A study of the transcriptional response to a single double-strand break in Bacillus subtilis
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ABSTRACT:
ORGANISM(S): Bacillus subtilis
PROVIDER: GSE13694 | GEO | 2008/11/21
SECONDARY ACCESSION(S): PRJNA110447
REPOSITORIES: GEO
Similar Datasets
Project description:Bacillus subtilis has been a model for gram-positive bacteria and it has long been exploited for industrial and biotechnological applications. However, the availability of facile genetic tools for physiological analysis has generally lagged substantially behind traditional genetic models such as Escherichia coli and Saccharomyces cerevisiae. In this work, we have developed an efficient, precise and scarless method for rapid multiple genetic modifications without altering the chromosome of B. subtilis. This method employs upp gene as a counter-selectable marker, double-strand break (DSB) repair caused by exogenous endonuclease I-SceI and comK overexpression for fast preparation of competent cell. Foreign dsDNA can be simply and efficiently integrated into the chromosome by double-crossover homologous recombination. The DSB repair is a potent inducement for stimulating the second intramolecular homologous recombination, which not only enhances the frequency of resolution by one to two orders of magnitude, but also selects for the resolved product. This method has been successfully and reiteratively used in B. subtilis to deliver point mutations, to generate in-frame deletions, and to construct large-scale deletions. Experimental results proved that it allowed repeated use of the selectable marker gene for multiple modifications and could be a useful technique for B. subtilis.
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Project description:Bacillus subtilis and most Gram positive bacteria possess four SMC like proteins: SMC, SbcC, RecN and the product of the yhaN gene, termed SbcE. SbcE is most similar to SbcC but contains a unique central domain. We show that SbcE plays a role during transformation in competent cells and in DNA double-strand break (DSB) repair. The phenotypes were strongly exacerbated by the additional deletion of recN or of sbcC, suggesting that all three proteins act upstream of RecA and provide distinct avenues for presynapsis. SbcE accumulated at the cell poles in competent cells, and localized as a discrete focus on the nucleoids in 10% of growing cells. This number moderately increased after treatment with DNA damaging agents and in the absence of RecN or of SbcC. Damage-induced foci of SbcE arose early after induction of DNA damage and rarely colocalized with the replication machinery. Our work shows that SMC-like proteins in B. subtilis play roles at different subcellular sites during DNA repair. SbcC operates at breaks occurring at the replication machinery, whereas RecN and SbcE function mainly, but not exclusively, at DSBs arising elsewhere on the chromosome. In agreement with this idea, we found that RecN-YFP damage-induced assemblies also arise in the absence of ongoing replication.
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Project description:Most proteins involved in the DNA double-strand break response (DSBR) accumulate at the damage sites, where they perform functions related to damage signaling, chromatin remodeling and repair. Over the last two decades, studying the accumulation of many DSBR proteins provided information about their functionality and underlying mechanisms of action. However, comparison and systemic interpretation of these data is challenging due to their scattered nature and differing experimental approaches. Here, we extracted, analyzed and compared the available results describing accumulation of 79 DSBR proteins at sites of DNA damage, which can be further explored using Cumulus (http://www.dna-repair.live/cumulus/)-the accompanying interactive online application. Despite large inter-study variability, our analysis revealed that the accumulation of most proteins starts immediately after damage induction, occurs in parallel and peaks within 15-20 min. Various DSBR pathways are characterized by distinct accumulation kinetics with major non-homologous end joining proteins being generally faster than those involved in homologous recombination, and signaling and chromatin remodeling factors accumulating with varying speeds. Our meta-analysis provides, for the first time, comprehensive overview of the temporal organization of the DSBR in mammalian cells and could serve as a reference for future mechanistic studies of this complex process.
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Project description:FANCA is a component of the Fanconi anemia (FA) core complex that activates DNA interstrand crosslink repair by monoubiquitination of FANCD2. Here, we report that purified FANCA protein catalyzes bidirectional single-strand annealing (SA) and strand exchange (SE) at a level comparable to RAD52, while a disease-causing FANCA mutant, F1263Δ, is defective in both activities. FANCG, which directly interacts with FANCA, dramatically stimulates its SA and SE activities. Alternatively, FANCB, which does not directly interact with FANCA, does not stimulate this activity. Importantly, five other patient-derived FANCA mutants also exhibit deficient SA and SE, suggesting that the biochemical activities of FANCA are relevant to the etiology of FA. A cell-based DNA double-strand break (DSB) repair assay demonstrates that FANCA plays a direct role in the single-strand annealing sub-pathway (SSA) of DSB repair by catalyzing SA, and this role is independent of the canonical FA pathway and RAD52.
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Project description:During meiosis, self-inflicted DNA double-strand breaks (DSBs) are created by the protein Spo11 and repaired by homologous recombination leading to gene conversions and crossovers. Crossover formation is vital for the segregation of homologous chromosomes during the first meiotic division and requires the RecA orthologue, Dmc1. We analyzed repair during meiosis of site-specific DSBs created by another nuclease, VMA1-derived endonuclease (VDE), in cells lacking Dmc1 strand-exchange protein. Turnover and resection of the VDE-DSBs was assessed in two different reporter cassettes that can repair using flanking direct repeat sequences, thereby obviating the need for a Dmc1-dependent DNA strand invasion step. Access of the single-strand binding complex replication protein A, which is normally used in all modes of DSB repair, was checked in chromatin immunoprecipitation experiments, using antibody against Rfa1. Repair of the VDE-DSBs was severely inhibited in dmc1Delta cells, a defect that was associated with a reduction in the long tract resection required to initiate single-strand annealing between the flanking repeat sequences. Mutants that either reduce Spo11-DSB formation or abolish resection at Spo11-DSBs rescued the repair block. We also found that a replication protein A component, Rfa1, does not accumulate to expected levels at unrepaired single-stranded DNA (ssDNA) in dmc1Delta cells. The requirement of Dmc1 for VDE-DSB repair using flanking repeats appears to be caused by the accumulation of large quantities of ssDNA that accumulate at Spo11-DSBs when Dmc1 is absent. We propose that these resected DSBs sequester both resection machinery and ssDNA binding proteins, which in wild-type cells would normally be recycled as Spo11-DSBs repair. The implication is that repair proteins are in limited supply, and this could reflect an underlying mechanism for regulating DSB repair in wild-type cells, providing protection from potentially harmful effects of overabundant repair proteins.
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Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series
2016-05-19 | E-GEOD-77184 | biostudies-arrayexpress
Project description:Bacillus subtilis RecA is important for spore resistance to DNA damage, even though spores contain a single non-replicating genome. We report that inactivation of RecA or its accessory factors, RecF, RecO, RecR and RecX, drastically reduce survival of mature dormant spores to ultrahigh vacuum desiccation and ionizing radiation that induce single strand (ss) DNA nicks and double-strand breaks (DSBs). The presence of non-cleavable LexA renders spores less sensitive to DSBs, and spores impaired in DSB recognition or end-processing show sensitivities to X-rays similar to wild-type. In vitro RecA cannot compete with SsbA for nucleation onto ssDNA in the presence of ATP. RecO is sufficient, at least in vitro, to overcome SsbA inhibition and stimulate RecA polymerization on SsbA-coated ssDNA. In the presence of SsbA, RecA slightly affects DNA replication in vitro, but addition of RecO facilitates RecA-mediated inhibition of DNA synthesis. We propose that repairing of the DNA lesions generates a replication stress to germinating spores, and the RecA·ssDNA filament might act by preventing potentially dangerous forms of DNA repair occurring during replication. RecA might stabilize a stalled fork or prevent or promote dissolution of reversed forks rather than its cleavage that should require end-processing.
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Project description:In response to genotoxic stress-induced DNA damage, TopBP1 mediates ATR activation for signaling transduction and DNA damage repair. However, the detailed molecular mechanism remains elusive. Here, using unbiased protein affinity purification and RNA sequencing, it is found that TopBP1 is associated with pre-ribosomal RNA (pre-rRNA). Pre-rRNA co-localized with TopBP1 at DNA double-strand breaks (DSBs). Similar to pre-rRNA, ribosomal proteins also colocalize with TopBP1 at DSBs. The recruitment of TopBP1 to DSBs is suppressed when cells are transiently treated with RNA polymerase I inhibitor (Pol I-i) to suppress pre-rRNA biogenesis but not protein translation. Moreover, the BRCT4-5 of TopBP1 recognizes pre-rRNA and forms liquid-liquid phase separation (LLPS) with pre-rRNA, which may be the molecular basis of DSB-induced foci of TopBP1. Finally, Pol I-i treatment impairs TopBP1-associated cell cycle checkpoint activation and homologous recombination repair. Collectively, this study reveals that pre-rRNA plays a key role in the TopBP1-dependent DNA damage response.
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Project description:DNA double strand breaks (DSBs) can be repaired by non-homologous end joining (NHEJ) or homology-directed repair (HR). HR requires nucleolytic degradation of 5' DNA ends to generate tracts of single-stranded DNA (ssDNA), which are also important for the activation of DNA damage checkpoints. Here we describe a quantitative analysis of DSB processing in the budding yeast Saccharomyces cerevisiae. We show that resection of an HO endonuclease-induced DSB is less extensive than previously estimated and provide evidence for significant instability of the 3' ssDNA tails. We show that both DSB resection and checkpoint activation are dose-dependent, especially during the G1 phase of the cell cycle. During G1, processing near the break is inhibited by competition with NHEJ, but extensive resection is regulated by an NHEJ-independent mechanism. DSB processing and checkpoint activation are more efficient in G2/M than in G1 phase, but are most efficient at breaks encountered by DNA replication forks during S phase. Our findings identify unexpected complexity of DSB processing and its regulation, and provide a framework for further mechanistic insights.
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Project description:Hereditary mutations in polynucleotide kinase-phosphatase (PNKP) result in a spectrum of neurological pathologies ranging from neurodevelopmental dysfunction in microcephaly with early onset seizures (MCSZ) to neurodegeneration in ataxia oculomotor apraxia-4 (AOA4) and Charcot-Marie-Tooth disease (CMT2B2). Consistent with this, PNKP is implicated in the repair of both DNA single-strand breaks (SSBs) and DNA double-strand breaks (DSBs); lesions that can trigger neurodegeneration and neurodevelopmental dysfunction, respectively. Surprisingly, however, we did not detect a significant defect in DSB repair (DSBR) in primary fibroblasts from PNKP patients spanning the spectrum of PNKP-mutated pathologies. In contrast, the rate of SSB repair (SSBR) is markedly reduced. Moreover, we show that the restoration of SSBR in patient fibroblasts collectively requires both the DNA kinase and DNA phosphatase activities of PNKP, and the fork-head associated (FHA) domain that interacts with the SSBR protein, XRCC1. Notably, however, the two enzymatic activities of PNKP appear to affect different aspects of disease pathology, with reduced DNA phosphatase activity correlating with neurodevelopmental dysfunction and reduced DNA kinase activity correlating with neurodegeneration. In summary, these data implicate reduced rates of SSBR, not DSBR, as the source of both neurodevelopmental and neurodegenerative pathology in PNKP-mutated disease, and the extent and nature of this reduction as the primary determinant of disease severity.
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