CSN5/Jab1 mutations affect axis formation in theDrosophila oocyte by activating a meiotic checkpoint

Development ◽  
2002 ◽  
Vol 129 (21) ◽  
pp. 5053-5064 ◽  
Author(s):  
Sergey Doronkin ◽  
Inna Djagaeva ◽  
Steven K. Beckendorf
Keyword(s):  
Signaling Pathways ◽  
Translational Control ◽  
Mammalian Cells ◽  
Strand Break ◽  
Cop9 Signalosome ◽  
Axis Formation ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Egfr Ligand ◽  
Dependent Protein

The COP9 signalosome (CSN) is linked to signaling pathways and ubiquitin-dependent protein degradation in yeast, plant and mammalian cells,but its roles in Drosophila development are just beginning to be understood. We show that during oogenesis CSN5/JAB1, one subunit of the CSN,is required for meiotic progression and for establishment of both the AP and DV axes of the Drosophila oocyte. The EGFR ligand Gurken is essential for both axes, and our results show that CSN5 mutations block the accumulation of Gurken protein in the oocyte. CSN5 mutations also cause the modification of Vasa, which is known to be required for Gurken translation. This CSN5 phenotype — defective axis formation, reduced Gurken accumulation and modification of Vasa — is very similar to the phenotype of the spindle-class genes that are required for the repair of meiotic recombination-induced, DNA double-strand breaks. When these breaks are not repaired, a DNA damage checkpoint mediated by mei-41 is activated. Accordingly, the CSN5 phenotype is suppressed by mutations inmei-41 or by mutations in mei-W68, which is required for double strand break formation. These results suggest that, like thespindle-class genes, CSN5 regulates axis formation by checkpoint-dependent, translational control of Gurken. They also reveal a link between DNA repair, axis formation and the COP9 signalosome, a protein complex that acts in multiple signaling pathways by regulating protein stability.

Double-strand-break-induced homologous recombination in mammalian cells

2001 ◽  
Vol 29 (2) ◽  
pp. 196-201 ◽  
Author(s):  
R. D. Johnson ◽  
M. Jasin
Keyword(s):  
Homologous Recombination ◽  
Sister Chromatid ◽  
Mammalian Cells ◽  
Indirect Evidence ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Crossing Over ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Strand Breaks

In mammalian cells, the repair of DNA double-strand breaks (DSBs) occurs by both homologous and non-homologous mechanisms. Indirect evidence, including that from gene targeting and random integration experiments, had suggested that non-homologous mechanisms were significantly more frequent than homologous ones. However, more recent experiments indicate that homologous recombination is also a prominent DSB repair pathway. These experiments show that mammalian cells use homologous sequences located at multiple positions throughout the genome to repair a DSB. However, template preference appears to be biased, with the sister chromatid being preferred by 2–3 orders of magnitude over a homologous or heterologous chromosome. The outcome of homologous recombination in mammalian cells is predominantly gene conversion that is not associated with crossing-over. The preference for the sister chromatid and the bias against crossing-over seen in mitotic mammalian cells may have developed in order to reduce the potential for genome alterations that could occur when other homologous repair templates are utilized. In attempts to understand further the mechanism of homologous recombination, the proteins that promote this process are beginning to be identified. To date, four mammalian proteins have been demonstrated conclusively to be involved in DSB repair by homologous recombination: Rad54, XRCC2, XRCC3 and BRCAI. This paper summarizes results from a number of recent studies.

Regulation of the cellular DNA double-strand break response

2007 ◽  
Vol 85 (6) ◽  
pp. 663-674 ◽  
Author(s):  
Kendra L. Cann ◽  
Geoffrey G. Hicks
Keyword(s):  
Mammalian Cells ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Breaks ◽  
Cell Cycle Checkpoints ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Dna Double Strand Break ◽  
Cellular Dna

DNA double-strand breaks occur frequently in cycling cells, and are also induced by exogenous sources, including ionizing radiation. Cells have developed integrated double-strand break response pathways to cope with these lesions, including pathways that initiate DNA repair (either via homologous recombination or nonhomologous end joining), the cell-cycle checkpoints (G1–S, intra-S phase, and G2–M) that provide time for repair, and apoptosis. However, before any of these pathways can be activated, the damage must first be recognized. In this review, we will discuss how the response of mammalian cells to DNA double-strand breaks is regulated, beginning with the activation of ATM, the pinnacle kinase of the double-strand break signalling cascade.

scholarly journals DNA Substrate Dependence of p53-Mediated Regulation of Double-Strand Break Repair

2002 ◽  
Vol 22 (17) ◽  
pp. 6306-6317 ◽  
Author(s):  
Nuray Akyüz ◽  
Gisa S. Boehden ◽  
Silke Süsse ◽  
Andreas Rimek ◽  
Ute Preuss ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Strand Break ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Dsb Repair ◽  
P53 Expression ◽  
Strand Breaks ◽  
Single Strand Breaks ◽  
Multistep Process ◽  
Non Homologous End Joining

ABSTRACT DNA double-strand breaks (DSBs) arise spontaneously after the conversion of DNA adducts or single-strand breaks by DNA repair or replication and can be introduced experimentally by expression of specific endonucleases. Correct repair of DSBs is central to the maintenance of genomic integrity in mammalian cells, since errors give rise to translocations, deletions, duplications, and expansions, which accelerate the multistep process of tumor progression. For p53 direct regulatory roles in homologous recombination (HR) and in non-homologous end joining (NHEJ) were postulated. To systematically analyze the involvement of p53 in DSB repair, we generated a fluorescence-based assay system with a series of episomal and chromosomally integrated substrates for I-SceI meganuclease-triggered repair. Our data indicate that human wild-type p53, produced either stably or transiently in a p53-negative background, inhibits HR between substrates for conservative HR (cHR) and for gene deletions. NHEJ via microhomologies flanking the I-SceI cleavage site was also downregulated after p53 expression. Interestingly, the p53-dependent downregulation of homology-directed repair was maximal during cHR between sequences with short homologies. Inhibition was minimal during recombination between substrates that support reporter gene reconstitution by HR and NHEJ. p53 with a hotspot mutation at codon 281, 273, 248, 175, or 143 was severely defective in regulating DSB repair (frequencies elevated up to 26-fold). For the transcriptional transactivation-inactive variant p53(138V) a defect became apparent with short homologies only. These results suggest that p53 plays a role in restraining DNA exchange between imperfectly homologous sequences and thereby in suppressing tumorigenic genome rearrangements.

scholarly journals Kinesin Kif2C in regulation of DNA double strand break dynamics and repair

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Songli Zhu ◽  
Mohammadjavad Paydar ◽  
Feifei Wang ◽  
Yanqiu Li ◽  
Ling Wang ◽  
...  
Keyword(s):  
Dna Damage ◽  
Mammalian Cells ◽  
Strand Break ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Dna Double Strand Break ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

DNA double strand breaks (DSBs) have detrimental effects on cell survival and genomic stability, and are related to cancer and other human diseases. In this study, we identified microtubule-depolymerizing kinesin Kif2C as a protein associated with DSB-mimicking DNA templates and known DSB repair proteins in Xenopus egg extracts and mammalian cells. The recruitment of Kif2C to DNA damage sites was dependent on both PARP and ATM activities. Kif2C knockdown or knockout led to accumulation of endogenous DNA damage, DNA damage hypersensitivity, and reduced DSB repair via both NHEJ and HR. Interestingly, Kif2C depletion, or inhibition of its microtubule depolymerase activity, reduced the mobility of DSBs, impaired the formation of DNA damage foci, and decreased the occurrence of foci fusion and resolution. Taken together, our study established Kif2C as a new player of the DNA damage response, and presented a new mechanism that governs DSB dynamics and repair.

scholarly journals The Deubiquitylating Enzyme USP44 Counteracts the DNA Double-strand Break Response Mediated by the RNF8 and RNF168 Ubiquitin Ligases

2013 ◽  
Vol 288 (23) ◽  
pp. 16579-16587 ◽  
Author(s):  
Anna Mosbech ◽  
Claudia Lukas ◽  
Simon Bekker-Jensen ◽  
Niels Mailand
Keyword(s):  
Ring Finger ◽  
Ubiquitin Ligases ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Histone H2a ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Dna Double Strand Break ◽  
Dependent Protein ◽  
Protein Recruitment

Protein recruitment to DNA double-strand breaks (DSBs) relies on ubiquitylation of the surrounding chromatin by the RING finger ubiquitin ligases RNF8 and RNF168. Flux through this pathway is opposed by several deubiquitylating enzymes (DUBs), including OTUB1 and USP3. By analyzing the effect of individually overexpressing the majority of human DUBs on RNF8/RNF168-mediated 53BP1 retention at DSB sites, we found that USP44 and USP29 powerfully inhibited this response at the level of RNF168 accrual. Both USP44 and USP29 promoted efficient deubiquitylation of histone H2A, but unlike USP44, USP29 displayed nonspecific reactivity toward ubiquitylated substrates. Moreover, USP44 but not other H2A DUBs was recruited to RNF168-generated ubiquitylation products at DSB sites. Individual depletion of these DUBs only mildly enhanced accumulation of ubiquitin conjugates and 53BP1 at DSBs, suggesting considerable functional redundancy among cellular DUBs that restrict ubiquitin-dependent protein assembly at DSBs. Our findings implicate USP44 in negative regulation of the RNF8/RNF168 pathway and illustrate the usefulness of DUB overexpression screens for identification of antagonizers of ubiquitin-dependent cellular responses.

The Homologous Chromosome Is an Effective Template for the Repair of Mitotic DNA Double-Strand Breaks in Drosophila

Genetics ◽  
2003 ◽  
Vol 165 (4) ◽  
pp. 1831-1842 ◽  
Author(s):  
Yikang S Rong ◽  
Kent G Golic
Keyword(s):  
Gene Conversion ◽  
Mammalian Cells ◽  
Homologous Chromosome ◽  
Germ Line ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Yeast Saccharomyces Cerevisiae ◽  
Strand Breaks ◽  
Mitotic Cells

AbstractIn recombinational DNA double-strand break repair a homologous template for gene conversion may be located at several different genomic positions: on the homologous chromosome in diploid organisms, on the sister chromatid after DNA replication, or at an ectopic position. The use of the homologous chromosome in mitotic gene conversion is thought to be limited in the yeast Saccharomyces cerevisiae and mammalian cells. In contrast, by studying the repair of double-strand breaks generated by the I-SceI rare-cutting endonuclease, we find that the homologous chromosome is frequently used in Drosophila melanogaster, which we suggest is attributable to somatic pairing of homologous chromosomes in mitotic cells of Drosophila. We also find that Drosophila mitotic cells of the germ line, like yeast, employ the homologous recombinational repair pathway more often than imperfect nonhomologous end joining.

scholarly journals CoincidentIn VitroAnalysis of DNA-PK-Dependent and -Independent Nonhomologous End Joining

2010 ◽  
Vol 2010 ◽  
pp. 1-11 ◽  
Author(s):  
Cynthia L. Hendrickson ◽  
Shubhadeep Purkayastha ◽  
Elzbieta Pastwa ◽  
Ronald D. Neumann ◽  
Thomas A. Winters
Keyword(s):  
Mammalian Cells ◽  
Current Model ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Ligase Iv ◽  
Dependent Protein ◽  
Regulatory Functions

In mammalian cells, DNA double-strand breaks (DSBs) are primarily repaired by nonhomologous end joining (NHEJ). The current model suggests that the Ku 70/80 heterodimer binds to DSB ends and recruits DNA-PKcsto form the active DNA-dependent protein kinase, DNA-PK. Subsequently, XRCC4, DNA ligase IV, XLF and most likely, other unidentified components participate in the final DSB ligation step. Therefore, DNA-PK plays a key role in NHEJ due to its structural and regulatory functions that mediate DSB end joining. However, recent studies show that additional DNA-PK-independent NHEJ pathways also exist. Unfortunately, the presence of DNA-PKcsappears to inhibit DNA-PK-independent NHEJ, andin vitroanalysis of DNA-PK-independent NHEJ in the presence of the DNA-PKcsprotein remains problematic. We have developed anin vitroassay that is preferentially active for DNA-PK-independent DSB repair based solely on its reaction conditions, facilitating coincident differential biochemical analysis of the two pathways. The results indicate the biochemically distinct nature of the end-joining mechanisms represented by the DNA-PK-dependent and -independent NHEJ assays as well as functional differences between the two pathways.

scholarly journals Structural basis for proficient oxidized ribonucleotide insertion in double strand break repair

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Joonas A. Jamsen ◽  
Akira Sassa ◽  
Lalith Perera ◽  
David D. Shock ◽  
William A. Beard ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Time Lapse ◽  
Strand Break ◽  
Structural Basis ◽  
End Joining ◽  
Strand Breaks ◽  
Nucleotide Pools ◽  
Nucleotide Insertion ◽  
Non Homologous End Joining

AbstractReactive oxygen species (ROS) oxidize cellular nucleotide pools and cause double strand breaks (DSBs). Non-homologous end-joining (NHEJ) attaches broken chromosomal ends together in mammalian cells. Ribonucleotide insertion by DNA polymerase (pol) μ prepares breaks for end-joining and this is required for successful NHEJ in vivo. We previously showed that pol μ lacks discrimination against oxidized dGTP (8-oxo-dGTP), that can lead to mutagenesis, cancer, aging and human disease. Here we reveal the structural basis for proficient oxidized ribonucleotide (8-oxo-rGTP) incorporation during DSB repair by pol μ. Time-lapse crystallography snapshots of structural intermediates during nucleotide insertion along with computational simulations reveal substrate, metal and side chain dynamics, that allow oxidized ribonucleotides to escape polymerase discrimination checkpoints. Abundant nucleotide pools, combined with inefficient sanitization and repair, implicate pol μ mediated oxidized ribonucleotide insertion as an emerging source of widespread persistent mutagenesis and genomic instability.

scholarly journals A stapled peptide mimetic of the CtIP tetramerization motif interferes with double-strand break repair and replication fork protection

2021 ◽  
Vol 7 (8) ◽  
pp. eabc6381
Author(s):  
Anika Kuster ◽  
Nour L. Mozaffari ◽  
Oliver J. Wilkinson ◽  
Jessica L. Wojtaszek ◽  
Christina Zurfluh ◽  
...  
Keyword(s):  
Strand Break ◽  
Amino Acid Residues ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Stapled Peptide ◽  
Patients With Cancer ◽  
Peptide Mimetic ◽  
Dna Damaging Agents ◽  
Stalled Replication Forks

Cancer cells display high levels of DNA damage and replication stress, vulnerabilities that could be exploited by drugs targeting DNA repair proteins. Human CtIP promotes homology-mediated repair of DNA double-strand breaks (DSBs) and protects stalled replication forks from nucleolytic degradation, thus representing an attractive candidate for targeted cancer therapy. Here, we establish a peptide mimetic of the CtIP tetramerization motif that inhibits CtIP activity. The hydrocarbon-stapled peptide encompassing amino acid residues 18 to 28 of CtIP (SP18–28) stably binds to CtIP tetramers in vitro and facilitates their aggregation into higher-order structures. Efficient intracellular uptake of SP18–28 abrogates CtIP localization to damaged chromatin, impairs DSB repair, and triggers extensive fork degradation. Moreover, prolonged SP18–28 treatment causes hypersensitivity to DNA-damaging agents and selectively reduces the viability of BRCA1-mutated cancer cell lines. Together, our data provide a basis for the future development of CtIP-targeting compounds with the potential to treat patients with cancer.

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