Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 8 de 8
Filter
Add more filters











Database
Language
Publication year range
1.
Nucleic Acids Res ; 50(11): 6235-6250, 2022 06 24.
Article in English | MEDLINE | ID: mdl-35670662

ABSTRACT

The integrity and proper expression of genomes are safeguarded by DNA and RNA surveillance pathways. While many RNA surveillance factors have additional functions in the nucleus, little is known about the incidence and physiological impact of converging RNA and DNA signals. Here, using genetic screens and genome-wide analyses, we identified unforeseen SMG-1-dependent crosstalk between RNA surveillance and DNA repair in living animals. Defects in RNA processing, due to viable THO complex or PNN-1 mutations, induce a shift in DNA repair in dividing and non-dividing tissues. Loss of SMG-1, an ATM/ATR-like kinase central to RNA surveillance by nonsense-mediated decay (NMD), restores DNA repair and radio-resistance in THO-deficient animals. Mechanistically, we find SMG-1 and its downstream target SMG-2/UPF1, but not NMD per se, to suppress DNA repair by non-homologous end-joining in favour of single strand annealing. We postulate that moonlighting proteins create short-circuits in vivo, allowing aberrant RNA to redirect DNA repair.


Subject(s)
DNA End-Joining Repair , Nonsense Mediated mRNA Decay , Protein Serine-Threonine Kinases , RNA , Animals , DNA/genetics , Genome-Wide Association Study , Protein Serine-Threonine Kinases/metabolism , RNA/genetics , RNA Helicases/genetics
2.
J Cell Sci ; 126(Pt 4): 889-903, 2013 Feb 15.
Article in English | MEDLINE | ID: mdl-23264744

ABSTRACT

Ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) arising in native chromatin elicit an RNF8/RNF168-dependent ubiquitylation response, which triggers the recruitment of various repair factors. Precisely how this response is regulated in the context of chromatin remains largely unexplored. Here, we show that SMARCA5/SNF2H, the catalytic subunit of ISWI chromatin remodeling complexes, is recruited to DSBs in a poly(ADP-ribose) polymerase 1 (PARP1)-dependent manner. Remarkably, PARP activity, although dispensable for the efficient spreading of γH2AX into damaged chromatin, selectively promotes spreading of SMARCA5, the E3 ubiquitin ligase RNF168, ubiquitin conjugates and the ubiquitin-binding factors RAD18 and the RAP80-BRCA1 complex throughout DSB-flanking chromatin. This suggests that PARP regulates the spatial organization of the RNF168-driven ubiquitin response to DNA damage. In support of this, we show that SMARCA5 and RNF168 interact in a DNA damage- and PARP-dependent manner. RNF168 became poly(ADP-ribosyl)ated after DNA damage, while RNF168 and poly(ADP-ribose) chains were required for SMARCA5 binding in vivo, explaining how SMARCA5 is linked to the RNF168 ubiquitin cascade. Moreover, SMARCA5 was found to regulate the ubiquitin response by promoting RNF168 accumulation at DSBs, which subsequently facilitates efficient ubiquitin conjugation and BRCA1 assembly. Underlining the importance of these findings, we show that SMARCA5 depletion renders cells sensitive to IR and results in DSB repair defects. Our study unveils a functional link between DNA damage-induced poly(ADP-ribosyl)ation, SMARCA5-mediated chromatin remodeling and RNF168-dependent signaling and repair of DSBs.


Subject(s)
Adenosine Triphosphatases/metabolism , Chromatin Assembly and Disassembly/physiology , Chromosomal Proteins, Non-Histone/metabolism , DNA Damage/physiology , Ubiquitin-Protein Ligases/metabolism , Adenosine Triphosphatases/genetics , Cell Line , Cell Line, Tumor , Cell Survival/genetics , Cell Survival/physiology , Chromatin Assembly and Disassembly/genetics , Chromosomal Proteins, Non-Histone/genetics , DNA Damage/genetics , DNA Repair/genetics , DNA Repair/physiology , HeLa Cells , Humans , Poly(ADP-ribose) Polymerases/genetics , Poly(ADP-ribose) Polymerases/metabolism , Signal Transduction/genetics , Signal Transduction/physiology , Ubiquitin-Protein Ligases/genetics
3.
PLoS Genet ; 8(6): e1002800, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22761594

ABSTRACT

Translesion synthesis (TLS) polymerases are specialized DNA polymerases capable of inserting nucleotides opposite DNA lesions that escape removal by dedicated DNA repair pathways. TLS polymerases allow cells to complete DNA replication in the presence of damage, thereby preventing checkpoint activation, genome instability, and cell death. Here, we characterize functional knockouts for polh-1 and polk-1, encoding the Caenorhabditis elegans homologs of the Y-family TLS polymerases η and κ. POLH-1 acts at many different DNA lesions as it protects cells against a wide range of DNA damaging agents, including UV, γ-irradiation, cisplatin, and methyl methane sulphonate (MMS). POLK-1 acts specifically but redundantly with POLH-1 in protection against methylation damage. Importantly, both polymerases play a prominent role early in embryonic development to allow fast replication of damaged genomes. Contrary to observations in mammalian cells, we show that neither POLH-1 nor POLK-1 is required for homologous recombination (HR) repair of DNA double-strand breaks. A genome-wide RNAi screen for genes that protect the C. elegans genome against MMS-induced DNA damage identified novel components in DNA damage bypass in the early embryo. Our data suggest SUMO-mediated regulation of both POLH-1 and POLK-1, and point towards a previously unrecognized role of the nuclear pore in regulating TLS.


Subject(s)
Caenorhabditis elegans , DNA Damage , DNA Repair/genetics , DNA-Directed DNA Polymerase , Animals , Caenorhabditis elegans/embryology , Caenorhabditis elegans/genetics , Caenorhabditis elegans Proteins/metabolism , Cisplatin/pharmacology , DNA Damage/drug effects , DNA Damage/genetics , DNA Damage/radiation effects , DNA-Directed DNA Polymerase/genetics , DNA-Directed DNA Polymerase/metabolism , Embryonic Development/genetics , Gamma Rays , Gene Knockout Techniques , Homologous Recombination/drug effects , Homologous Recombination/genetics , Homologous Recombination/radiation effects , Methyl Methanesulfonate/pharmacology , Nuclear Pore/genetics , Porins/genetics , Porins/metabolism , Radiation-Protective Agents/metabolism , Small Ubiquitin-Related Modifier Proteins/genetics , Small Ubiquitin-Related Modifier Proteins/metabolism , Sumoylation/genetics , Ultraviolet Rays
4.
Mutat Res ; 689(1-2): 50-8, 2010 Jul 07.
Article in English | MEDLINE | ID: mdl-20471405

ABSTRACT

Homologous recombination is essential for repair of DNA interstrand cross-links and double-strand breaks. The Rad51C protein is one of the five Rad51 paralogs in vertebrates implicated in homologous recombination. A previously described hamster cell mutant defective in Rad51C (CL-V4B) showed increased sensitivity to DNA damaging agents and displayed genomic instability. Here, we identified a splice donor mutation at position +5 of intron 5 of the Rad51C gene in this mutant, and generated mice harboring an analogous base pair alteration. Rad51C(splice) heterozygous animals are viable and do not display any phenotypic abnormalities, however homozygous Rad51C(splice) embryos die during early development (E8.5). Detailed analysis of two CL-V4B revertants, V4B-MR1 and V4B-MR2, that have reduced levels of full-length Rad51C transcript when compared to wild type hamster cells, showed increased sensitivity to mitomycin C (MMC) in clonogenic survival, suggesting haploinsufficiency of Rad51C. Similarly, mouse Rad51C(splice/neo) heterozygous ES cells also displayed increased MMC sensitivity. Moreover, in both hamster revertants, Rad51C haploinsufficiency gives rise to increased frequencies of spontaneous and MMC-induced chromosomal aberrations, impaired sister chromatid cohesion and reduced cloning efficiency. These results imply that adequate expression of Rad51C in mammalian cells is essential for maintaining genomic stability and sister chromatid cohesion to prevent malignant transformation.


Subject(s)
DNA Damage , DNA-Binding Proteins/genetics , Embryonic Development/genetics , Genomic Instability , Animals , Chromosome Aberrations , Cricetinae , Cricetulus , Female , Haploidy , Mice , Mice, Inbred C57BL , Mitomycin/pharmacology , Mutation , Pregnancy , Sister Chromatid Exchange
5.
Genetics ; 169(2): 795-806, 2005 Feb.
Article in English | MEDLINE | ID: mdl-15545651

ABSTRACT

Site-specific double-strand breaks (DSBs) were generated in the white gene located on the X chromosome of Drosophila by excision of the w(hd) P-element. To investigate the role of nonhomologous end joining (NHEJ) and homologous recombination (HR) in the repair of these breaks, the w(hd) P-element was mobilized in flies carrying mutant alleles of either lig4 or rad54. The survival of both lig4- and rad54-deficient males was reduced to 25% in comparison to the wild type, indicating that both NHEJ and HR are involved in the repair P-induced gaps in males. Survival of lig4-deficient females was not affected at all, implying that HR using the homologous chromosome as a template can partially compensate for the impaired NHEJ pathway. In rad54 mutant females survival was reduced to 70% after w(hd) excision. PCR analysis indicated that the undamaged homologous chromosome may compensate for the potential loss of the broken chromosome in rad54 mutant females after excision. Molecular analysis of the repair junctions revealed microhomology (2-8 bp)-dependent DSB repair in most products. In the absence of Lig4, the 8-bp target site duplication is used more frequently for repair. Our data indicate the presence of efficient alternative end-joining mechanisms, which partly depend on the presence of microhomology but do not require Lig4.


Subject(s)
DNA Damage , DNA Ligases/physiology , DNA Repair , DNA Transposable Elements/physiology , DNA-Binding Proteins/physiology , Drosophila Proteins/physiology , Drosophila/genetics , Egg Proteins/physiology , Insect Proteins/physiology , Alleles , Animals , Crosses, Genetic , DNA Helicases , Female , Gene Deletion , Genes, Insect , Male , Mutation , Survival Rate , X Chromosome
6.
DNA Repair (Amst) ; 3(6): 603-15, 2004 Jun 03.
Article in English | MEDLINE | ID: mdl-15135728

ABSTRACT

The Rad50/Mre11/Nbs1 protein complex has a crucial role in DNA metabolism, in particular in double-strand break (DSB) repair through homologous recombination (HR). To elucidate the role of the Rad50 protein complex in DSB repair in a multicellular eukaryote, we generated a Rad50 deficient Drosophila strain by P-element mediated mutagenesis. Disruption of Rad50 causes retarded development and pupal lethality. To investigate the mechanism of pupal death, brains and wing imaginal discs from third instar larvae were studied in more detail. Wing imaginal discs from Rad50 mutant larvae displayed a 3.5-fold increase in the induction of spontaneous apoptotic cells in comparison to their heterozygous siblings. This finding correlates with increased levels of phosphorylated histone H2Av, indicating an accumulation of DSBs in Rad50 mutant larvae. A 45-fold increase in the frequency of anaphase bridges was detected in the brains of Rad50 deficient larvae, consistent with a role for Rad50 in telomere maintenance and/or replication of DNA. The induction of DSBs and defects in chromosome segregation are in agreement with a role of Drosophila Rad50 in repairing the DSBs that arise during replication.


Subject(s)
Apoptosis , DNA Damage , DNA/genetics , Drosophila Proteins/physiology , Drosophila melanogaster/genetics , Endodeoxyribonucleases/physiology , Exodeoxyribonucleases/physiology , Genes, Lethal , Amino Acid Sequence , Animals , Brain/physiology , DNA Repair Enzymes , DNA Replication , DNA-Binding Proteins , Escherichia coli Proteins , Female , Gene Expression Regulation, Developmental , Heterozygote , Histones/metabolism , Larva/growth & development , Larva/metabolism , Male , Molecular Sequence Data , Mutagenesis , Sequence Homology, Amino Acid , Wings, Animal/physiology
7.
Genetics ; 165(4): 1929-41, 2003 Dec.
Article in English | MEDLINE | ID: mdl-14704177

ABSTRACT

DNA Ligase IV has a crucial role in double-strand break (DSB) repair through nonhomologous end joining (NHEJ). Most notably, its inactivation leads to embryonic lethality in mammals. To elucidate the role of DNA Ligase IV (Lig4) in DSB repair in a multicellular lower eukaryote, we generated viable Lig4-deficient Drosophila strains by P-element-mediated mutagenesis. Embryos and larvae of mutant lines are hypersensitive to ionizing radiation but hardly so to methyl methanesulfonate (MMS) or the crosslinking agent cis-diamminedichloroplatinum (cisDDP). To determine the relative contribution of NHEJ and homologous recombination (HR) in Drosophila, Lig4; Rad54 double-mutant flies were generated. Survival studies demonstrated that both HR and NHEJ have a major role in DSB repair. The synergistic increase in sensitivity seen in the double mutant, in comparison with both single mutants, indicates that both pathways partially overlap. However, during the very first hours after fertilization NHEJ has a minor role in DSB repair after exposure to ionizing radiation. Throughout the first stages of embryogenesis of the fly, HR is the predominant pathway in DSB repair. At late stages of development NHEJ also becomes less important. The residual survival of double mutants after irradiation strongly suggests the existence of a third pathway for the repair of DSBs in Drosophila.


Subject(s)
DNA Damage , DNA Ligases/physiology , DNA Repair , DNA-Binding Proteins/physiology , DNA/radiation effects , Drosophila Proteins/physiology , Drosophila melanogaster/genetics , Egg Proteins/physiology , Amino Acid Sequence , Animals , Animals, Genetically Modified , Cisplatin/toxicity , Cross-Linking Reagents/toxicity , DNA Helicases , DNA Ligase ATP , Drosophila melanogaster/drug effects , Drosophila melanogaster/enzymology , Drosophila melanogaster/radiation effects , Female , Homozygote , Male , Methyl Methanesulfonate/toxicity , Molecular Sequence Data , Mutagens/toxicity , Sequence Homology, Amino Acid , Survival Rate
8.
Mol Cell Biol ; 22(7): 2159-69, 2002 Apr.
Article in English | MEDLINE | ID: mdl-11884603

ABSTRACT

The Rev3 gene of Saccharomyces cerevisiae encodes the catalytic subunit of DNA polymerase zeta that is implicated in mutagenic translesion synthesis of damaged DNA. To investigate the function of its mouse homologue, we have generated mouse embryonic stem cells and mice carrying a targeted disruption of Rev3. Although some strain-dependent variation was observed, Rev3(-/-) embryos died around midgestation, displaying retarded growth in the absence of consistent developmental abnormalities. Rev3(-/-) cell lines could not be established, indicating a cell-autonomous requirement of Rev3 for long-term viability. Histochemical analysis of Rev3(-/-) embryos did not reveal aberrant replication or cellular proliferation but demonstrated massive apoptosis in all embryonic lineages. Although increased levels of p53 are detected in Rev3(-/-) embryos, the embryonic phenotype was not rescued by the absence of p53. A significant increase in double-stranded DNA breaks as well as chromatid and chromosome aberrations was observed in cells from Rev3(-/-) embryos. The inner cell mass of cultured Rev3(-/-) blastocysts dies of a delayed apoptotic response after exposure to a low dose of N-acetoxy-2-acetylaminofluorene. These combined data are compatible with a model in which, in the absence of polymerase zeta, double-stranded DNA breaks accumulate at sites of unreplicated DNA damage, eliciting a p53-independent apoptotic response. Together, these data are consistent with involvement of polymerase zeta in translesion synthesis of endogenously and exogenously induced DNA lesions.


Subject(s)
DNA Damage , DNA-Directed DNA Polymerase , Fungal Proteins/metabolism , Saccharomyces cerevisiae Proteins , Acetoxyacetylaminofluorene/pharmacology , Animals , Apoptosis , Blastocyst/drug effects , Blastocyst/metabolism , Cell Division , Cells, Cultured , Chromosome Aberrations , Crosses, Genetic , Embryo Loss , Embryo, Mammalian/cytology , Embryo, Mammalian/drug effects , Embryo, Mammalian/metabolism , Female , Fungal Proteins/genetics , Gene Deletion , In Situ Nick-End Labeling , Male , Mice , Mice, Knockout , Mutagenesis, Site-Directed , Polymerase Chain Reaction , Stem Cells/metabolism , Tumor Suppressor Protein p53/metabolism
SELECTION OF CITATIONS
SEARCH DETAIL