo, F, and Longhese, M
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checkpoint ,Genetics ,Molecular Medicine ,DNA damage ,cell cycle ,protein kinase ,yeast ,Genetics (clinical) - Abstract
Studies performed in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe have led the way in defining the DNA damage checkpoint and in identifying most of the proteins involved in this regulatory network, which turned out to have structural and functional equivalents in humans. Subsequent experiments revealed that the checkpoint is an elaborate signal transduction pathway that has the ability to sense and signal the presence of damaged DNA and transduce this information to influence a multifaceted cellular response that is essential for cancer avoidance. This review focuses on the work that was done in Saccharomyces cerevisiae to articulate the checkpoint concept, to identify its players and the mechanisms of activation and deactivation.
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- 2022
20. How do cells sense DNA lesions?
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Chiara Vittoria Colombo, Elisa Gobbini, Marco Gnugnoli, Maria Pia Longhese, Colombo, C, Gnugnoli, M, Gobbini, E, and Longhese, M
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DNA Replication ,Saccharomyces cerevisiae Proteins ,DNA recombination ,DNA damage ,DNA repair ,DNA, Single-Stranded ,BIO/18 - GENETICA ,Ataxia Telangiectasia Mutated Proteins ,Saccharomyces cerevisiae ,single-stranded DNA ,Protein Serine-Threonine Kinases ,Biology ,DNA damage response ,Biochemistry ,law.invention ,Xenopus laevis ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,law ,Schizosaccharomyces ,Sense (molecular biology) ,Animals ,Humans ,Phosphorylation ,030304 developmental biology ,0303 health sciences ,Proto-Oncogene Proteins c-ets ,Cell Cycle ,Intracellular Signaling Peptides and Proteins ,DNA replication ,DNA ,Cell cycle ,Cell biology ,Repressor Proteins ,body regions ,ATR ,chemistry ,ATM ,Recombinant DNA ,Exogenous DNA ,030217 neurology & neurosurgery ,DNA Damage ,Signal Transduction - Abstract
DNA is exposed to both endogenous and exogenous DNA damaging agents that chemically modify it. To counteract the deleterious effects exerted by DNA lesions, eukaryotic cells have evolved a network of cellular pathways, termed DNA damage response (DDR). The DDR comprises both mechanisms devoted to repair DNA lesions and signal transduction pathways that sense DNA damage and transduce this information to specific cellular targets. These targets, in turn, impact a wide range of cellular processes including DNA replication, DNA repair and cell cycle transitions. The importance of the DDR is highlighted by the fact that DDR inactivation is commonly found in cancer and causes many different human diseases. The protein kinases ATM and ATR, as well as their budding yeast orthologs Tel1 and Mec1, act as master regulators of the DDR. The initiating events in the DDR entail both DNA lesion recognition and assembly of protein complexes at the damaged DNA sites. Here, we review what is known about the early steps of the DDR.
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- 2020
21. Functional and structural insights into the MRX/MRN complex, a key player in recognition and repair of DNA double-strand breaks
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Maria Pia Longhese, Jacopo Vertemara, Renata Tisi, Giuseppe Zampella, Tisi, R, Vertemara, J, Zampella, G, and Longhese, M
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CHIM/03 - CHIMICA GENERALE ED INORGANICA ,DNA damage ,lcsh:Biotechnology ,Biophysics ,BIO/18 - GENETICA ,MRX/MRN ,Review Article ,Biology ,Biochemistry ,DNA sequencing ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Structural Biology ,lcsh:TP248.13-248.65 ,Mre11 ,Genetics ,Homologous chromosome ,ComputingMethodologies_COMPUTERGRAPHICS ,030304 developmental biology ,0303 health sciences ,Xrs2/NBS1 ,BIO/11 - BIOLOGIA MOLECOLARE ,Double-strand break (DSB) ,Computer Science Applications ,Cell biology ,enzymes and coenzymes (carbohydrates) ,MRX complex ,MRN complex ,chemistry ,030220 oncology & carcinogenesis ,Rad50 ,Homologous recombination ,DNA ,Biotechnology - Abstract
Graphical abstract, Chromosomal DNA double-strand breaks (DSBs) are potentially lethal DNA lesions that pose a significant threat to genome stability and therefore need to be repaired to preserve genome integrity. Eukaryotic cells possess two main mechanisms for repairing DSBs: non-homologous end-joining (NHEJ) and homologous recombination (HR). HR requires that the 5′ terminated strands at both DNA ends are nucleolytically degraded by a concerted action of nucleases in a process termed DNA-end resection. This degradation leads to the formation of 3′-ended single-stranded DNA (ssDNA) ends that are essential to use homologous DNA sequences for repair. The evolutionarily conserved Mre11-Rad50-Xrs2/NBS1 complex (MRX/MRN) has enzymatic and structural activities to initiate DSB resection and to maintain the DSB ends tethered to each other for their repair. Furthermore, it is required to recruit and activate the protein kinase Tel1/ATM, which plays a key role in DSB signaling. All these functions depend on ATP-regulated DNA binding and nucleolytic activities of the complex. Several structures have been obtained in recent years for Mre11 and Rad50 subunits from archaea, and a few from the bacterial and eukaryotic orthologs. Nevertheless, the mechanism of activation of this protein complex is yet to be fully elucidated. In this review, we focused on recent biophysical and structural insights on the MRX complex and their interplay.
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- 2020
22. To Fix or Not to Fix: Maintenance of Chromosome Ends Versus Repair of DNA Double-Strand Breaks
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Erika Casari, Marco Gnugnoli, Carlo Rinaldi, Paolo Pizzul, Chiara Vittoria Colombo, Diego Bonetti, Maria Pia Longhese, Casari, E, Gnugnoli, M, Rinaldi, C, Pizzul, P, Colombo, C, Bonetti, D, and Longhese, M
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double-strand break ,senescence ,DNA End-Joining Repair ,Carcinogenesis ,S. cerevisiae ,Saccharomyces cerevisiae ,General Medicine ,Telomere ,checkpoint ,cancer ,Animals ,Humans ,DNA Breaks, Double-Stranded ,Telomerase ,Telomere Shortening - Abstract
Early work by Muller and McClintock discovered that the physical ends of linear chromosomes, named telomeres, possess an inherent ability to escape unwarranted fusions. Since then, extensive research has shown that this special feature relies on specialized proteins and structural properties that confer identity to the chromosome ends, thus allowing cells to distinguish them from intrachromosomal DNA double-strand breaks. Due to the inability of conventional DNA replication to fully replicate the chromosome ends and the downregulation of telomerase in most somatic human tissues, telomeres shorten as cells divide and lose this protective capacity. Telomere attrition causes the activation of the DNA damage checkpoint that leads to a cell-cycle arrest and the entering of cells into a nondividing state, called replicative senescence, that acts as a barrier against tumorigenesis. However, downregulation of the checkpoint overcomes this barrier and leads to further genomic instability that, if coupled with re-stabilization of telomeres, can drive tumorigenesis. This review focuses on the key experiments that have been performed in the model organism Saccharomyces cerevisiae to uncover the mechanisms that protect the chromosome ends from eliciting a DNA damage response, the conservation of these pathways in mammals, as well as the consequences of their loss in human cancer.
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- 2022
23. The chromatin remodeler Chd1 supports MRX and Exo1 functions in resection of DNA double-strand breaks
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Marco Gnugnoli, Maria Pia Longhese, Erika Casari, Gnugnoli, M, Casari, E, and Longhese, M
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Cancer Research ,genetic processes ,Cancer Treatment ,Gene Expression ,QH426-470 ,Biochemistry ,DNA annealing ,Histones ,chemistry.chemical_compound ,Medicine and Health Sciences ,Genetic annealing ,DNA Breaks, Double-Stranded ,Genetics (clinical) ,Double strand ,biology ,Chromosome Biology ,Physics ,Tumor Resection ,Chromatin ,Cell biology ,Nucleosomes ,Nucleic acids ,Physical sciences ,Histone ,Surgical Oncology ,Oncology ,Nucleic acid thermodynamics ,Epigenetics ,biological phenomena, cell phenomena, and immunity ,Research Article ,Clinical Oncology ,Saccharomyces cerevisiae Proteins ,DNA repair ,DNA recombination ,Saccharomyces cerevisiae ,Genes, Fungal ,Biophysics ,Surgical and Invasive Medical Procedures ,DNA repair, Chd1, chromatin, DSBs, resection ,Resection ,DNA-binding proteins ,Genetics ,Molecular Biology ,Ecology, Evolution, Behavior and Systematics ,Biology and life sciences ,Surgical Resection ,Proteins ,DNA ,Cell Biology ,biology.organism_classification ,enzymes and coenzymes (carbohydrates) ,Exodeoxyribonucleases ,chemistry ,biology.protein ,DNA damage ,Clinical Medicine ,Homologous recombination - Abstract
Repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) requires that the 5’-terminated DNA strands are resected to generate single-stranded DNA overhangs. This process is initiated by a short-range resection catalyzed by the MRX (Mre11-Rad50-Xrs2) complex, which is followed by a long-range step involving the nucleases Exo1 and Dna2. Here we show that the Saccharomyces cerevisiae ATP-dependent chromatin-remodeling protein Chd1 participates in both short- and long-range resection by promoting MRX and Exo1 association with the DSB ends. Furthermore, Chd1 reduces histone occupancy near the DSB ends and promotes DSB repair by HR. All these functions require Chd1 ATPase activity, supporting a role for Chd1 in the opening of chromatin at the DSB site to facilitate MRX and Exo1 processing activities., Author summary DNA double strand breaks (DSBs) are among the most severe types of damage occurring in the genome because their faulty repair can result in chromosome instability, commonly associated with carcinogenesis. Efficient and accurate repair of DSBs relies on several proteins required to process them. However, eukaryotic genomes are compacted into chromatin, which restricts the access to DNA of the enzymes devoted to repair DNA DSBs. To overcome this natural barrier, eukaryotes have evolved chromatin remodeling enzymes that use energy derived from ATP hydrolysis to modulate chromatin structure. Here, we examine the role in DSB repair of the ATP-dependent chromatin remodeler Chd1, which is frequently mutated in prostate cancer. We find that Chd1 is important to repair DNA DSBs by homologous recombination (HR) because it promotes the association with a damaged site of the MRX complex and Exo1, which are necessary to initiate HR. This Chd1 function requires its ATPase activity, suggesting that Chd1 increases the accessibility to chromatin to initiate repair of DNA lesions.
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- 2021
24. Interplay between Sae2 and Rif2 in the regulation of Mre11-Rad50 activities at DNA ends
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Diego Bonetti, Michela Clerici, Maria Pia Longhese, Bonetti, D, Clerici, M, and Longhese, M
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Saccharomyces cerevisiae Proteins ,DNA Repair ,Saccharomyces cerevisiae ,Telomere-Binding Proteins ,BIO/18 - GENETICA ,Protein Serine-Threonine Kinases ,chemistry.chemical_compound ,Genetics ,Protein kinase A ,MRX/N, Sae2, Rif2, DNA end ,chemistry.chemical_classification ,Endodeoxyribonucleases ,biology ,fungi ,Intracellular Signaling Peptides and Proteins ,Chromosome ,DNA ,biology.organism_classification ,Endonucleases ,Cell biology ,Telomere ,DNA-Binding Proteins ,enzymes and coenzymes (carbohydrates) ,Enzyme ,Exodeoxyribonucleases ,chemistry ,Rad50 ,biological phenomena, cell phenomena, and immunity ,Homologous recombination ,Developmental Biology - Abstract
DNA double-strand breaks (DSBs) can be repaired by non-homologous end-joining (NHEJ) or homologous recombination (HR). HR is initiated by nucleolytic degradation of the DSB ends in a process termed resection. The Mre11-Rad50-Xrs2/NBS1 (MRX/N) complex is a multifunctional enzyme that, aided by the Sae2/CtIP protein, promotes DSB resection and maintains the DSB ends tethered to each other to facilitate their re-ligation. Furthermore, it activates the protein kinase Tel1/ATM, which initiates DSB signaling. In Saccharomyces cerevisiae, these MRX functions are inhibited by the Rif2 protein, which is enriched at telomeres and protects telomeric DNA from being sensed and processed as a DSB. The present review focuses on recent data showing that Sae2 and Rif2 regulate MRX functions in opposite manners by interacting with Rad50 and influencing ATP-dependent Mre11-Rad50 conformational changes. As Sae2 is enriched at DSBs whereas Rif2 is predominantly present at telomeres, the relative abundance of these two MRX regulators can provide an effective mechanism to activate or inactivate MRX depending on the nature of chromosome ends.
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- 2021
25. Uncoupling Sae2 Functions in Downregulation of Tel1 and Rad53 Signaling Activities
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Luca Menin, Chiara Vittoria Colombo, Riccardo Ranieri, Maria Pia Longhese, Michela Clerici, Diego Bonetti, Colombo, C, Menin, L, Ranieri, R, Bonetti, D, Clerici, M, and Longhese, M
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Saccharomyces cerevisiae Proteins ,DNA Repair ,DNA damage ,Mutant ,Down-Regulation ,BIO/18 - GENETICA ,Cell Cycle Proteins ,Saccharomyces cerevisiae ,Protein Serine-Threonine Kinases ,Investigations ,Biology ,MRX ,Resection ,03 medical and health sciences ,Endonuclease ,chemistry.chemical_compound ,0302 clinical medicine ,Downregulation and upregulation ,Rad9 ,Dsb repair ,Genetics ,DNA Breaks, Double-Stranded ,Sae2 ,030304 developmental biology ,0303 health sciences ,Nuclease ,Endodeoxyribonucleases ,Tel1 ,fungi ,Intracellular Signaling Peptides and Proteins ,Endonucleases ,Cell biology ,Checkpoint Kinase 2 ,enzymes and coenzymes (carbohydrates) ,Exodeoxyribonucleases ,chemistry ,Rad53 ,Mutation ,biology.protein ,biological phenomena, cell phenomena, and immunity ,030217 neurology & neurosurgery ,DNA ,Signal Transduction - Abstract
The Mre11-Rad50-Xrs2 (MRX) complex acts together with the Sae2 protein to initiate resection of DNA double-strand breaks (DSBs) and to regulate a checkpoint response that couples cell cycle progression with DSB repair. Sae2 supports resistance to DNA damage and downregulates the signaling activities of MRX, Tel1, and Rad53 checkpoint proteins at the sites of damage. How these functions are connected to each other is not known. Here, we describe the separation-of-function sae2-ms mutant that, similar to SAE2 deletion, upregulates MRX and Tel1 signaling activities at DSBs by reducing Mre11 endonuclease activity. However, unlike SAE2 deletion, Sae2-ms causes neither DNA damage sensitivity nor enhanced Rad53 activation, indicating that DNA damage resistance depends mainly on Sae2-mediated Rad53 inhibition. The lack of Sae2, but not the presence of Sae2-ms, impairs long-range resection and increases both Rad9 accumulation at DSBs and Rad53–Rad9 interaction independently of Mre11 nuclease activity. Altogether, these data lead to a model whereby Sae2 plays distinct functions in limiting MRX-Tel1 and Rad9 abundance at DSBs, with the control on Rad9 association playing the major role in supporting DNA damage resistance and in regulating long-range resection and checkpoint activation.
- Published
- 2018
26. Dpb4 promotes resection of DNA double-strand breaks and checkpoint activation by acting in two different protein complexes
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Erika Casari, Michela Clerici, Elisa Gobbini, Marco Gnugnoli, Marco Mangiagalli, Maria Pia Longhese, Casari, E, Gobbini, E, Gnugnoli, M, Mangiagalli, M, Clerici, M, and Longhese, M
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Genomic instability ,Saccharomyces cerevisiae Proteins ,DNA Repair ,DNA damage ,DNA polymerase ,DNA repair ,Science ,genetic processes ,Saccharomyces cerevisiae ,General Physics and Astronomy ,Article ,General Biochemistry, Genetics and Molecular Biology ,Histones ,chemistry.chemical_compound ,DNA Breaks, Double-Stranded ,DNA damage checkpoints ,Adenosine Triphosphatases ,Multidisciplinary ,biology ,Chemistry ,fungi ,DNA ,DNA Polymerase II ,General Chemistry ,Chromatin Assembly and Disassembly ,biology.organism_classification ,Chromatin ,Cell biology ,enzymes and coenzymes (carbohydrates) ,Histone ,Mutation ,Histone fold ,biology.protein ,DNA damage, Chromatin, Checkpoint, Resection, S. cerevisiae ,biological phenomena, cell phenomena, and immunity ,DNA Damage ,Transcription Factors - Abstract
Budding yeast Dpb4 (POLE3/CHRAC17 in mammals) is a highly conserved histone fold protein that is shared by two protein complexes: the chromatin remodeler ISW2/hCHRAC and the DNA polymerase ε (Pol ε) holoenzyme. In Saccharomyces cerevisiae, Dpb4 forms histone-like dimers with Dls1 in the ISW2 complex and with Dpb3 in the Pol ε complex. Here, we show that Dpb4 plays two functions in sensing and processing DNA double-strand breaks (DSBs). Dpb4 promotes histone removal and DSB resection by interacting with Dls1 to facilitate the association of the Isw2 ATPase to DSBs. Furthermore, it promotes checkpoint activation by interacting with Dpb3 to facilitate the association of the checkpoint protein Rad9 to DSBs. Persistence of both Isw2 and Rad9 at DSBs is enhanced by the A62S mutation that is located in the Dpb4 histone fold domain and increases Dpb4 association at DSBs. Thus, Dpb4 exerts two distinct functions at DSBs depending on its interactors., The histone folding protein Dpb4 forms histone-like dimers within the ISW2 complex and the Pol ε complex in S. cerevisiae. Here the authors reveal insights into two distinct functions that Dpb4 exerts at DSBs depending on its interactors.
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- 2021
27. Sensing R-Loop-Associated DNA Damage to Safeguard Genome Stability
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Maria Pia Longhese, Diego Bonetti, Carlo Rinaldi, Paolo Pizzul, Rinaldi, C, Pizzul, P, Longhese, M, and Bonetti, D
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Genome instability ,R-loop ,DNA damage ,replication stress ,Review ,Biology ,Genome ,DSB ,03 medical and health sciences ,chemistry.chemical_compound ,Cell and Developmental Biology ,0302 clinical medicine ,Transcription (biology) ,replication stre ,lcsh:QH301-705.5 ,030304 developmental biology ,0303 health sciences ,RNA ,Cell Biology ,R-loops ,Cell biology ,ATR ,chemistry ,lcsh:Biology (General) ,ATM ,Nucleic acid ,DSBs ,030217 neurology & neurosurgery ,DNA ,Developmental Biology - Abstract
DNA transcription and replication are two essential physiological processes that can turn into a threat for genome integrity when they compete for the same DNA substrate. During transcription, the nascent RNA strongly binds the template DNA strand, leading to the formation of a peculiar RNA–DNA hybrid structure that displaces the non-template single-stranded DNA. This three-stranded nucleic acid transition is called R-loop. Although a programed formation of R-loops plays important physiological functions, these structures can turn into sources of DNA damage and genome instability when their homeostasis is altered. Indeed, both R-loop level and distribution in the genome are tightly controlled, and the list of factors involved in these regulatory mechanisms is continuously growing. Over the last years, our knowledge of R-loop homeostasis regulation (formation, stabilization, and resolution) has definitely increased. However, how R-loops affect genome stability and how the cellular response to their unscheduled formation is orchestrated are still not fully understood. In this review, we will report and discuss recent findings about these questions and we will focus on the role of ATM- and Rad3-related (ATR) and Ataxia–telangiectasia-mutated (ATM) kinases in the activation of an R-loop-dependent DNA damage response.
- Published
- 2020
28. The Rad53CHK1/CHK2-Spt21NPAT and Tel1ATM axes couple glucose tolerance to histone dosage and subtelomeric silencing
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Bruhn, Christopher, Ajazi, Arta, Ferrari, Elisa, Lanz, Michael Charles, Lanz, Michael, Batrin, Renaud, Choudhary, Ramveer, Walvekar, Adhish, Laxman, Sunil, Longhese, Maria Pia, Fabre, Emmanuelle, Bustamente Smolka, Marcus, Foiani, Marco, IFOM, Istituto FIRC di Oncologia Molecolare (IFOM), Cornell University [New York], Génomes, biologie cellulaire et thérapeutiques (GenCellDi (UMR_S_944)), Collège de France (CdF (institution))-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Institute for Stem Cell Science and Regenerative Medicine [Bangalore, Inde] (inStem), Università degli Studi di Milano-Bicocca [Milano] (UNIMIB), Università degli Studi di Milano [Milano] (UNIMI), C.B. was supported by fellowships from Associazione Italiana per la Ricerca sul Cancro (AIRC) Fellowship i-Care (Marie Curie co-funded by the European Union, ID 16173) and the European Commission (EC-FP7-SIPOD, ID PCOFUND-GA-2012-600399). This work was supported by grants from Fondazione AIRC under IG 2015 (M.F., ID 16770), IG 2018 (M.F., ID 21416), and IG 2017 (M.P.L., ID 19783), by the Ministero dell'Istruzione/Ministero dell'Università e della Ricerca (M.F., MIUR-PRIN-15-FOIANI) and by Progetti di Ricerca di Interesse Nazionale (PRIN) 2015 (M.P.L.). E. Fabre acknowledges Labex 'Who am I?' (ANR-11-LABX-0071, Idex ANR-11-IDEX-0005-02) and Cancéropôle Ile de France (ORFOCRISE PME-2015)., ANR-11-IDEX-0005,USPC,Université Sorbonne Paris Cité(2011), Bruhn, C, Ajazi, A, Ferrari, E, Lanz, M, Batrin, R, Choudhary, R, Walvekar, A, Laxman, S, Longhese, M, Fabre, E, Smolka, M, Foiani, M, Génomes, biologie cellulaire et thérapeutiques (GenCellDi (U944 / UMR7212)), Collège de France (CdF (institution))-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Università degli Studi di Milano-Bicocca = University of Milano-Bicocca (UNIMIB), Università degli Studi di Milano = University of Milan (UNIMI), Bodescot, Myriam, and Université Sorbonne Paris Cité - - USPC2011 - ANR-11-IDEX-0005 - IDEX - VALID
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0301 basic medicine ,DNA Repair ,DNA damage ,DNA repair ,[SDV]Life Sciences [q-bio] ,Science ,General Physics and Astronomy ,Saccharomyces cerevisiae ,[SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC] ,02 engineering and technology ,Protein-Serine-Threonine Kinase ,General Biochemistry, Genetics and Molecular Biology ,Ataxia Telangiectasia Mutated Protein ,03 medical and health sciences ,Histone Deacetylase ,Cell Cycle Protein ,[SDV.BC.BC] Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC] ,Serine ,Gene Silencing ,Epigenetics ,Phosphorylation ,lcsh:Science ,Transcription factor ,ComputingMilieux_MISCELLANEOUS ,Multidisciplinary ,biology ,Chemistry ,Acetylation ,General Chemistry ,Telomere ,021001 nanoscience & nanotechnology ,Subtelomere ,Chromatin ,Cell biology ,Checkpoint Kinase 2 ,Histone ,Glucose ,030104 developmental biology ,Intracellular Signaling Peptides and Protein ,Mutation ,biology.protein ,lcsh:Q ,0210 nano-technology ,Saccharomyces cerevisiae Protein ,DNA Damage ,Transcription Factors - Abstract
The DNA damage response (DDR) coordinates DNA metabolism with nuclear and non-nuclear processes. The DDR kinase Rad53CHK1/CHK2 controls histone degradation to assist DNA repair. However, Rad53 deficiency causes histone-dependent growth defects in the absence of DNA damage, pointing out unknown physiological functions of the Rad53-histone axis. Here we show that histone dosage control by Rad53 ensures metabolic homeostasis. Under physiological conditions, Rad53 regulates histone levels through inhibitory phosphorylation of the transcription factor Spt21NPAT on Ser276. Rad53-Spt21 mutants display severe glucose dependence, caused by excess histones through two separable mechanisms: dampening of acetyl-coenzyme A-dependent carbon metabolism through histone hyper-acetylation, and Sirtuin-mediated silencing of starvation-induced subtelomeric domains. We further demonstrate that repression of subtelomere silencing by physiological Tel1ATM and Rpd3HDAC activities coveys tolerance to glucose restriction. Our findings identify DDR mutations, histone imbalances and aberrant subtelomeric chromatin as interconnected causes of glucose dependence, implying that DDR kinases coordinate metabolism and epigenetic changes.
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- 2020
29. The 9-1-1 Complex Controls Mre11 Nuclease and Checkpoint Activation during Short-Range Resection of DNA Double-Strand Breaks
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Chiara Vittoria Colombo, Maria Pia Longhese, Elisa Gobbini, Erika Casari, Diego Bonetti, Gobbini, E, Casari, E, Colombo, C, Bonetti, D, and Longhese, M
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0301 basic medicine ,double-strand break ,Saccharomyces cerevisiae Proteins ,DNA Repair ,DNA damage ,S. cerevisiae ,MRX/MRN ,Saccharomyces cerevisiae ,General Biochemistry, Genetics and Molecular Biology ,Resection ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,checkpoint ,resection ,Homologous Recombination ,lcsh:QH301-705.5 ,Double strand ,double-strand breaks ,Nuclease ,Endodeoxyribonucleases ,biology ,RecQ Helicases ,DNA Helicases ,DNA ,Endonucleases ,Cell biology ,DNA-Binding Proteins ,enzymes and coenzymes (carbohydrates) ,030104 developmental biology ,Exodeoxyribonucleases ,lcsh:Biology (General) ,chemistry ,biology.protein ,biological phenomena, cell phenomena, and immunity ,Homologous recombination ,Dpb11/TopBP1 ,Rad9/53BP1 ,030217 neurology & neurosurgery ,9-1-1 ,DNA Damage - Abstract
Summary Homologous recombination is initiated by nucleolytic degradation (resection) of DNA double-strand breaks (DSBs). DSB resection is a two-step process in which an initial short-range step is catalyzed by the Mre11-Rad50-Xrs2 (MRX) complex and limited to the vicinity of the DSB end. Then the two long-range resection Exo1 and Dna2-Sgs1 nucleases extend the resected DNA tracts. How short-range resection is regulated and contributes to checkpoint activation remains to be determined. Here, we show that abrogation of long-range resection induces a checkpoint response that decreases DNA damage resistance. This checkpoint depends on the 9-1-1 complex, which recruits Dpb11 and Rad9 at damaged DNA. Furthermore, the 9-1-1 complex, independently of Dpb11 and Rad9, restricts short-range resection by negatively regulating Mre11 nuclease. We propose that 9-1-1, which is loaded at the leading edge of resection, plays a key function in regulating Mre11 nuclease and checkpoint activation once DSB resection is initiated.
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- 2020
30. DNA binding modes influence Rap1 activity in the regulation of telomere length and MRX functions at DNA ends
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Jacopo Vertemara, Renata Tisi, Diego Bonetti, Marco Notaro, Giuseppe Zampella, Paolo Pizzul, Carlo Rinaldi, Maria Pia Longhese, Bonetti, D, Rinaldi, C, Vertemara, J, Notaro, M, Pizzul, P, Tisi, R, Zampella, G, and Longhese, M
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endocrine system ,Saccharomyces cerevisiae Proteins ,Transcription, Genetic ,DNA damage ,Telomere-Binding Proteins ,Mutant ,Saccharomyces cerevisiae ,Genome Integrity, Repair and Replication ,Biology ,medicine.disease_cause ,Models, Biological ,Shelterin Complex ,Turn (biochemistry) ,chemistry.chemical_compound ,Genetics ,medicine ,DNA Breaks, Double-Stranded ,DSB, telomere, genome stability, Rap1, MRX ,DNA, Fungal ,Alleles ,Mutation ,fungi ,Telomere Homeostasis ,Telomere ,Cell biology ,enzymes and coenzymes (carbohydrates) ,chemistry ,Multiprotein Complexes ,Rap1 ,Function (biology) ,DNA ,DNA Damage ,Protein Binding ,Transcription Factors - Abstract
The cellular response to DNA double-strand breaks (DSBs) is initiated by the Mre11–Rad50–Xrs2 (MRX) complex that has structural and catalytic functions. MRX association at DSBs is counteracted by Rif2, which is known to interact with Rap1 that binds telomeric DNA through two tandem Myb-like domains. Whether and how Rap1 acts at DSBs is unknown. Here we show that Rif2 inhibits MRX association to DSBs in a manner dependent on Rap1, which binds to DSBs and promotes Rif2 association to them. Rap1 in turn can negatively regulate MRX function at DNA ends also independently of Rif2. In fact, a characterization of Rap1 mutant variants shows that Rap1 binding to DNA through both Myb-like domains results in formation of Rap1-DNA complexes that control MRX functions at both DSBs and telomeres primarily through Rif2. By contrast, Rap1 binding to DNA through a single Myb-like domain results in formation of high stoichiometry complexes that act at DNA ends mostly in a Rif2-independent manner. Altogether these findings indicate that the DNA binding modes of Rap1 influence its functional properties, thus highlighting the structural plasticity of this protein.
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- 2020
31. Structure–function relationships of the Mre11 protein in the control of DNA end bridging and processing
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Erika Casari, Maria Pia Longhese, Renata Tisi, Corinne Cassani, Antonio Marsella, Marsella, A, Cassani, C, Casari, E, Tisi, R, and Longhese, M
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Saccharomyces cerevisiae Proteins ,DNA damage ,BIO/18 - GENETICA ,Saccharomyces cerevisiae ,Biology ,Proteomics ,Resection ,Conserved sequence ,03 medical and health sciences ,chemistry.chemical_compound ,Mre11 ,Genetics ,Allele ,DNA, Fungal ,Sae2 ,030304 developmental biology ,0303 health sciences ,Endodeoxyribonucleases ,Tel1 ,030302 biochemistry & molecular biology ,General Medicine ,BIO/11 - BIOLOGIA MOLECOLARE ,Endonucleases ,Cell biology ,DNA-Binding Proteins ,enzymes and coenzymes (carbohydrates) ,Exodeoxyribonucleases ,MRX complex ,chemistry ,Rad50 ,DNA ,DNA Damage - Abstract
The evolutionarily conserved Mre11-Rad50-Xrs2 (MRX) complex cooperates with the Sae2 protein in initiating resection of DNA double-strand breaks (DSBs) and in maintaining the DSB ends tethered to each other for their accurate repair. How these MRX-Sae2 functions contribute to DNA damage resistance is not understood. By taking advantage of mre11 alleles that suppress the hypersensitivity of sae2∆ cells to genotoxic agents, we have recently found that Mre11 can be divided in two structurally distinct domains that support resistance to genotoxic agents by mediating different processes. While the Mre11 N-terminal domain impacts on the resection activity of long-range resection nucleases by mediating MRX and Tel1/ATM association to DNA DSBs, the C-terminus influences the MRX-tethering activity by its virtue to interact with Rad50. Given the evolutionary conservation of the MRX complex, our results have implications for understanding the consequences of its dysfunctions in human diseases.
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- 2018
32. Rad9/53 <scp>BP</scp> 1 protects stalled replication forks from degradation in Mec1/ <scp>ATR</scp> ‐defective cells
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Diego Bonetti, Matteo Villa, Maria Pia Longhese, Massimo Carraro, Villa, M, Bonetti, D, Carraro, M, and Longhese, M
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DNA Replication ,0301 basic medicine ,Saccharomyces cerevisiae Proteins ,BIO/18 - GENETICA ,Cell Cycle Proteins ,Saccharomyces cerevisiae ,Protein Serine-Threonine Kinases ,Biochemistry ,Resection ,03 medical and health sciences ,chemistry.chemical_compound ,Stress, Physiological ,Rad9 ,Genetics ,Mec1 ,resection ,Viability assay ,Molecular Biology ,Nuclease ,Sgs1 ,Microbial Viability ,biology ,fungi ,Intracellular Signaling Peptides and Proteins ,Articles ,Yeast ,Replication (computing) ,Cell biology ,030104 developmental biology ,chemistry ,biology.protein ,Degradation (geology) ,replication fork ,biological phenomena, cell phenomena, and immunity ,Tumor Suppressor p53-Binding Protein 1 ,DNA - Abstract
Nucleolytic processing by nucleases can be a relevant mechanism to allow repair/restart of stalled replication forks. However, nuclease action needs to be controlled to prevent overprocessing of damaged replication forks that can be detrimental to genome stability. The checkpoint protein Rad9/53BP1 is known to limit nucleolytic degradation (resection) of DNA double-strand breaks (DSBs) in both yeast and mammals. Here, we show that loss of the inhibition that Rad9 exerts on resection exacerbates the sensitivity to replication stress of Mec1/ATR-defective yeast cells by exposing stalled replication forks to Dna2-dependent degradation. This Rad9 protective function is independent of checkpoint activation and relies mainly on Rad9-Dpb11 interaction. We propose that Rad9/53BP1 supports cell viability by protecting stalled replication forks from extensive resection when the intra-S checkpoint is not fully functional.
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- 2018
33. Tel1/ATM Signaling to the Checkpoint Contributes to Replicative Senescence in the Absence of Telomerase
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Chiara Vittoria Colombo, Giorgia Maestrini, Luca Menin, Michela Clerici, Maria Pia Longhese, Menin, L, Colombo, C, Maestrini, G, Longhese, M, and Clerici, M
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Senescence ,DNA Replication ,Telomerase ,Saccharomyces cerevisiae Proteins ,Cell division ,Mutant ,DNA, Single-Stranded ,Ataxia Telangiectasia Mutated Proteins ,Saccharomyces cerevisiae ,Biology ,Protein Serine-Threonine Kinases ,Investigations ,replicative senescence ,03 medical and health sciences ,chemistry.chemical_compound ,checkpoint ,0302 clinical medicine ,Genetics ,Cellular Senescence ,Telomere Shortening ,030304 developmental biology ,0303 health sciences ,Kinase ,Tel1 ,DNA replication ,Intracellular Signaling Peptides and Proteins ,Cell Cycle Checkpoints ,Telomere ,Cell biology ,DNA-Binding Proteins ,chemistry ,Amino Acid Substitution ,Mutant Proteins ,030217 neurology & neurosurgery ,DNA ,Cell Division ,DNA Damage - Abstract
Mecl/ATR and Tell/ATM trigger replicative senescence when telomeres become critically short in the absence of telomerase, but how Tell/ATM promotes senescence is still unclear. Menin et al. studied the functions of Saccharomyces cerevisiae Tel1 in senescence by using... Telomeres progressively shorten at every round of DNA replication in the absence of telomerase. When they become critically short, telomeres trigger replicative senescence by activating a DNA damage response that is governed by the Mec1/ATR and Tel1/ATM protein kinases. While Mec1/ATR is known to block cell division when extended single-stranded DNA (ssDNA) accumulates at eroded telomeres, the molecular mechanism by which Tel1/ATM promotes senescence is still unclear. By characterizing a Tel1–hy184 mutant variant that compensates for the lack of Mec1 functions, we provide evidence that Tel1 promotes senescence by signaling to a Rad9-dependent checkpoint. Tel1–hy184 anticipates senescence onset in telomerase-negative cells, while the lack of Tel1 or the expression of a kinase-defective (kd) Tel1 variant delays it. Both Tel1–hy184 and Tel1–kd do not alter ssDNA generation at telomeric DNA ends. Furthermore, Rad9 and (only partially) Mec1 are responsible for the precocious senescence promoted by Tel1–hy184. This precocious senescence is mainly caused by the F1751I, D1985N, and E2133K amino acid substitutions, which are located in the FRAP–ATM–TRAPP domain of Tel1 and also increase Tel1 binding to DNA ends. Altogether, these results indicate that Tel1 induces replicative senescence by directly signaling dysfunctional telomeres to the checkpoint machinery.
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- 2019
34. Processing of DNA double-strand breaks by the MRX complex in a chromatin context
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Marco Gnugnoli, Carlo Rinaldi, Maria Pia Longhese, Antonio Marsella, Erika Casari, Diego Bonetti, Chiara Vittoria Colombo, Casari, E, Rinaldi, C, Marsella, A, Gnugnoli, M, Colombo, C, Bonetti, D, and Longhese, M
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Programmed cell death ,genetic processes ,Context (language use) ,BIO/18 - GENETICA ,Review ,Biochemistry, Genetics and Molecular Biology (miscellaneous) ,Biochemistry ,Sae2/CtIP ,chemistry.chemical_compound ,Mre11 ,Molecular Biosciences ,Molecular Biology ,lcsh:QH301-705.5 ,MRX complex ,Chemistry ,Kinase ,fungi ,Xrs2/NBS1 ,Resection ,Cell biology ,Chromatin ,enzymes and coenzymes (carbohydrates) ,MRN complex ,lcsh:Biology (General) ,Double-strand break ,Rad50 ,Tel1/ATM ,biological phenomena, cell phenomena, and immunity ,DNA - Abstract
DNA double-strand breaks (DSBs) are highly cytotoxic lesions that must be repaired to ensure genomic stability and avoid cell death. The cellular response to DSBs is initiated by the evolutionarily conserved Mre11-Rad50-Xrs2/NBS1 (MRX/MRN) complex that has structural and catalytic functions. Furthermore, it is responsible for DSB signaling through the activation of the checkpoint kinase Tel1/ATM. Here, we review functions and regulation of the MRX/MRN complex in DSB processing in a chromatin context, as well as its interplay with Tel1/ATM.
- Published
- 2019
35. The ATP-bound conformation of the Mre11-Rad50 complex is essential for Tel1/ATM activation
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Matteo Bassani, Antonio Marsella, Giuseppe Zampella, Corinne Cassani, Renata Tisi, Maria Pia Longhese, Jacopo Vertemara, Cassani, C, Vertemara, J, Bassani, M, Marsella, A, Tisi, R, Zampella, G, and Longhese, M
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Transcriptional Activation ,Cell cycle checkpoint ,Saccharomyces cerevisiae Proteins ,DNA Repair ,Dimer ,Molecular Conformation ,Ataxia Telangiectasia Mutated Proteins ,Saccharomyces cerevisiae ,Biology ,Genome Integrity, Repair and Replication ,Protein Serine-Threonine Kinases ,medicine.disease_cause ,DNA, DNA double strand breaks, Rad50, MRX, ATP, Tel1, ATM ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Adenosine Triphosphate ,Genetics ,medicine ,DNA, Fungal ,030304 developmental biology ,0303 health sciences ,Mutation ,Endodeoxyribonucleases ,Transition (genetics) ,Fungal genetics ,Wild type ,Intracellular Signaling Peptides and Proteins ,DNA-Binding Proteins ,enzymes and coenzymes (carbohydrates) ,Exodeoxyribonucleases ,chemistry ,Rad50 ,Multiprotein Complexes ,Biophysics ,biological phenomena, cell phenomena, and immunity ,Protein Multimerization ,030217 neurology & neurosurgery ,DNA ,DNA Damage ,Protein Binding ,Signal Transduction - Abstract
Activation of the checkpoint protein Tel1 requires the Mre11–Rad50–Xrs2 (MRX) complex, which recruits Tel1 at DNA double-strand breaks (DSBs) through direct interaction between Tel1 and Xrs2. However, in vitro Tel1 activation by MRX requires ATP binding to Rad50, suggesting a role also for the MR subcomplex in Tel1 activation. Here we describe two separation-of-functions alleles, mre11-S499P and rad50-A78T, which we show to specifically affect Tel1 activation without impairing MRX functions in DSB repair. Both Mre11-S499P and Rad50-A78T reduce Tel1–MRX interaction leading to poor Tel1 association at DSBs and consequent loss of Tel1 activation. The Mre11-S499P variant reduces Mre11–Rad50 interaction, suggesting an important role for MR complex formation in Tel1 activation. Molecular dynamics simulations show that the wild type MR subcomplex bound to ATP lingers in a tightly ‘closed’ conformation, while ADP presence leads to the destabilization of Rad50 dimer and of Mre11–Rad50 association, both events being required for MR conformational transition to an open state. By contrast, MRA78T undertakes complex opening even if Rad50 is bound to ATP, indicating that defective Tel1 activation caused by MRA78T results from destabilization of the ATP-bound conformational state.
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- 2019
36. Sae2 and Rif2 regulate MRX endonuclease activity at DNA double-strand breaks in opposite manners
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Elda Cannavo, Renata Tisi, Antonio Marsella, Maria Pia Longhese, Petr Cejka, Giordano Reginato, Elisa Gobbini, Corinne Cassani, Marsella, A, Gobbini, E, Cassani, C, Tisi, R, Cannavo, E, Reginato, G, Cejka, P, and Longhese, M
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double-strand break ,0301 basic medicine ,Saccharomyces cerevisiae Proteins ,Telomere-Binding Proteins ,BIO/18 - GENETICA ,Saccharomyces cerevisiae ,Models, Biological ,Article ,MRX ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,Endonuclease ,chemistry.chemical_compound ,Residue (chemistry) ,checkpoint ,0302 clinical medicine ,Dna cleavage ,Rif2 ,DNA Breaks, Double-Stranded ,lcsh:QH301-705.5 ,Sae2 ,Mre11-Rad50 ,Double strand ,double-strand breaks ,Nuclease ,biology ,Tel1 ,Cell Cycle Checkpoints ,Endonucleases ,3. Good health ,Cell biology ,enzymes and coenzymes (carbohydrates) ,030104 developmental biology ,lcsh:Biology (General) ,chemistry ,Multiprotein Complexes ,Rad53 ,Rad50 ,Mutation ,biology.protein ,biological phenomena, cell phenomena, and immunity ,Checkpoint ,Double-strand breaks ,Gene Deletion ,030217 neurology & neurosurgery ,Function (biology) ,DNA ,DNA Damage ,Protein Binding - Abstract
Summary The Mre11-Rad50-Xrs2 (MRX) complex detects and processes DNA double-strand breaks (DSBs). Its DNA binding and processing activities are regulated by transitions between an ATP-bound state and a post-hydrolysis cutting state that is nucleolytically active. Mre11 endonuclease activity is stimulated by Sae2, whose lack increases MRX persistence at DSBs and checkpoint activation. Here we show that the Rif2 protein inhibits Mre11 endonuclease activity and is responsible for the increased MRX retention at DSBs in sae2Δ cells. We identify a Rad50 residue that is important for Rad50-Rif2 interaction and Rif2 inhibition of Mre11 nuclease. This residue is located near a Rad50 surface that binds Sae2 and is important in stabilizing the Mre11-Rad50 (MR) interaction in the cutting state. We propose that Sae2 stimulates Mre11 endonuclease activity by stabilizing a post-hydrolysis MR conformation that is competent for DNA cleavage, whereas Rif2 antagonizes this Sae2 function and stabilizes an endonuclease inactive MR conformation., Graphical abstract, Highlights • Sae2 stimulates Mre11 endonuclease activity by stabilizing the MRX cutting state • Rif2 inhibits Sae2-mediated stimulation of Mre11 endonuclease activity • The rad50-N18S mutation escapes Rif2-mediated inhibition of Mre11 nuclease • Rif2 stabilizes an endonuclease inactive MR conformation that persistently binds DSBs, Phosphorylated Sae2 interacts with Rad50 and stimulates Mre11 endonuclease activity through an unknown mechanism. Marsella et al. show that Sae2 binding to the Rad50-Mre11 interface stabilizes a post-hydrolysis, endonuclease-active Mre11-Rad50 conformation. Rif2 interferes with the adoption of this conformation and inhibits Mre11 endonuclease activity.
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- 2021
37. Coupling end resection with the checkpoint response at DNA double-strand breaks
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Diego Bonetti, Matteo Villa, Corinne Cassani, Elisa Gobbini, Maria Pia Longhese, Villa, M, Cassani, C, Gobbini, E, Bonetti, D, and Longhese, M
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0301 basic medicine ,Saccharomyces cerevisiae Proteins ,Cell cycle checkpoint ,genetic processes ,Saccharomyces cerevisiae ,BIO/18 - GENETICA ,Biology ,Models, Biological ,MRX ,Resection ,Nuclease ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,chemistry.chemical_compound ,0302 clinical medicine ,Mec1 ,DNA Breaks, Double-Stranded ,Molecular Biology ,Pharmacology ,Genetics ,Double strand ,Tel1 ,fungi ,Cell Cycle Checkpoints ,Cell Biology ,G2-M DNA damage checkpoint ,Endonucleases ,biology.organism_classification ,Cell biology ,Coupling (electronics) ,enzymes and coenzymes (carbohydrates) ,030104 developmental biology ,chemistry ,Double-strand break ,health occupations ,Molecular Medicine ,biological phenomena, cell phenomena, and immunity ,Homologous recombination ,030217 neurology & neurosurgery ,DNA - Abstract
DNA double-strand breaks (DSBs) are a nasty form of damage that needs to be repaired to ensure genome stability. The DSB ends can undergo a strand-biased nucleolytic processing (resection) to generate 3′-ended single-stranded DNA (ssDNA) that channels DSB repair into homologous recombination. Generation of ssDNA also triggers the activation of the DNA damage checkpoint, which couples cell cycle progression with DSB repair. The checkpoint response is intimately linked to DSB resection, as some checkpoint proteins regulate the resection process. The present review will highlight recent works on the mechanism and regulation of DSB resection and its interplays with checkpoint activation/inactivation in budding yeast.
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- 2016
38. The <scp>MRX</scp> complex regulates Exo1 resection activity by altering <scp>DNA</scp> end structure
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Maria Pia Longhese, Patrick Sung, Corinne Cassani, Elisa Gobbini, Fabiana Mambretti, Weibin Wang, Erika Casari, Giuseppe Zampella, Renata Tisi, Jacopo Vertemara, Gobbini, E, Cassani, C, Vertemara, J, Wang, W, Mambretti, F, Casari, E, Sung, P, Tisi, R, Zampella, G, and Longhese, M
- Subjects
0301 basic medicine ,Saccharomyces cerevisiae Proteins ,genetic processes ,Mutant ,Saccharomyces cerevisiae ,Biology ,Exo1 ,MRX ,General Biochemistry, Genetics and Molecular Biology ,Resection ,03 medical and health sciences ,chemistry.chemical_compound ,Protein Domains ,DNA Breaks, Double-Stranded ,resection ,DNA, Fungal ,Sae2 ,Molecular Biology ,Nuclease ,Endodeoxyribonucleases ,General Immunology and Microbiology ,General Neuroscience ,fungi ,Articles ,double‐strand break ,Cell biology ,enzymes and coenzymes (carbohydrates) ,Exodeoxyribonucleases ,030104 developmental biology ,MRX complex ,chemistry ,Multiprotein Complexes ,health occupations ,biology.protein ,biological phenomena, cell phenomena, and immunity ,Homologous recombination ,Function (biology) ,DNA - Abstract
Homologous recombination is triggered by nucleolytic degradation (resection) of DNA double‐strand breaks (DSBs). DSB resection requires the Mre11‐Rad50‐Xrs2 (MRX) complex, which promotes the activity of Exo1 nuclease through a poorly understood mechanism. Here, we describe the Mre11‐R10T mutant variant that accelerates DSB resection compared to wild‐type Mre11 by potentiating Exo1‐mediated processing. This increased Exo1 resection activity leads to a decreased association of the Ku complex to DSBs and an enhanced DSB resection in G1, indicating that Exo1 has a direct function in preventing Ku association with DSBs. Molecular dynamics simulations show that rotation of the Mre11 capping domains is able to induce unwinding of double‐strand DNA (dsDNA). The R10T substitution causes altered orientation of the Mre11 capping domain that leads to persistent melting of the dsDNA end. We propose that MRX creates a specific DNA end structure that promotes Exo1 resection activity by facilitating the persistence of this nuclease on the DSB ends, uncovering a novel MRX function in DSB resection.
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- 2018
39. Processing of DNA ends in the maintenance of genome stability
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Michela Clerici, Maria Pia Longhese, Diego Bonetti, Chiara Vittoria Colombo, Bonetti, D, Colombo, C, Clerici, M, and Longhese, M
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0301 basic medicine ,lcsh:QH426-470 ,ved/biology.organism_classification_rank.species ,Saccharomyces cerevisiae ,Biology ,DNA replication ,medicine.disease_cause ,MRX ,03 medical and health sciences ,chemistry.chemical_compound ,Nuclease ,Genetic ,Genetics ,medicine ,Model organism ,Genetics (clinical) ,ved/biology ,Checkpoint ,fungi ,Chromosome ,biology.organism_classification ,Resection ,Cell biology ,lcsh:Genetics ,030104 developmental biology ,MRX complex ,chemistry ,Double-strand break ,Molecular Medicine ,nucleases ,Homologous recombination ,Carcinogenesis ,DNA - Abstract
DNA double-strand breaks (DSBs) are particularly hazardous lesions as their inappropriate repair can result in chromosome rearrangements, an important driving force of tumorigenesis. DSBs can be repaired by end joining mechanisms or by homologous recombination (HR). HR requires the action of several nucleases that preferentially remove the 5'-terminated strands at both DSB ends in a process called DNA end resection. The same nucleases are also involved in the processing of replication fork structures. Much of our understanding of these pathways has come from studies in the model organism Saccharomyces cerevisiae. Here, we review the current knowledge of the mechanism of resection at DNA DSBs and replication forks.
- Published
- 2018
40. Structurally distinct Mre11 domains mediate MRX functions in resection, end-tethering and DNA damage resistance
- Author
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Antonio Marsella, Giuseppe Zampella, Maria Pia Longhese, Renata Tisi, Weibin Wang, Corinne Cassani, Elisa Gobbini, Jacopo Vertemara, Patrick Sung, Cassani, C, Gobbini, E, Vertemara, J, Wang, W, Marsella, A, Sung, P, Tisi, R, Zampella, G, and Longhese, M
- Subjects
0301 basic medicine ,Models, Molecular ,DNA Repair ,Double-Strand-Break ,Genome Integrity, Repair and Replication ,medicine.disease_cause ,Exo1 ,chemistry.chemical_compound ,DNA Breaks, Double-Stranded ,Endodeoxyribonucleases ,Mutation ,Topoisomerase-I ,Fungal genetics ,Cell biology ,biological phenomena, cell phenomena, and immunity ,medicine.drug ,Saccharomyces cerevisiae Proteins ,DNA repair ,DNA damage ,Nuclease Activitie ,Antineoplastic Agents ,Phleomycins ,Saccharomyces cerevisiae ,Biology ,03 medical and health sciences ,Protein Domains ,Drug Resistance, Fungal ,Genetics ,medicine ,Saccharomyces-Cerevisiae ,Sae2 ,DNA Helicases ,Complex Function ,Endonucleases ,Recombination ,enzymes and coenzymes (carbohydrates) ,030104 developmental biology ,Exodeoxyribonucleases ,chemistry ,Rad50 ,Multiprotein Complexes ,biology.protein ,Camptothecin ,Protein Multimerization ,DNA ,Repair - Abstract
Sae2 cooperates with the Mre11–Rad50-Xrs2 (MRX) complex to initiate resection of DNA double-strand breaks (DSBs) and to maintain the DSB ends in close proximity to allow their repair. How these diverse MRX-Sae2 functions contribute to DNA damage resistance is not known. Here, we describe mre11 alleles that suppress the hypersensitivity of sae2Δ cells to genotoxic agents. By assessing the impact of these mutations at the cellular and structural levels, we found that all the mre11 alleles that restore sae2Δ resistance to both camptothecin and phleomycin affect the Mre11 N-terminus and suppress the resection defect of sae2Δ cells by lowering MRX and Tel1 association to DSBs. As a consequence, the diminished Tel1 persistence potentiates Sgs1-Dna2 resection activity by decreasing Rad9 association to DSBs. By contrast, the mre11 mutations restoring sae2Δ resistance only to phleomycin are located in Mre11 C-terminus and bypass Sae2 function in end-tethering but not in DSB resection, possibly by destabilizing the Mre11–Rad50 open conformation. These findings unmask the existence of structurally distinct Mre11 domains that support resistance to genotoxic agents by mediating different processes.
- Published
- 2017
41. Escape of Sgs1 from Rad9 inhibition reduces the requirement for Sae2 and functional <scp>MRX</scp> in <scp>DNA</scp> end resection
- Author
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Matteo Villa, Giulia Tedeschi, Diego Bonetti, Maria Pia Longhese, Corinne Cassani, Elisa Gobbini, Bonetti, D, Villa, M, Gobbini, E, Cassani, C, Tedeschi, G, and Longhese, M
- Subjects
double-strand break ,Saccharomyces cerevisiae Proteins ,genetic processes ,Mutant ,Saccharomyces cerevisiae ,BIO/18 - GENETICA ,Cell Cycle Proteins ,Biology ,Models, Biological ,Biochemistry ,Resection ,chemistry.chemical_compound ,Genetic ,Rad9 ,Genetics ,resection ,Molecular Biology ,Sgs1 ,Endodeoxyribonucleases ,RecQ Helicases ,Scientific Reports ,fungi ,Recombinational DNA Repair ,Endonucleases ,biology.organism_classification ,Molecular biology ,Cell biology ,DNA-Binding Proteins ,enzymes and coenzymes (carbohydrates) ,Exodeoxyribonucleases ,MRX complex ,chemistry ,Multiprotein Complexes ,Extensive resection ,biological phenomena, cell phenomena, and immunity ,Homologous recombination ,DNA - Abstract
Homologous recombination requires nucleolytic degradation (resection) of DNA double-strand break (DSB) ends. In Saccharomyces cerevisiae, the MRX complex and Sae2 are involved in the onset of DSB resection, whereas extensive resection requires Exo1 and the concerted action of Dna2 and Sgs1. Here, we show that the checkpoint protein Rad9 limits the action of Sgs1/Dna2 in DSB resection by inhibiting Sgs1 binding/persistence at the DSB ends. When inhibition by Rad9 is abolished by the Sgs1-ss mutant variant or by deletion of RAD9, the requirement for Sae2 and functional MRX in DSB resection is reduced. These results provide new insights into how early and long-range resection is coordinated. Synopsis The checkpoint protein Rad9 inhibits the Sgs1/Dna2 long-range resection machinery and thereby increases the requirement for MRX/Sae2 activities in DSB resection. The Sgs1-ss mutant variant suppresses the sensitivity to DNA damaging agents and the resection defect of sae2 cells. Rad9 limits the action of Sgs1/Dna2 in DSB resection by inhibiting Sgs1 binding/persistence at the DSB ends. The escape of Sgs1 from Rad9 inhibition reduces the requirement for Sae2 and functional MRX in DSB resection. The checkpoint protein Rad9 inhibits the Sgs1/Dna2 long-range resection machinery and thereby increases the requirement for MRX-Sae2 activities in DSB resection.
- Published
- 2015
42. Regulation of telomere metabolism by the RNA processing protein Xrn1
- Author
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Michael Lisby, Daniele Cesena, Emanuela Rizzo, Diego Bonetti, Corinne Cassani, Maria Pia Longhese, Cesena, D, Cassani, C, Rizzo, E, Lisby, M, Bonetti, D, and Longhese, M
- Subjects
0301 basic medicine ,Telomerase ,Saccharomyces cerevisiae Proteins ,Telomere-Binding Proteins ,DNA repair ,DNA, Single-Stranded ,BIO/18 - GENETICA ,CST complex ,Genome Integrity, Repair and Replication ,Biology ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Genetics ,RNA Processing, Post-Transcriptional ,Xrn1 ,Uncapping ,Telomere-binding protein ,Exosome Multienzyme Ribonuclease Complex ,Temperature ,Telomere Homeostasis ,Telomere ,G2-M DNA damage checkpoint ,Repressor Proteins ,030104 developmental biology ,RNA processing ,chemistry ,Exoribonucleases ,Mutation ,030217 neurology & neurosurgery ,DNA ,Function (biology) - Abstract
Telomeric DNA consists of repetitive G-rich sequences that terminate with a 3΄-ended single stranded overhang (G-tail), which is important for telomere extension by telomerase. Several proteins, including the CST complex, are necessary to maintain telomere structure and length in both yeast and mammals. Emerging evidence indicates that RNA processing factors play critical, yet poorly understood, roles in telomere metabolism. Here, we show that the lack of the RNA processing proteins Xrn1 or Rrp6 partially bypasses the requirement for the CST component Cdc13 in telomere protection by attenuating the activation of the DNA damage checkpoint. Xrn1 is necessary for checkpoint activation upon telomere uncapping because it promotes the generation of single-stranded DNA. Moreover, Xrn1 maintains telomere length by promoting the association of Cdc13 to telomeres independently of ssDNA generation and exerts this function by downregulating the transcript encoding the telomerase inhibitor Rif1. These findings reveal novel roles for RNA processing proteins in the regulation of telomere metabolism with implications for genome stability in eukaryotes.
- Published
- 2017
43. Tel1 and Rif2 Regulate MRX Functions in End-Tethering and Repair of DNA Double-Strand Breaks
- Author
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Corinne Cassani, Hengyao Niu, Weibin Wang, Patrick Sung, Michela Clerici, Elisa Gobbini, Maria Pia Longhese, Cassani, C, Gobbini, E, Wang, W, Niu, H, Clerici, M, Sung, P, and Longhese, M
- Subjects
0301 basic medicine ,DNA End-Joining Repair ,genetic processes ,Biochemistry ,Conformational-Change ,Damage Response ,Adenosine Triphosphate ,Atm Activation ,0302 clinical medicine ,Chemical reactions ,Medicine and Health Sciences ,Short Telomere ,DNA Breaks, Double-Stranded ,Biology (General) ,Homologous Recombination ,Genetics ,Organic Compounds ,Hydrolysis ,General Neuroscience ,Monosaccharides ,Intracellular Signaling Peptides and Proteins ,3. Good health ,Cell biology ,DNA-Binding Proteins ,Nucleic acids ,Physical sciences ,Non-homologous end joining ,Chemistry ,MRX complex ,Mating-Type ,Biological Cultures ,biological phenomena, cell phenomena, and immunity ,General Agricultural and Biological Sciences ,Research Article ,Saccharomyces cerevisiae Proteins ,DNA damage ,DNA repair ,QH301-705.5 ,Molecular Sequence Data ,Telomere-Binding Proteins ,Immunology ,Carbohydrates ,Nuclease Activitie ,Surgical and Invasive Medical Procedures ,BIO/18 - GENETICA ,Saccharomyces cerevisiae ,Protein Serine-Threonine Kinases ,Biology ,Research and Analysis Methods ,Mre11 Complex ,Non-Homologous End Joining ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,Hypersensitivity ,Saccharomyces-Cerevisiae ,Amino Acid Sequence ,Binding Domain ,Endodeoxyribonucleases ,Biology and life sciences ,Surgical Resection ,General Immunology and Microbiology ,Organic Chemistry ,fungi ,Chemical Compounds ,Galactose ,DNA ,Cell Cultures ,enzymes and coenzymes (carbohydrates) ,Exodeoxyribonucleases ,030104 developmental biology ,MRN complex ,ATP hydrolysis ,Rad50 ,health occupations ,Clinical Immunology ,Clinical Medicine ,Homologous recombination ,030217 neurology & neurosurgery - Abstract
The cellular response to DNA double-strand breaks (DSBs) is initiated by the MRX/MRN complex (Mre11-Rad50-Xrs2 in yeast; Mre11-Rad50-Nbs1 in mammals), which recruits the checkpoint kinase Tel1/ATM to DSBs. In Saccharomyces cerevisiae, the role of Tel1 at DSBs remains enigmatic, as tel1Δ cells do not show obvious hypersensitivity to DSB-inducing agents. By performing a synthetic phenotype screen, we isolated a rad50-V1269M allele that sensitizes tel1Δ cells to genotoxic agents. The MRV1269MX complex associates poorly to DNA ends, and its retention at DSBs is further reduced by the lack of Tel1. As a consequence, tel1Δ rad50-V1269M cells are severely defective both in keeping the DSB ends tethered to each other and in repairing a DSB by either homologous recombination (HR) or nonhomologous end joining (NHEJ). These data indicate that Tel1 promotes MRX retention to DSBs and this function is important to allow proper MRX-DNA binding that is needed for end-tethering and DSB repair. The role of Tel1 in promoting MRX accumulation to DSBs is counteracted by Rif2, which is recruited to DSBs. We also found that Rif2 enhances ATP hydrolysis by MRX and attenuates MRX function in end-tethering, suggesting that Rif2 can regulate MRX activity at DSBs by modulating ATP-dependent conformational changes of Rad50., This study reveals novel roles for the checkpoint kinase Tel1/ATM and Rif2 in regulating the function of the MRX complex during repair of DNA double-strand breaks by nonhomologous end joining and homologous recombination., Author Summary Many tumors contain mutations that confer defects in repairing DNA double-strand breaks (DSBs). In both yeast and mammals, the MRX/MRN complex (Mre11-Rad50-Xrs2 in yeast; Mre11-Rad50-Nbs1 in mammals) plays critical functions in repairing a DSB by either nonhomologous end joining (NHEJ) or homologous recombination (HR). Furthermore, it recruits the checkpoint kinase Tel1/ATM. Although ATM is considered to be a tumor suppressor, up-regulation of ATM signaling promotes chemoresistance, radioresistance and metastasis. For this reason, cancer therapies targeting ATM have been developed to increase the effectiveness of standard genotoxic treatments and/or to set up synthetic lethal approaches in cancers with DNA repair defects. We aimed to identify the precise role of ATM/Tel1 in these processes. By performing a synthetic phenotype screen, we identified a mutation (rad50-V1269M) altering the Rad50 subunit of the MRX complex, which sensitizes cells lacking Tel1 to genotoxic agents. Genetic and biochemical characterization of MRV1269MX protein complex revealed that Tel1 promotes MRX association at DSBs to allow proper MRX-DNA binding that is needed for DSB repair. The role of Tel1 in promoting MRX retention on DSBs is counteracted by Rif2, which can regulate MRX activity at DSBs by modulating ATP-dependent conformational changes in Rad50. Our finding that MRX dysfunctions can be synthetically lethal with Tel1 loss in the presence of genotoxic agents suggests that ATM inhibitors could be beneficial in patients whose tumors have defective MRN functions.
- Published
- 2016
44. Functions and regulation of the MRX complex at DNA double-strand breaks
- Author
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Diego Bonetti, Matteo Villa, Corinne Cassani, Maria Pia Longhese, Elisa Gobbini, Gobbini, E, Cassani, C, Villa, M, Bonetti, D, and Longhese, M
- Subjects
0301 basic medicine ,double-strand break ,Applied Microbiology ,Saccharomyces cerevisiae ,BIO/18 - GENETICA ,Biology ,Biochemistry, Genetics and Molecular Biology (miscellaneous) ,Applied Microbiology and Biotechnology ,Microbiology ,MRX ,Resection ,03 medical and health sciences ,chemistry.chemical_compound ,Nuclease ,Rif2 ,Virology ,Genetics ,resection ,Molecular Biology ,lcsh:QH301-705.5 ,Sae2 ,Double strand ,Kinase ,fungi ,Tel1 ,Cell Biology ,biology.organism_classification ,enzymes and coenzymes (carbohydrates) ,030104 developmental biology ,MRX complex ,chemistry ,lcsh:Biology (General) ,Parasitology ,nucleases ,biological phenomena, cell phenomena, and immunity ,Homologous recombination ,DNA ,Function (biology) - Abstract
DNA double-strand breaks (DSBs) pose a serious threat to genome stability and cell survival. Cells possess mechanisms that recognize DSBs and promote their repair through either homologous recombination (HR) or non-homologous end joining (NHEJ). The evolutionarily conserved Mre11-Rad50-Xrs2 (MRX) complex plays a central role in the cellular response to DSBs, as it is implicated in controlling end resection and in maintaining the DSB ends tethered to each other. Furthermore, it is responsible for DSB signaling by activating the checkpoint kinase Tel1 that, in turn, supports MRX function in a positive feedback loop. The present review focuses mainly on recent works in the budding yeast Saccharomyces cerevisiae to highlight structure and regulation of MRX as well as its interplays with Tel1.
- Published
- 2016
45. Sae2 Function at DNA Double-Strand Breaks Is Bypassed by Dampening Tel1 or Rad53 Activity
- Author
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Matteo Villa, Michela Clerici, Elisa Gobbini, Luca Menin, Maria Pia Longhese, Marco Gnugnoli, Gobbini, E, Villa, M, Gnugnoli, M, Menin, L, Clerici, M, and Longhese, M
- Subjects
double-strand break ,Genome instability ,Cancer Research ,Cell cycle checkpoint ,Saccharomyces cerevisiae Proteins ,lcsh:QH426-470 ,DNA damage ,BIO/18 - GENETICA ,Cell Cycle Proteins ,Saccharomyces cerevisiae ,yeast ,Biology ,Protein Serine-Threonine Kinases ,Genomic Instability ,checkpoint ,Genetics ,Hypersensitivity ,DNA Breaks, Double-Stranded ,Phosphorylation ,Homologous Recombination ,Sae2 ,Molecular Biology ,Checkpoint Kinase 2 ,Genetics (clinical) ,Ecology, Evolution, Behavior and Systematics ,RecQ Helicases ,fungi ,Cell Cycle ,DNA Helicases ,Intracellular Signaling Peptides and Proteins ,G2-M DNA damage checkpoint ,Cell cycle ,Endonucleases ,lcsh:Genetics ,MRX complex ,Cancer research ,biological phenomena, cell phenomena, and immunity ,Homologous recombination ,Research Article ,DNA Damage - Abstract
The MRX complex together with Sae2 initiates resection of DNA double-strand breaks (DSBs) to generate single-stranded DNA (ssDNA) that triggers homologous recombination. The absence of Sae2 not only impairs DSB resection, but also causes prolonged MRX binding at the DSBs that leads to persistent Tel1- and Rad53-dependent DNA damage checkpoint activation and cell cycle arrest. Whether this enhanced checkpoint signaling contributes to the DNA damage sensitivity and/or the resection defect of sae2Δ cells is not known. By performing a genetic screen, we identify rad53 and tel1 mutant alleles that suppress both the DNA damage hypersensitivity and the resection defect of sae2Δ cells through an Sgs1-Dna2-dependent mechanism. These suppression events do not involve escaping the checkpoint-mediated cell cycle arrest. Rather, defective Rad53 or Tel1 signaling bypasses Sae2 function at DSBs by decreasing the amount of Rad9 bound at DSBs. As a consequence, reduced Rad9 association to DNA ends relieves inhibition of Sgs1-Dna2 activity, which can then compensate for the lack of Sae2 in DSB resection and DNA damage resistance. We propose that persistent Tel1 and Rad53 checkpoint signaling in cells lacking Sae2 increases the association of Rad9 at DSBs, which in turn inhibits DSB resection by limiting the activity of the Sgs1-Dna2 resection machinery., Author Summary Genome instability is one of the most pervasive characteristics of cancer cells and can be due to DNA repair defects and failure to arrest the cell cycle. Among the many types of DNA damage, the DNA double strand break (DSB) is one of the most severe, because it can cause mutations and chromosomal rearrangements. Generation of DSBs triggers a highly conserved mechanism, known as DNA damage checkpoint, which arrests the cell cycle until DSBs are repaired. DSBs can be repaired by homologous recombination, which requires the DSB ends to be nucleolytically processed (resected) to generate single-stranded DNA. In Saccharomyces cerevisiae, DSB resection is initiated by the MRX complex together with Sae2, whereas more extensive resection is catalyzed by both Exo1 and Dna2-Sgs1. The absence of Sae2 not only impairs DSB resection, but also leads to the hyperactivation of the checkpoint proteins Tel1/ATM and Rad53, leading to persistent cell cycle arrest. In this manuscript we show that persistent Tel1 and Rad53 signaling activities in sae2Δ cells cause DNA damage hypersensitivity and defective DSB resection by increasing the amount of Rad9 bound at the DSBs, which in turn inhibits the Sgs1-Dna2 resection machinery. As ATM inhibition has been proposed as a strategy for cancer treatment, the finding that defective Tel1 signaling activity restores DNA damage resistance in sae2Δ cells might have implications in cancer therapies that use ATM inhibitors for synthetic lethal approaches that are devised to kill tumor cells with defective DSB repair.
- Published
- 2015
46. Local unwinding of double-strand DNA ends by the MRX complex promotes Exo1 processing activity
- Author
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Maria Pia Longhese, Jacopo Vertemara, Elisa Gobbini, Gobbini, E, Vertemara, J, and Longhese, M
- Subjects
double-strand break ,0301 basic medicine ,Cancer Research ,S. cerevisiae ,BIO/18 - GENETICA ,Exo1 ,MRX ,Resection ,03 medical and health sciences ,Exonuclease 1 ,chemistry.chemical_compound ,0302 clinical medicine ,Author’s Views ,resection ,Double strand ,Cell biology ,enzymes and coenzymes (carbohydrates) ,030104 developmental biology ,MRX complex ,chemistry ,Molecular Medicine ,biological phenomena, cell phenomena, and immunity ,Homologous recombination ,Novel mutation ,030217 neurology & neurosurgery ,DNA - Abstract
Homologous recombination is initiated by nucleolytic degradation (resection) of DNA double-strand breaks (DSBs), which involves different nucleases including the Mre11-Rad50-Xrs2 (MRX) complex and the Exonuclease 1 (Exo1). The characterization of a novel mutation in Mre11 causing accelerated DSB resection has allowed to show that MRX facilitates DNA end processing by Exo1 through local unwinding of double-stranded DNA ends.
- Published
- 2018
47. Resection is responsible for loss of transcription around a double-strand break in Saccharomyces cerevisiae
- Author
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Maria Pia Longhese, Michela Clerici, Maxime Wery, Antonin Morillon, Nicola Manfrini, Fabrizio d'Adda di Fagagna, Chiara Vittoria Colombo, Marc Descrimes, Università degli Studi di Milano-Bicocca = University of Milano-Bicocca (UNIMIB), Université Pierre et Marie Curie - Paris 6 (UPMC), Dynamics of genetic information : fundamental basis and cancer, Institut Curie [Paris], FIRC, Institute of Molecular Oncology Foundation, National Research Council of Italy | Consiglio Nazionale delle Ricerche (CNR), Università degli Studi di Milano-Bicocca [Milano] (UNIMIB), Consiglio Nazionale delle Ricerche [Roma] (CNR), Manfrini, N, Clerici, M, Wery, M, Colombo, C, Descrimes, M, Morillon, A, d’Adda di fagagna, F, and Longhese, M
- Subjects
Transcription, Genetic ,Immunology and Microbiology (all) ,QH301-705.5 ,DNA repair ,DNA damage ,Science ,genetic processes ,S. cerevisiae ,BIO/18 - GENETICA ,[SDV.CAN]Life Sciences [q-bio]/Cancer ,Saccharomyces cerevisiae ,Biology ,General Biochemistry, Genetics and Molecular Biology ,DNA double-strand breaks ,DNA Breaks, Double-Stranded ,chromosome ,resection ,Biology (General) ,gene ,Transcription bubble ,chemistry.chemical_classification ,DNA ligase ,Biochemistry, Genetics and Molecular Biology (all) ,Neuroscience (all) ,DNA clamp ,General Immunology and Microbiology ,Medicine (all) ,General Neuroscience ,fungi ,DNA replication ,General Medicine ,Molecular biology ,Cell biology ,enzymes and coenzymes (carbohydrates) ,DNA Repair Enzymes ,chemistry ,Genes and Chromosomes ,RNA polymerase ,DNA double-strand break ,health occupations ,Medicine ,DNA supercoil ,biological phenomena, cell phenomena, and immunity ,transcription ,In vitro recombination ,Research Article - Abstract
Emerging evidence indicate that the mammalian checkpoint kinase ATM induces transcriptional silencing in cis to DNA double-strand breaks (DSBs) through a poorly understood mechanism. Here we show that in Saccharomyces cerevisiae a single DSB causes transcriptional inhibition of proximal genes independently of Tel1/ATM and Mec1/ATR. Since the DSB ends undergo nucleolytic degradation (resection) of their 5′-ending strands, we investigated the contribution of resection in this DSB-induced transcriptional inhibition. We discovered that resection-defective mutants fail to stop transcription around a DSB, and the extent of this failure correlates with the severity of the resection defect. Furthermore, Rad9 and generation of γH2A reduce this DSB-induced transcriptional inhibition by counteracting DSB resection. Therefore, the conversion of the DSB ends from double-stranded to single-stranded DNA, which is necessary to initiate DSB repair by homologous recombination, is responsible for loss of transcription around a DSB in S. cerevisiae. DOI: http://dx.doi.org/10.7554/eLife.08942.001, eLife digest DNA is constantly under assault from harmful chemicals; some of which are produced inside the cell, while others come from outside of the cell. Breaks that form across both strands in a DNA double helix are considered the most dangerous type of DNA damage, and can cause a cell to die or become cancerous if they are not repaired accurately. ‘Homologous recombination’ is one of the main mechanisms used by cells to repair DNA double-strand breaks. This mechanism requires enzymes to eat away at the end of one of the DNA strands on each side of the double-strand break. This process is called ‘resection’ and it exposes single strands of DNA. These single-stranded DNA ‘tails’ are then free to interact with an intact copy of the same DNA sequence from elsewhere in the cell's nucleus, which is used as a guide when repairing the damage. The proteins involved in homologous recombination have to work around other processes that go on inside the nucleus, such as the transcription of DNA in genes into RNA molecules. Previous research has reported that forming a double-strand break in the DNA reduces the levels of transcription for the genes that surround the break, but it was not clear how this occurred. In mammalian cells, inhibiting the transcription of genes around a double-strand DNA break depends on a signaling pathway that is activated whenever DNA damage is detected. Manfrini et al. now show that this is not the case for budding yeast (Saccharomyces cerevisiae). Instead, the experiments indicate that it is the resection of the DNA around a double-strand break to form single-stranded tails that inhibits transcription in budding yeast. One of the next challenges will be to see if the resection process makes any contribution to changes in the transcription of genes that surround a double-strand break in mammals as well. DOI: http://dx.doi.org/10.7554/eLife.08942.002
- Published
- 2015
48. The Saccharomyces cerevisiae 14-3-3 Proteins Are Required for the G1/S Transition, Actin Cytoskeleton Organization and Cell Wall Integrity
- Author
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Francisca Lottersberger, Giovanna Lucchini, Simonetta Piatti, Andrea Panza, Maria Pia Longhese, Lottersberger, F, Panza, A, Lucchini, G, Piatti, S, and Longhese, M
- Subjects
Osmosis ,Saccharomyces cerevisiae Proteins ,actin cytoskeleton ,Genes, Fungal ,Saccharomyces cerevisiae ,Gene Dosage ,S. cerevisiae ,BIO/18 - GENETICA ,Investigations ,Actin cytoskeleton organization ,S Phase ,Calcium-binding protein ,Genetics ,DNA, Fungal ,Genes, Suppressor ,Cytoskeleton ,Protein Kinase C ,14-3-3 ,Membrane Glycoproteins ,Base Sequence ,biology ,Calcium-Binding Proteins ,G1 Phase ,Intracellular Signaling Peptides and Proteins ,Temperature ,DNA replication ,Membrane Proteins ,G1/S transition ,biology.organism_classification ,Actin cytoskeleton ,Actins ,Cell biology ,14-3-3 Proteins ,Membrane protein ,Mutation ,cell wall ,cell cycle - Abstract
14-3-3 proteins are highly conserved polypeptides that participate in many biological processes by binding phosphorylated target proteins. The Saccharomyces cerevisiae BMH1 and BMH2 genes, whose concomitant deletion is lethal, encode two functionally redundant 14-3-3 isoforms. To gain insights into the essential function(s) shared by these proteins, we searched for high-dosage suppressors of the growth defects of temperature-sensitive bmh mutants. Both the protein kinase C1 (Pkc1) and its upstream regulators Wsc2 and Mid2 were found to act as high dosage suppressors of bmh mutants' temperature sensitivity, indicating a functional interaction between 14-3-3 and Pkc1. Consistent with a role of 14-3-3 proteins in Pkc1-dependent cellular processes, shift to the restrictive temperature of bmh mutants severely impaired initiation of DNA replication, polarization of the actin cytoskeleton, and budding, as well as cell wall integrity. Because Pkc1 acts in concert with the Swi4-Swi6 (SBF) transcriptional activator to control all these processes, the defective G1/S transition of bmh mutants might be linked to impaired SBF activity. Indeed, the levels of the G1 cyclin CLN2 transcripts, which are positively regulated by SBF, were dramatically reduced in bmh mutants. Remarkably, budding and DNA replication defects of bmh mutants were suppressed by CLN2 expression from an SBF-independent promoter, suggesting that 14-3-3 proteins might contribute to regulating the late G1 transcriptional program.
- Published
- 2006
49. The cellular response to chromosome breakage
- Author
-
Maria Pia Longhese, Michela Clerici, Davide Mantiero, Longhese, M, Mantiero, D, and Clerici, M
- Subjects
double-strand break ,DNA Repair ,DNA damage ,DNA repair ,S. cerevisiae ,BIO/18 - GENETICA ,Biology ,Microbiology ,Histones ,Animals ,Humans ,DNA Breaks, Double-Stranded ,Mec1 ,Molecular Biology ,Recombination, Genetic ,Genetics ,Models, Genetic ,Checkpoint ,Cell Cycle ,Tel1 ,fungi ,DNA replication ,Chromosome Breakage ,Telomere ,Chromatin ,Histone ,biology.protein ,Chromosome breakage ,Homologous recombination ,Signal Transduction - Abstract
DNA double-strand breaks (DSBs) are among the most deleterious types of damage that can occur in the genome of eukaryotic cells because failure to repair them can lead to loss of genetic information and chromosome rearrangements. DSBs can arise by failures in DNA replication and by exposure to environmental factors, such as ionizing radiations and radiomimetic chemicals. Moreover, they might arise when telomeres undergo extensive erosion, leading to the activation of the DNA damage response pathways and the onset of apoptosis and/or senescence. Importantly, DSBs can also form in a programmed manner during development. For example, meiotic recombination and rearrangement of the immunoglobulin genes in lymphocytes require the generation of site- or region-specific DSBs through the action of specific endonucleases. Efficient DSB repair is crucial in safeguarding genome integrity, whose maintenance in the face of DSBs involves branched signalling networks that switch on DNA damage checkpoints, activate DNA repair, induce chromatin reorganization and modulate numerous cellular processes. Not surprisingly, defects in these networks result in a variety of diseases ranging from severe genetic disorders to cancer predisposition and accelerated ageing.
- Published
- 2006
50. The Saccharomyces cerevisiae Sae2 protein negatively regulates DNA damage checkpoint signalling
- Author
-
Davide Mantiero, Michela Clerici, Giovanna Lucchini, Maria Pia Longhese, Clerici, M, Mantiero, D, Lucchini, G, and Longhese, M
- Subjects
Saccharomyces cerevisiae Proteins ,Time Factors ,Cell cycle checkpoint ,DNA Repair ,DNA repair ,DNA damage ,Blotting, Western ,Genes, Fungal ,Scientific Report ,BIO/18 - GENETICA ,Saccharomyces cerevisiae ,Protein Serine-Threonine Kinases ,Biology ,Biochemistry ,Fungal Proteins ,Mitotic cell cycle ,Genetics ,CHEK1 ,Phosphorylation ,DNA, Fungal ,Molecular Biology ,Sae2, checkpoint, MRX, Mec1, Tel1 ,Endodeoxyribonucleases ,Cell Cycle ,Intracellular Signaling Peptides and Proteins ,G2-M DNA damage checkpoint ,Cell cycle ,Endonucleases ,Molecular biology ,Cell biology ,Exodeoxyribonucleases ,MRX complex ,biological phenomena, cell phenomena, and immunity ,DNA Damage ,Signal Transduction - Abstract
Double-strand breaks (DSBs) elicit a DNA damage response, resulting in checkpoint-mediated cell-cycle delay and DNA repair. The Saccharomyces cerevisiae Sae2 protein is known to act together with the MRX complex in meiotic DSB processing, as well as in DNA damage response during the mitotic cell cycle. Here, we report that cells lacking Sae2 fail to turn off both Mec1- and Tel1-dependent checkpoints activated by a single irreparable DSB, and delay Mre11 foci disassembly at DNA breaks, indicating that Sae2 may negatively regulate checkpoint signalling by modulating MRX association at damaged DNA. Consistently, high levels of Sae2 prevent checkpoint activation and impair MRX foci formation in response to unrepaired DSBs. Mec1- and Tel1-dependent Sae2 phosphorylation is necessary for these Sae2 functions, suggesting that the two kinases, once activated, may regulate checkpoint switch off through Sae2-mediated inhibition of MRX signalling.
- Published
- 2006
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