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1.
Int J Mol Sci ; 25(6)2024 Mar 09.
Article in English | MEDLINE | ID: mdl-38542135

ABSTRACT

G-quadruplexes or G4s are non-canonical secondary structures of nucleic acids characterized by guanines arranged in stacked tetraplex arrays. Decades of research into these peculiar assemblies of DNA and RNA, fueled by the development and optimization of a vast array of techniques and assays, has resulted in a large amount of information regarding their structure, stability, localization, and biological significance in native systems. A plethora of articles have reported the roles of G-quadruplexes in multiple pathways across several species, ranging from gene expression regulation to RNA biogenesis and trafficking, DNA replication, and genome maintenance. Crucially, a large amount of experimental evidence has highlighted the roles of G-quadruplexes in cancer biology and other pathologies, pointing at these structurally unique guanine assemblies as amenable drug targets. Given the rapid expansion of this field of research, this review aims at summarizing all the relevant aspects of G-quadruplex biology by combining and discussing results from seminal works as well as more recent and cutting-edge experimental evidence. Additionally, the most common methodologies used to study G4s are presented to aid the reader in critically interpreting and integrating experimental data.


Subject(s)
G-Quadruplexes , DNA/genetics , DNA/chemistry , RNA/genetics , RNA/chemistry , Gene Expression Regulation , DNA Replication
2.
Cell Rep ; 42(2): 112060, 2023 02 28.
Article in English | MEDLINE | ID: mdl-36735534

ABSTRACT

The nucleolytic processing (resection) of a DNA double-strand break (DSB) is a critical step to repair the lesion by homologous recombination (HR). PARylation, which is the attachment of poly(ADP-ribose) (PAR) units to specific targets by PAR polymerases (PARPs), regulates many steps of HR, including resection. Here, we show that preventing PARylation of the oncosuppressor BRCA1 induces hyper-resection of DSBs through BRCA2 and the EXO1 nuclease. Upon expression of the unPARylatable variant of BRCA1, we observe a reduced 53BP1-RIF1 barrier for resection accompanied by an increase in the recruitment of the RAD51 recombinase. Similar results are observed when cells are treated with the clinically approved PARP inhibitor olaparib. We propose that PARylation of BRCA1 is important to limit the formation of excessively extended DNA filaments, thereby reducing illegitimate chromosome rearrangements. Our results shed light on molecular aspects of HR and on the mechanisms of PARP inhibitor treatment.


Subject(s)
Poly ADP Ribosylation , Poly(ADP-ribose) Polymerase Inhibitors , BRCA1 Protein/metabolism , DNA/metabolism , DNA Breaks, Double-Stranded , DNA Repair , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Tumor Suppressor p53-Binding Protein 1/metabolism , Humans , Cell Line
3.
Int J Mol Sci ; 21(18)2020 Sep 12.
Article in English | MEDLINE | ID: mdl-32932697

ABSTRACT

Alterations in DNA repair pathways are one of the main drivers of cancer insurgence. Nevertheless, cancer cells are more susceptible to DNA damage than normal cells and they rely on specific functional repair pathways to survive. Thanks to advances in genome sequencing, we now have a better idea of which genes are mutated in specific cancers and this prompted the development of inhibitors targeting DNA repair players involved in pathways essential for cancer cells survival. Currently, the pivotal concept is that combining the inhibition of mechanisms on which cancer cells viability depends is the most promising way to treat tumorigenesis. Numerous inhibitors have been developed and for many of them, efficacy has been demonstrated either alone or in combination with chemo or radiotherapy. In this review, we will analyze the principal pathways involved in cell cycle checkpoint and DNA repair focusing on how their alterations could predispose to cancer, then we will explore the inhibitors developed or in development specifically targeting different proteins involved in each pathway, underscoring the rationale behind their usage and how their combination and/or exploitation as adjuvants to classic therapies could help in patients clinical outcome.


Subject(s)
Antineoplastic Agents/therapeutic use , DNA Repair/drug effects , DNA Repair/genetics , Neoplasms/drug therapy , Neoplasms/genetics , Animals , Carcinogenesis/drug effects , Cell Cycle Checkpoints/drug effects , Cell Cycle Checkpoints/genetics , Cell Survival/drug effects , Cell Survival/genetics , DNA Damage/drug effects , DNA Damage/genetics , Humans , Signal Transduction/drug effects , Signal Transduction/genetics
4.
Nat Commun ; 11(1): 3181, 2020 06 23.
Article in English | MEDLINE | ID: mdl-32576832

ABSTRACT

The DNA damage checkpoint (DDC) is often robustly activated during the homologous recombination (HR) repair of DNA double strand breaks (DSBs). DDC activation controls several HR repair factors by phosphorylation, preventing premature segregation of entangled chromosomes formed during HR repair. The DDC mediator 53BP1/Rad9 limits the nucleolytic processing (resection) of a DSB, controlling the formation of the 3' single-stranded DNA (ssDNA) filament needed for recombination, from yeast to human. Here we show that Rad9 promotes stable annealing between the recombinogenic filament and the donor template in yeast, limiting strand rejection by the Sgs1 and Mph1 helicases. This regulation allows repair by long tract gene conversion, crossover recombination and break-induced replication (BIR), only after DDC activation. These findings shed light on how cells couple DDC with the choice and effectiveness of HR sub-pathways, with implications for genome instability and cancer.


Subject(s)
Cell Cycle Proteins/metabolism , DNA Repair/genetics , DNA Repair/physiology , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Tumor Suppressor p53-Binding Protein 1/metabolism , Cell Cycle Proteins/genetics , Cell Survival , DEAD-box RNA Helicases/metabolism , DNA Breaks, Double-Stranded , DNA, Single-Stranded , DNA-Binding Proteins/metabolism , Gene Conversion , Genomic Instability , Homologous Recombination , Humans , Rad52 DNA Repair and Recombination Protein/metabolism , RecQ Helicases/metabolism , Recombinational DNA Repair , Saccharomyces cerevisiae Proteins/genetics , Tumor Suppressor p53-Binding Protein 1/genetics
5.
Mutat Res Rev Mutat Res ; 784: 108300, 2020.
Article in English | MEDLINE | ID: mdl-32430097

ABSTRACT

RNA:DNA hybrids form when nascent transcripts anneal to the DNA template strand or any homologous DNA region. Co-transcriptional RNA:DNA hybrids, organized in R-loop structures together with the displaced non-transcribed strand, assist gene expression, DNA repair and other physiological cellular functions. A dark side of the matter is that RNA:DNA hybrids are also a cause of DNA damage and human diseases. In this review, we summarize recent advances in the understanding of the mechanisms by which the impairment of hybrid turnover promotes DNA damage and genome instability via the interference with DNA replication and DNA double-strand break repair. We also discuss how hybrids could contribute to cancer, neurodegeneration and susceptibility to viral infections, focusing on dysfunctions associated with the anti-R-loop helicase Senataxin.


Subject(s)
DNA Damage , DNA Repair , DNA/chemistry , Genomic Instability , RNA/chemistry , Transcription, Genetic , Animals , DNA/genetics , Humans , RNA/genetics
6.
Cell Rep ; 31(5): 107603, 2020 05 05.
Article in English | MEDLINE | ID: mdl-32375052

ABSTRACT

An important but still enigmatic function of DNA:RNA hybrids is their role in DNA double-strand break (DSB) repair. Here, we show that Sen1, the budding yeast ortholog of the human helicase Senataxin, is recruited at an HO endonuclease-induced DSB and limits the local accumulation of DNA:RNA hybrids. In the absence of Sen1, hybrid accumulation proximal to the DSB promotes increased binding of the Ku70-80 (KU) complex at the break site, mutagenic non-homologous end joining (NHEJ), micro-homology-mediated end joining (MMEJ), and chromosome translocations. We also show that homology-directed recombination (HDR) by gene conversion is mostly proficient in sen1 mutants after single DSB. However, in the absence of Sen1, DNA:RNA hybrids, Mre11, and Dna2 initiate resection through a non-canonical mechanism. We propose that this resection mechanism through local DNA:RNA hybrids acts as a backup to prime HDR when canonical pathways are altered, but at the expense of genome integrity.


Subject(s)
DNA Breaks, Double-Stranded , DNA End-Joining Repair/physiology , DNA Repair/physiology , DNA/metabolism , Exodeoxyribonucleases/metabolism , DNA-Binding Proteins/metabolism , Homologous Recombination/physiology , Humans , Nuclear Proteins/metabolism
7.
Int J Mol Sci ; 21(5)2020 Mar 02.
Article in English | MEDLINE | ID: mdl-32131532

ABSTRACT

In the last decade, it has become evident that RNA is frequently found in DNA. It is now well established that single embedded ribonucleoside monophosphates (rNMPs) are primarily introduced by DNA polymerases and that longer stretches of RNA can anneal to DNA, generating RNA:DNA hybrids. Among them, the most studied are R-loops, peculiar three-stranded nucleic acid structures formed upon the re-hybridization of a transcript to its template DNA. In addition, polyribonucleotide chains are synthesized to allow DNA replication priming, double-strand breaks repair, and may as well result from the direct incorporation of consecutive rNMPs by DNA polymerases. The bright side of RNA into DNA is that it contributes to regulating different physiological functions. The dark side, however, is that persistent RNA compromises genome integrity and genome stability. For these reasons, the characterization of all these structures has been under growing investigation. In this review, we discussed the origin of single and multiple ribonucleotides in the genome and in the DNA of organelles, focusing on situations where the aberrant processing of RNA:DNA hybrids may result in multiple rNMPs embedded in DNA. We concluded by providing an overview of the currently available strategies to study the presence of single and multiple ribonucleotides in DNA in vivo.


Subject(s)
DNA/chemistry , Genomic Instability , Nucleic Acid Heteroduplexes/chemistry , Ribonucleotides/chemistry , Animals , DNA/genetics , DNA Replication , Humans , Nucleic Acid Heteroduplexes/genetics , R-Loop Structures , Ribonucleotides/genetics
8.
Front Mol Biosci ; 6: 55, 2019.
Article in English | MEDLINE | ID: mdl-31380392

ABSTRACT

In all the eukaryotic cells, nucleolytic processing (resection) of a double strand DNA break (DSB) is a key step to channel the repair of the lesion toward the homologous recombination, at the expenses of the non-homologous end joining (NHEJ). The coordinated action of several nucleases and helicases generates 3' single strand (ss) DNA, which is covered by RPA and recombination factors. Molecular details of the process have been first dissected in the model organism Saccharomyces cerevisiae. When DSB ends are occupied by KU, a central component of the NHEJ, the Mre11-Rad50-Xrs2 (MRX) nuclease complex (MRN in human), aided by the associated factors Sae2 (CTIP in human), initiates the resection process, inducing a nick close to the DSB ends. Then, starting from the nick, the nucleases Mre11, Exo1, Dna2, in cooperation with Sgs1 helicase (BLM in human), degrade DNA strand in both the directions, creating the 3' ssDNA filament. Multiple levels of regulation of the break processing ensure faithful DSB repair, preventing chromosome rearrangements, and genome instability. Here we review the DSB resection process and its regulation in the context of chromatin. Particularly, we focus on proteins that limit DSB resection, acting as physical barriers toward nucleases and helicases. Moreover, we also take into consideration recent evidence regarding functional interplay between DSB repair and RNA molecules nearby the break site.

9.
DNA Repair (Amst) ; 68: 68-74, 2018 08.
Article in English | MEDLINE | ID: mdl-30017059

ABSTRACT

Cas9 endonuclease from S. pyogenes is widely used to induce controlled double strand breaks (DSB) at desired genomic loci for gene editing. Here, we describe a droplet digital PCR (ddPCR) method to precisely quantify the kinetic of formation and 5'-end nucleolytic processing of Cas9-induced DSB in different human cells lines. Notably, DSB processing is a finely regulated process, which dictates the choice between non-homologous end joining (NHEJ) and homology directed repair (HDR). This step of DSB repair is also a relevant point to be taken into consideration to improve Cas9-mediated technology. Indeed, by this protocol, we show that processing of Cas9-induced DSB is impaired by CTIP or BRCA1 depletion, while it is accelerated after down-regulation of DNA-PKcs and 53BP1, two DSB repair key factors. In conclusion, the method we describe here can be used to study DSB repair mechanisms, with direct utility for molecularly optimising the knock-out/in outcomes in genome manipulation.


Subject(s)
CRISPR-Associated Proteins/metabolism , DNA Breaks, Double-Stranded , DNA End-Joining Repair , DNA/metabolism , Polymerase Chain Reaction/methods , Recombinational DNA Repair , CRISPR-Associated Proteins/pharmacology , CRISPR-Associated Proteins/toxicity , Cell Line , DNA/drug effects , Gene Editing , Humans , Kinetics
10.
Methods Mol Biol ; 1672: 119-129, 2018.
Article in English | MEDLINE | ID: mdl-29043621

ABSTRACT

The nucleolytic degradation of the 5'-ending strand of a Double-Strand DNA break (DSB) is necessary to initiate homologous recombination to correctly repair the break. This process is called DNA end resection and it is finely regulated to prevent genome rearrangements. Here, we describe a protocol to quantify DSB resection rate by qPCR, which could be applied to every organisms whenever the break site and its flanking region sequences are known.


Subject(s)
DNA Breaks, Double-Stranded , Real-Time Polymerase Chain Reaction , Computational Biology/methods , DNA, Fungal , Real-Time Polymerase Chain Reaction/methods , Yeasts/genetics
11.
J Cell Biol ; 216(3): 623-639, 2017 03 06.
Article in English | MEDLINE | ID: mdl-28228534

ABSTRACT

Genome maintenance and cancer suppression require homologous recombination (HR) DNA repair. In yeast and mammals, the scaffold protein TOPBP1Dpb11 has been implicated in HR, although its precise function and mechanism of action remain elusive. In this study, we show that yeast Dpb11 plays an antagonistic role in recombination control through regulated protein interactions. Dpb11 mediates opposing roles in DNA end resection by coordinating both the stabilization and exclusion of Rad9 from DNA lesions. The Mec1 kinase promotes the pro-resection function of Dpb11 by mediating its interaction with the Slx4 scaffold. Human TOPBP1Dpb11 engages in interactions with the anti-resection factor 53BP1 and the pro-resection factor BRCA1, suggesting that TOPBP1 also mediates opposing functions in HR control. Hyperstabilization of the 53BP1-TOPBP1 interaction enhances the recruitment of 53BP1 to nuclear foci in the S phase, resulting in impaired HR and the accumulation of chromosomal aberrations. Our results support a model in which TOPBP1Dpb11 plays a conserved role in mediating a phosphoregulated circuitry for the control of recombinational DNA repair.


Subject(s)
Cell Cycle Proteins/genetics , DNA-Binding Proteins/genetics , Homologous Recombination/genetics , Nuclear Proteins/genetics , Recombinational DNA Repair/genetics , Tumor Suppressor p53-Binding Protein 1/genetics , DNA Damage/genetics , Fungal Proteins/genetics , HEK293 Cells , Humans , S Phase/genetics , Yeasts
12.
Chromosoma ; 126(1): 45-58, 2017 02.
Article in English | MEDLINE | ID: mdl-27165041

ABSTRACT

Homologous recombination-mediated DNA repair is essential for maintaining genome integrity. It is a multi-step process that involves resection of DNA ends, strand invasion, DNA synthesis and/or DNA end ligation, and finally, the processing of recombination intermediates such as Holliday junctions or other joint molecules. Over the last 15 years, it has been established that the Slx4 protein plays key roles in the processing of recombination intermediates, functioning as a scaffold to coordinate the action of structure-specific endonucleases. Recent work in budding yeast has uncovered unexpected roles for Slx4 in the initial step of DNA-end resection and in the modulation of DNA damage checkpoint signaling. Here we review these latest findings and discuss the emerging role of yeast Slx4 as an important coordinator of DNA damage signaling responses and a regulator of multiple steps in homologous recombination-mediated repair.


Subject(s)
Cell Cycle Checkpoints , Endodeoxyribonucleases/metabolism , Homologous Recombination , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Carrier Proteins/chemistry , Carrier Proteins/metabolism , Cell Cycle Proteins/metabolism , DNA Damage , DNA Repair , Endodeoxyribonucleases/genetics , Phosphorylation , Protein Binding , Protein Interaction Domains and Motifs , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Signal Transduction
13.
Cell Cycle ; 15(21): 2906-2919, 2016 Nov.
Article in English | MEDLINE | ID: mdl-27565373

ABSTRACT

Polo-like kinases (PLKs) control several aspects of eukaryotic cell division and DNA damage response. Remarkably, PLKs are overexpressed in several types of cancer, being therefore a marker of bad prognosis. As such, specific PLK kinase activity inhibitors are already used in clinical trials and the regulation of PLK activation is a relevant topic of cancer research. Phosphorylation of threonine residues in the T-loop of the kinase domain is pivotal for PLKs activation. Here, we show that T238A substitution in the T-loop reduces the kinase activity of Cdc5, the only PLK in Saccharomyces cerevisiae, with minor effect on cell growth in unperturbed conditions. However, the cdc5-T238A cells have increased rate of chromosome loss and gross chromosomal rearrangements, indicating altered genome stability. Moreover, the T238A mutation affects timely localization of Cdc5 to the spindle pole bodies and blocks cell cycle restart after one irreparable double-strand break. In cells responding to alkylating agent metylmethane sulfonate (MMS), the cdc5-T238A mutation reduces the phosphorylation of Mus81-Mms4 resolvase and exacerbates the MMS sensitivity of sgs1Δ cells that accumulate Holliday junctions. Of importance, the previously described checkpoint adaptation defective allele, cdc5-ad does not show reduced kinase activity, defective Mms4 phosphorylation and genetic interaction with sgs1Δ. Our data define the importance of regulating Cdc5 activity through T-loop phosphorylation to preserve genome integrity and respond to DNA damage.


Subject(s)
Cell Cycle Proteins/metabolism , Chromosomal Instability , Chromosomes, Fungal/metabolism , DNA Damage , Protein Serine-Threonine Kinases/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Adenoviridae/metabolism , Amino Acid Sequence , Cell Cycle Checkpoints/genetics , Cell Cycle Proteins/chemistry , DNA Breaks, Double-Stranded , DNA Repair , Gene Rearrangement , Genomic Instability , Microbial Viability , Models, Biological , Mutant Proteins/metabolism , Phosphorylation , Protein Serine-Threonine Kinases/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Spindle Poles/metabolism , Telomere/metabolism , Threonine/metabolism
14.
Nucleic Acids Res ; 44(2): 669-82, 2016 Jan 29.
Article in English | MEDLINE | ID: mdl-26490958

ABSTRACT

The DNA damage checkpoint pathway is activated in response to DNA lesions and replication stress to preserve genome integrity. However, hyper-activation of this surveillance system is detrimental to the cell, because it might prevent cell cycle re-start after repair, which may also lead to senescence. Here we show that the scaffold proteins Slx4 and Rtt107 limit checkpoint signalling at a persistent double-strand DNA break (DSB) and at uncapped telomeres. We found that Slx4 is recruited within a few kilobases of an irreparable DSB, through the interaction with Rtt107 and the multi-BRCT domain scaffold Dpb11. In the absence of Slx4 or Rtt107, Rad9 binding near the irreparable DSB is increased, leading to robust checkpoint signalling and slower nucleolytic degradation of the 5' strand. Importantly, in slx4Δ sae2Δ double mutant cells these phenotypes are exacerbated, causing a severe Rad9-dependent defect in DSB repair. Our study sheds new light on the molecular mechanism that coordinates the processing and repair of DSBs with DNA damage checkpoint signalling, preserving genome integrity.


Subject(s)
DNA Breaks, Double-Stranded , Endodeoxyribonucleases/metabolism , Nuclear Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Camptothecin/pharmacology , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , DNA Repair Enzymes/genetics , DNA Repair Enzymes/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Endodeoxyribonucleases/genetics , Endonucleases/genetics , Endonucleases/metabolism , Methyl Methanesulfonate/pharmacology , Mutation , Nuclear Proteins/genetics , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Signal Transduction , Telomere/genetics , Telomere/metabolism , Topoisomerase I Inhibitors/pharmacology
15.
PLoS Genet ; 11(1): e1004928, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25569305

ABSTRACT

The Mre11-Rad50-Xrs2 nuclease complex, together with Sae2, initiates the 5'-to-3' resection of Double-Strand DNA Breaks (DSBs). Extended 3' single stranded DNA filaments can be exposed from a DSB through the redundant activities of the Exo1 nuclease and the Dna2 nuclease with the Sgs1 helicase. In the absence of Sae2, Mre11 binding to a DSB is prolonged, the two DNA ends cannot be kept tethered, and the DSB is not efficiently repaired. Here we show that deletion of the yeast 53BP1-ortholog RAD9 reduces Mre11 binding to a DSB, leading to Rad52 recruitment and efficient DSB end-tethering, through an Sgs1-dependent mechanism. As a consequence, deletion of RAD9 restores DSB repair either in absence of Sae2 or in presence of a nuclease defective MRX complex. We propose that, in cells lacking Sae2, Rad9/53BP1 contributes to keep Mre11 bound to a persistent DSB, protecting it from extensive DNA end resection, which may lead to potentially deleterious DNA deletions and genome rearrangements.


Subject(s)
Cell Cycle Proteins/genetics , DNA Breaks, Double-Stranded , DNA End-Joining Repair/genetics , Endodeoxyribonucleases/genetics , Exodeoxyribonucleases/genetics , Homologous Recombination/genetics , Saccharomyces cerevisiae Proteins/genetics , Cell Cycle Proteins/metabolism , DNA, Single-Stranded/genetics , DNA-Binding Proteins/genetics , Endodeoxyribonucleases/metabolism , Endonucleases/genetics , Endonucleases/metabolism , Exodeoxyribonucleases/metabolism , Mutation , Rad52 DNA Repair and Recombination Protein/genetics , Rad52 DNA Repair and Recombination Protein/metabolism , RecQ Helicases/genetics , RecQ Helicases/metabolism , Saccharomyces cerevisiae , Saccharomyces cerevisiae Proteins/metabolism
16.
Mol Cell Proteomics ; 13(2): 551-65, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24302356

ABSTRACT

The cell cycle checkpoint kinases play central roles in the genome maintenance of eukaryotes. Activation of the yeast checkpoint kinase Rad53 involves Rad9 or Mrc1 adaptor-mediated phospho-priming by Mec1 kinase, followed by auto-activating phosphorylation within its activation loop. However, the mechanisms by which these adaptors regulate priming phosphorylation of specific sites and how this then leads to Rad53 activation remain poorly understood. Here we used quantitative mass spectrometry to delineate the stepwise phosphorylation events in the activation of endogenous Rad53 in response to S phase alkylation DNA damage, and we show that the two Rad9 and Mrc1 adaptors, the four N-terminal Mec1-target TQ sites of Rad53 (Rad53-SCD1), and Rad53-FHA2 coordinate intimately for optimal priming phosphorylation to support substantial Rad53 auto-activation. Rad9 or Mrc1 alone can mediate surprisingly similar Mec1 target site phosphorylation patterns of Rad53, including previously undetected tri- and tetraphosphorylation of Rad53-SCD1. Reducing the number of TQ motifs turns the SCD1 into a proportionally poorer Mec1 target, which then requires the presence of both Mrc1 and Rad9 for sufficient priming and auto-activation. The phosphothreonine-interacting Rad53-FHA domains, particularly FHA2, regulate phospho-priming by interacting with the checkpoint mediators but do not seem to play a major role in the phospho-SCD1-dependent auto-activation step. Finally, mutation of all four SCD1 TQ motifs greatly reduces Rad53 activation but does not eliminate it, and residual Rad53 activity in this mutant is dependent on Rad9 but not Mrc1. Altogether, our results provide a paradigm for how phosphorylation site clusters and checkpoint mediators can be involved in the regulation of signaling relay in protein kinase cascades in vivo and elucidate an SCD1-independent Rad53 auto-activation mechanism through the Rad9 pathway. The work also demonstrates the power of mass spectrometry for in-depth analyses of molecular mechanisms in cellular signaling in vivo.


Subject(s)
Cell Cycle Proteins/metabolism , Checkpoint Kinase 2/metabolism , Mass Spectrometry/methods , Protein Kinases/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Alkylating Agents/pharmacology , Catalytic Domain , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/genetics , Checkpoint Kinase 2/chemistry , Checkpoint Kinase 2/genetics , DNA Damage/physiology , Enzyme Activation , Homeostasis , Organisms, Genetically Modified , Phosphorylation , Protein Interaction Mapping , Protein Processing, Post-Translational , S Phase/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics
17.
Nat Cell Biol ; 15(8): 883-5, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23907189

ABSTRACT

Common fragile sites (CFSs) are chromosomal regions that are prone to form breaks or gaps during mitosis, in particular following replication stress. The mechanisms modulating CFS expression and promoting safe chromatid transmission to daughter cells are not clear. Now CFS expression is shown to reflect the activity of the MUS81-EME1 resolvase complex which cooperates with the dissolving action of the BLM helicase to prevent uncontrolled chromosome breakage and to promote genome integrity.


Subject(s)
Chromatids/metabolism , Chromosome Fragile Sites/genetics , Chromosome Fragile Sites/physiology , DNA-Binding Proteins/metabolism , Endonucleases/metabolism , Gene Expression Regulation , Mitosis/physiology , Humans
18.
Mol Cell Biol ; 33(16): 3202-13, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23754745

ABSTRACT

The essential yeast kinases Mec1 and Rad53, or human ATR and Chk1, are crucial for checkpoint responses to exogenous genotoxic agents, but why they are also required for DNA replication in unperturbed cells remains poorly understood. Here we report that even in the absence of DNA-damaging agents, the rad53-4AQ mutant, lacking the N-terminal Mec1 phosphorylation site cluster, is synthetic lethal with a deletion of the RAD9 DNA damage checkpoint adaptor. This phenotype is caused by an inability of rad53-4AQ to activate the downstream kinase Dun1, which then leads to reduced basal deoxynucleoside triphosphate (dNTP) levels, spontaneous replication fork stalling, and constitutive activation of and dependence on S phase DNA damage checkpoints. Surprisingly, the kinase-deficient rad53-K227A mutant does not share these phenotypes but is rendered inviable by additional phosphosite mutations that prevent its binding to Dun1. The results demonstrate that ultralow Rad53 catalytic activity is sufficient for normal replication of undamaged chromosomes as long as it is targeted toward activation of the effector kinase Dun1. Our findings indicate that the essential S phase function of Rad53 is comprised by the combination of its role in regulating basal dNTP levels and its compensatory kinase function if dNTP levels are perturbed.


Subject(s)
Cell Cycle Proteins/metabolism , DNA Damage , Protein Serine-Threonine Kinases/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/genetics , Checkpoint Kinase 2 , DNA, Fungal/genetics , Enzyme Activation , Gene Deletion , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Mutation , Phosphorylation , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/genetics , Protein Structure, Tertiary , Proteolysis , S Phase , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics
19.
Cell Cycle ; 12(12): 1848-60, 2013 Jun 15.
Article in English | MEDLINE | ID: mdl-23708517

ABSTRACT

DNA double-strand breaks (DSBs) are the most cytotoxic form of DNA damage, since they can lead to genome instability and chromosome rearrangements, which are hallmarks of cancer cells. To face this kind of lesion, eukaryotic cells developed two alternative repair pathways, homologous recombination (HR) and non-homologous end joining (NHEJ). Repair pathway choice is influenced by the cell cycle phase and depends upon the 5'-3' nucleolytic processing of the break ends, since the generation of ssDNA tails strongly stimulates HR and inhibits NHEJ. A large amount of work has elucidated the key components of the DSBs repair machinery and how this crucial process is finely regulated. The emerging view suggests that besides endo/exonucleases and helicases activities required for end resection, molecular barrier factors are specifically loaded in the proximity of the break, where they physically or functionally limit DNA degradation, preventing excessive accumulation of ssDNA, which could be threatening for cell survival.


Subject(s)
DNA Breaks, Double-Stranded , DNA Repair/physiology , Animals , Chromatin/metabolism , DNA Repair/genetics , Genomic Instability/genetics , Homologous Recombination/genetics , Homologous Recombination/physiology , Humans
20.
DNA Repair (Amst) ; 12(5): 347-55, 2013 May 01.
Article in English | MEDLINE | ID: mdl-23473644

ABSTRACT

Saccharomyces cerevisiae cells with a single double-strand break (DSB) activate the ATR/Mec1-dependent checkpoint response as a consequence of extensive ssDNA accumulation. The recombination factor Tid1/Rdh54, a member of the Swi2-like family proteins, has an ATPase activity and may contribute to the remodelling of nucleosomes on DNA. Tid1 dislocates Rad51 recombinase from dsDNA, can unwind and supercoil DNA filaments, and has been implicated in checkpoint adaptation from a G2/M arrest induced by an unrepaired DSB. Here we show that both ATR/Mec1 and Chk2/Rad53 kinases are implicated in the phosphorylation of Tid1 in the presence of DNA damage, indicating that the protein is regulated during the DNA damage response. We show that Tid1 ATPase activity is dispensable for its phosphorylation and for its recruitment near a DSB, but it is required to switch off Rad53 activation and for checkpoint adaptation. Mec1 and Rad53 kinases, together with Rad51 recombinase, are also implicated in the hyper-phosphorylation of the ATPase defective Tid1-K318R variant and in the efficient binding of the protein to the DSB site. In summary, Tid1 is a novel target of the DNA damage checkpoint pathway that is also involved in checkpoint adaptation.


Subject(s)
Cell Cycle Proteins/metabolism , DNA Breaks, Double-Stranded , DNA Helicases/metabolism , DNA Topoisomerases/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Adaptation, Physiological/genetics , Cell Cycle Checkpoints/genetics , Cell Cycle Proteins/genetics , Checkpoint Kinase 2 , DNA Helicases/genetics , DNA Topoisomerases/genetics , Intracellular Signaling Peptides and Proteins/genetics , Mutation, Missense , Phosphorylation , Protein Serine-Threonine Kinases/genetics , Recombinational DNA Repair , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics
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