scholarly journals Bub3–BubR1-dependent sequestration of Cdc20Fizzy at DNA breaks facilitates the correct segregation of broken chromosomes

2015 ◽  
Vol 211 (3) ◽  
pp. 517-532 ◽  
Author(s):  
Nicolas Derive ◽  
Cedric Landmann ◽  
Emilie Montembault ◽  
Marie-Charlotte Claverie ◽  
Priscillia Pierre-Elies ◽  
...  
Keyword(s):  
Double Strand Breaks ◽  
Dependent Manner ◽  
Anaphase Promoting Complex ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
Strand Breaks ◽  
Daughter Cells ◽  
Local Inhibition ◽  
Chromosome Fragments ◽  
And Function

The presence of DNA double-strand breaks during mitosis is particularly challenging for the cell, as it produces broken chromosomes lacking a centromere. This situation can cause genomic instability resulting from improper segregation of the broken fragments into daughter cells. We recently uncovered a process by which broken chromosomes are faithfully transmitted via the BubR1-dependent tethering of the two broken chromosome ends. However, the mechanisms underlying BubR1 recruitment and function on broken chromosomes were largely unknown. We show that BubR1 requires interaction with Bub3 to localize on the broken chromosome fragments and to mediate their proper segregation. We also find that Cdc20, a cofactor of the E3 ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C), accumulates on DNA breaks in a BubR1 KEN box–dependent manner. A biosensor for APC/C activity demonstrates a BubR1-dependent local inhibition of APC/C around the segregating broken chromosome. We therefore propose that the Bub3–BubR1 complex on broken DNA inhibits the APC/C locally via the sequestration of Cdc20, thus promoting proper transmission of broken chromosomes.

scholarly journals Stepwise 5′ DNA end-specific resection of DNA breaks by the Mre11-Rad50-Xrs2 and Sae2 nuclease ensemble

2019 ◽  
Vol 116 (12) ◽  
pp. 5505-5513 ◽  
Author(s):  
Elda Cannavo ◽  
Giordano Reginato ◽  
Petr Cejka
Keyword(s):  
Atp Hydrolysis ◽  
Double Strand Breaks ◽  
Dependent Manner ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
Strand Breaks ◽  
Dna Strands ◽  
Dna Strand ◽  
The Individual

To repair DNA double-strand breaks by homologous recombination, the 5′-terminated DNA strands must first be resected to produce 3′ overhangs. Mre11 fromSaccharomyces cerevisiaeis a 3′ → 5′ exonuclease that is responsible for 5′ end degradation in vivo. Using plasmid-length DNA substrates and purified recombinant proteins, we show that the combined exonuclease and endonuclease activities of recombinant MRX-Sae2 preferentially degrade the 5′-terminated DNA strand, which extends beyond the vicinity of the DNA end. Mechanistically, Rad50 restricts the Mre11 exonuclease in an ATP binding-dependent manner, preventing 3′ end degradation. Phosphorylated Sae2, along with stimulating the MRX endonuclease as shown previously, also overcomes this inhibition to promote the 3′ → 5′ exonuclease of MRX, which requires ATP hydrolysis by Rad50. Our results support a model in which MRX-Sae2 catalyzes 5′-DNA end degradation by stepwise endonucleolytic DNA incisions, followed by exonucleolytic 3′ → 5′ degradation of the individual DNA fragments. This model explains how both exonuclease and endonuclease activities of Mre11 functionally integrate within the MRX-Sae2 ensemble to resect 5′-terminated DNA.

scholarly journals Autophosphorylation of DNA-PKCS regulates its dynamics at DNA double-strand breaks

2007 ◽  
Vol 177 (2) ◽  
pp. 219-229 ◽  
Author(s):  
Naoya Uematsu ◽  
Eric Weterings ◽  
Ken-ichi Yano ◽  
Keiko Morotomi-Yano ◽  
Burkhard Jakob ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Laser System ◽  
Double Strand Breaks ◽  
Dependent Manner ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Dna Ends

The DNA-dependent protein kinase catalytic subunit (DNA-PKCS) plays an important role during the repair of DNA double-strand breaks (DSBs). It is recruited to DNA ends in the early stages of the nonhomologous end-joining (NHEJ) process, which mediates DSB repair. To study DNA-PKCS recruitment in vivo, we used a laser system to introduce DSBs in a specified region of the cell nucleus. We show that DNA-PKCS accumulates at DSB sites in a Ku80-dependent manner, and that neither the kinase activity nor the phosphorylation status of DNA-PKCS influences its initial accumulation. However, impairment of both of these functions results in deficient DSB repair and the maintained presence of DNA-PKCS at unrepaired DSBs. The use of photobleaching techniques allowed us to determine that the kinase activity and phosphorylation status of DNA-PKCS influence the stability of its binding to DNA ends. We suggest a model in which DNA-PKCS phosphorylation/autophosphorylation facilitates NHEJ by destabilizing the interaction of DNA-PKCS with the DNA ends.

scholarly journals Genetic Separation of Sae2 Nuclease Activity from Mre11 Nuclease Functions in Budding Yeast

2017 ◽  
Vol 37 (24) ◽  
Author(s):  
Sucheta Arora ◽  
Rajashree A. Deshpande ◽  
Martin Budd ◽  
Judy Campbell ◽  
America Revere ◽  
...  
Keyword(s):  
Nuclease Activity ◽  
Double Strand Breaks ◽  
Endonuclease Activity ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
Content Type ◽  
Strand Breaks ◽  
Dna Ends

ABSTRACT Sae2 promotes the repair of DNA double-strand breaks in Saccharomyces cerevisiae. The role of Sae2 is linked to the Mre11/Rad50/Xrs2 (MRX) complex, which is important for the processing of DNA ends into single-stranded substrates for homologous recombination. Sae2 has intrinsic endonuclease activity, but the role of this activity has not been assessed independently from its functions in promoting Mre11 nuclease activity. Here we identify and characterize separation-of-function mutants that lack intrinsic nuclease activity or the ability to promote Mre11 endonucleolytic activity. We find that the ability of Sae2 to promote MRX nuclease functions is important for DNA damage survival, particularly in the absence of Dna2 nuclease activity. In contrast, Sae2 nuclease activity is essential for DNA repair when the Mre11 nuclease is compromised. Resection of DNA breaks is impaired when either Sae2 activity is blocked, suggesting roles for both Mre11 and Sae2 nuclease activities in promoting the processing of DNA ends in vivo. Finally, both activities of Sae2 are important for sporulation, indicating that the processing of meiotic breaks requires both Mre11 and Sae2 nuclease activities.

scholarly journals Poly(ADP-ribose) glycohydrolase promotes formation and homology-directed repair of meiotic DNA double-strand breaks independent of its catalytic activity

2020 ◽  
Author(s):  
Eva Janisiw ◽  
Marilina Raices ◽  
Fabiola Balmir ◽  
Luis Paulin Paz ◽  
Antoine Baudrimont ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Synaptonemal Complex ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Post Translational Modification ◽  
Dna Breaks ◽  
Strand Breaks ◽  
Homology Directed Repair ◽  
Damage Repair ◽  
Somatic Tissues

SummaryPoly(ADP-ribosyl)ation is a reversible post-translational modification synthetized by ADP-ribose transferases and removed by poly(ADP-ribose) glycohydrolase (PARG), which plays important roles in DNA damage repair. While well-studied in somatic tissues, much less is known about poly(ADP-ribosyl)ation in the germline, where DNA double-strand breaks are introduced by a regulated program and repaired by crossover recombination to establish a tether between homologous chromosomes. The interaction between the parental chromosomes is facilitated by meiotic specific adaptation of the chromosome axes and cohesins, and reinforced by the synaptonemal complex. Here, we uncover an unexpected role for PARG in promoting the induction of meiotic DNA breaks and their homologous recombination-mediated repair in Caenorhabditis elegans. PARG-1/PARG interacts with both axial and central elements of the synaptonemal complex, REC-8/Rec8 and the MRN/X complex. PARG-1 shapes the recombination landscape and reinforces the tightly regulated control of crossover numbers without requiring its catalytic activity. We unravel roles in regulating meiosis, beyond its enzymatic activity in poly(ADP-ribose) catabolism.

scholarly journals Wortmannin, a specific inhibitor of phosphatidylinositol-3-kinase, induces accumulation of DNA double-strand breaks

2020 ◽  
Vol 61 (2) ◽  
pp. 171-176 ◽  
Author(s):  
Makoto Ihara ◽  
Kazuko Shichijo ◽  
Satoshi Takeshita ◽  
Takashi Kudo
Keyword(s):  
Dna Damage ◽  
Specific Inhibitor ◽  
Double Strand Breaks ◽  
Dependent Manner ◽  
Dna Double Strand Breaks ◽  
Phosphatidylinositol 3 Kinase ◽  
Strand Breaks ◽  
Γ Ray ◽  
Scid Cells ◽  
Phosphatidylinositol 3

Abstract Wortmannin, a fungal metabolite, is a specific inhibitor of the phosphatidylinositol 3-kinase (PI3K) family, which includes double-stranded DNA dependent protein kinase (DNA-PK) and ataxia telangiectasia mutated kinase (ATM). We investigated the effects of wortmannin on DNA damage in DNA-PK-deficient cells obtained from severe combined immunodeficient mice (SCID cells). Survival of wortmannin-treated cells decreased in a concentration-dependent manner. After treatment with 50 μM wortmannin, survival decreased to 60% of that of untreated cells. We observed that treatment with 20 and 50 μM wortmannin induced DNA damage equivalent to that by 0.37 and 0.69 Gy, respectively, of γ-ray radiation. The accumulation of DNA double-strand breaks (DSBs) in wortmannin-treated SCID cells was assessed using pulsed-field gel electrophoresis. The maximal accumulation was observed 4 h after treatment. Moreover, the presence of DSBs was confirmed by the ability of nuclear extracts from γ-ray-irradiated SCID cells to produce in vitro phosphorylation of histone H2AX. These results suggest that wortmannin induces cellular toxicity by accumulation of spontaneous DSBs through inhibition of ATM.

scholarly journals Dynamics of RecA-mediated repair of replication-dependent DNA breaks

2018 ◽  
Vol 217 (7) ◽  
pp. 2299-2307 ◽  
Author(s):  
Vincent Amarh ◽  
Martin A. White ◽  
David R.F. Leach
Keyword(s):  
Temporal Dynamics ◽  
Chromosomal Rearrangements ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
Chromosomal Locus ◽  
Chromosomal Replication ◽  
Strand Breaks ◽  
Sister Chromosome ◽  
Temporal Efficiency

Chromosomal replication is the major source of spontaneous DNA double-strand breaks (DSBs) in living cells. Repair of these DSBs is essential for cell viability, and accuracy of repair is critical to avoid chromosomal rearrangements. Repair of replication-dependent DSBs occurs primarily by homologous recombination with a sister chromosome. However, this reaction has never been visualized at a defined chromosomal locus, so little is known about its spatial or temporal dynamics. Repair of a replication-independent DSB generated in Escherichia coli by a rare-cutting endonuclease leads to the formation of a bundle of RecA filaments. In this study, we show that in contrast, repair of a replication-dependent DSB involves a transient RecA focus localized in the central region of the cell in which the DNA is replicated. The recombining loci remain centrally located with restricted movement before segregating with little extension to the period of postreplicative sister-chromosome cohesion. The spatial and temporal efficiency of this reaction is remarkable.

scholarly journals Loss of SORCS2 is associated with neuronal DNA double-strand breaks

2021 ◽  
Author(s):  
Katerina O. Gospodinova ◽  
Ditte Olsen ◽  
Mathias Kaas ◽  
Susan M. Anderson ◽  
Jonathan Phillips ◽  
...  
Keyword(s):  
Dna Damage ◽  
Neuronal Activity ◽  
Neuronal Cell ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Neuronal Viability ◽  
Functional Studies ◽  
Neuronal Stimulation ◽  
And Function

AbstractSORCS2 is one of five proteins that constitute the Vps10p-domain receptor family. Members of this family play important roles in cellular processes linked to neuronal survival, differentiation and function. Genetic and functional studies implicate SORCS2 in cognitive function, as well as in neurodegenerative and psychiatric disorders. DNA damage and DNA repair deficits are linked to ageing and neurodegeneration, and transient neuronal DNA double-strand breaks (DSBs) also occur as a result of neuronal activity. Here, we report a novel role for SORCS2 in DSB formation. We show that SorCS2 loss is associated with elevated DSB levels in the mouse dentate gyrus and that knocking out SORCS2 in a human neuronal cell line increased Topoisomerase IIβ-dependent DSB formation and reduced neuronal viability. Neuronal stimulation had no impact on levels of DNA damage, suggesting that the observed differences are unlikely to be the result of aberrant neuronal activity. Our findings are consistent with studies linking the VPS10 receptors and DNA damage to neurodegenerative conditions.

Cytosolic Histone H1 Releasing under Different Apoptotic Stimuli in Chronic Lymphocytic Leukemia (CLL).

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4790-4790
Author(s):  
Eva Gine ◽  
Marta Crespo ◽  
Emili Montserrat ◽  
Francesc Bosch
Keyword(s):  
Functional Status ◽  
Peripheral Blood ◽  
Histone H1 ◽  
Double Strand Breaks ◽  
Fish Analysis ◽  
Dependent Manner ◽  
Wild Type ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Cells In Culture

Abstract Recently, it has been demonstrated that nuclear histone H1 could be released into cytoplasm when apoptosis is induced by DNA double-strand breaks, this process being dependent of p53 functional status. In addition, cytosolic histone H1.2 was shown to induce cytochrome C release in a Bak-dependent manner. The aims of this study were: 1) to analyze the presence of histone H1.2 in the cytosol of peripheral blood purified CLL cells during apoptosis induced by different chemotherapeutic drugs or ionizing radiation in CLL cells in culture; 2) to correlate the presence of cytosolic histone H1 with the p53 functional status. FISH analysis was used to select samples with (n=2) or without (n=4) p53 deletion. Peripheral blood purified CLL cells were cultured for 24 hours with no treatment, after irradiation (25 Gy), in the presence of fludarabine and mitoxantrone (FM) (1mg/ml and 0.5 mg/ml respectively), or with etoposide (5.8 mg/ml). Cell viability and analysis of apoptosis were performed by annexin V/PI staining and FACscan analysis. Cytosolic and nuclear fractions were separated with the Nuclear/Cytosol Fractionation kit (Biovision). The presence of histone H1 in the cytosolic fraction was assessed by Western Blott using the anti-histone H1 antibody (Upstate). Nuclear contamination of this fraction was detected by using anti-ribonucleoprotein (hnRNP) antibody (Abcam). In p53 wild-type CLL cases, increased apoptosis was observed under all stimuli, being the FM combination the most effective. In contrast, CLL cases with p53 deletion displayed a lower or no response to the different treatments. In p53 wild-type CLL cases, histone H1 was only observed in cytosolic fractions at 24 hours when irradiation or etoposide treatment was applied, but not with FM treatment. Interestingly, cytosolic histone H1 was also present in the control cells at 24h of culture, indicating that H1 was also involved in the spontaneous apoptosis observed due to the microambient deprivation of CLL cells in culture. This histone H1 traffic was also confirmed using immunofluorescence analysis. Conversely, in p53 deleted CLL cases histone H1 was not detected into cytosolic fractions with any treatment. In conclusion, cytosolic histone H1 appeared in CLL cases in a p53 dependent manner when DNA double-strand breaks were induced. These results suggest that histone H1 plays an important role in the p53-dependent cytosol apoptotic signaling.

scholarly journals Generation of Markerless Deletions in the Nosocomial PathogenClostridium difficileby Induction of DNA Double-Strand Breaks

2018 ◽  
Vol 85 (3) ◽  
Author(s):  
Elena-Stella Theophilou ◽  
Prerna Vohra ◽  
Maurice P. Gallagher ◽  
Ian R. Poxton ◽  
Garry W. Blakely
Keyword(s):  
Clostridium Difficile ◽  
Regulatory Elements ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
Gene Encoding ◽  
Strand Breaks ◽  
Allelic Exchange ◽  
Genes Encoding

ABSTRACTClostridium difficileis an important nosocomial pathogen associated with potentially fatal disease induced by the use of antibiotics. Genetic characterization of such clinically important bacteria is often hampered by lack of availability of suitable tools. Here, we describe the use of I-SceI to induce DNA double-strand breaks, which increase the frequency of allelic exchange and enable the generation of markerless deletions inC. difficile. The usefulness of the system is illustrated by the deletion of genes encoding putative AddAB homologues. The ΔaddABmutants are sensitive to ultraviolet light and the antibiotic metronidazole, indicating a role in homologous recombination and the repair of DNA breaks. Despite the impairment in recombination, the mutants are still proficient for induction of the SOS response. In addition, deletion of thefliCgene, and subsequent complementation, reveals the importance of potential regulatory elements required for expression of a downstream gene encoding the flagellin glycosyltransferase.IMPORTANCEMost sequenced bacterial genomes contain genes encoding proteins of unknown or hypothetical function. To identify a phenotype for mutations in such genes, deletion is the preferred method for mutagenesis because it reduces the likelihood of polar effects, although it does not eliminate the possibility. Allelic exchange to produce deletions is dependent on the length of homologous regions used to generate merodiploids. Shorter regions of homology resolve at lower frequencies. The work presented here demonstrates the utility of inducing DNA double-strand breaks to increase the frequency of merodiploid resolution inClostridium difficile. Using this approach, we reveal the roles of two genes, encoding homologues of AddAB, in survival following DNA damage. The method is readily applicable to the production of deletions inC. difficileand expands the toolbox available for genetic analysis of this important anaerobic pathogen.

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