In an effort to identify novel genes involved in recombination repair, we isolated fission yeast Schizosaccharomyces pombe mutants sensitive to methyl methanesulfonate (MMS) and a synthetic lethal with rad2. A gene that complements such mutations was isolated from the S. pombe genomic library, and subsequent analysis identified it as the fbh1 gene encoding the F-box DNA helicase, which is conserved in mammals but not conserved in Saccharomyces cerevisiae. An fbh1 deletion mutant is moderately sensitive to UV, MMS, and ␥ rays. The rhp51 (RAD51 ortholog) mutation is epistatic to fbh1. fbh1 is essential for viability in stationary-phase cells and in the absence of either Srs2 or Rqh1 DNA helicase. In each case, lethality is suppressed by deletion of the recombination gene rhp57. These results suggested that fbh1 acts downstream of rhp51 and rhp57. Following UV irradiation or entry into the stationary phase, nuclear chromosomal domains of the fbh1⌬ mutant shrank, and accumulation of some recombination intermediates was suggested by pulsed-field gel electrophoresis. Focus formation of Fbh1 protein was induced by treatment that damages DNA. Thus, the F-box DNA helicase appears to process toxic recombination intermediates, the formation of which is dependent on the function of Rhp51.Homologous recombination not only shuffles genetic information upon sexual reproduction but also repairs damaged DNA by use of the homologous information. Furthermore, it can regenerate replication forks when they become stalled or collapsed.Molecular mechanisms of homologous recombination in eukaryotes have been most extensively studied in the budding yeast Saccharomyces cerevisiae (reviewed in references3, 27, 42, 46, and 48). In this yeast, the MRX (Mre11 Rad50 Xrs2) complex is required for the processing of double-strand break ends to generate 3Ј-protruding ends. The resulting singlestrand regions are coated by single-strand-binding protein RPA (replication protein A). Rad52 stimulates loading of Rad51 on RPA-coated single-strand DNA to form Rad51 nucleoprotein filament. A complex of Rad55 and Rad57, which are Rad51 paralogs, is also implicated in the assembly and stabilization of Rad51 nucleoprotein filament. Rad51 nucleoprotein filament searches homologous sequences and catalyzes the exchange of strands to form a heteroduplex joint called a D loop. Rad54 facilitates D-loop formation by remodeling chromatin structures. The annealed 3Ј ends are then used as primers for repair DNA synthesis. The resulting junction molecules are resolved either by dissociation of the crossed strands or by cutting of the junction point. The Rad52 group proteins (Rad50, Rad51, Rad52, Rad54, Rad55, Rad57, Mre11, and Xrs2) are conserved throughout eukaryotes, indicating a conservation of the molecular mechanisms pertaining to homologous recombination.In addition to the aforementioned recombination factors,

Background:RecN is an SMC (structural maintenance of chromosomes) family protein that is required for DNA doublestrand breaks (DSBs) repair. Results: We identified a RecA mutant that is deficient in interacting with RecN. Conclusion: A functional interaction between RecN and RecA is required for assembly of RecN at the sites of DSBs. Significance: RecN is critical for protecting the structural integrity of chromosomes during DSBs repair.

Protein degradation in bacteria plays a dynamic and critical role in the cellular response to environmental stimuli such as heat shock and DNA damage and in removing damaged proteins or protein aggregates. Escherichia coli recN is a member of the structural maintenance of chromosomes family and is required for DNA double strand break (DSB) repair. This study shows that RecN protein has a short half-life and its degradation is dependent on the cytoplasmic protease ClpXP and a degradation signal at the C terminus of RecN. In cells with DNA DSBs, green fluorescent protein-RecN localized in discrete foci on nucleoids and formed visible aggregates in the cytoplasm, both of which disappeared rapidly in wild-type cells when DSBs were repaired. In contrast, in ⌬clpX cells, RecN aggregates persisted in the cytoplasm after release from DNA damage. Furthermore, analysis of cells experiencing chronic DNA damage revealed that proteolytic removal of RecN aggregates by ClpXP was important for cell viability. These data demonstrate that ClpXP is a critical factor in the cellular clearance of cytoplasmic RecN aggregates from the cell and therefore plays an important role in DNA damage tolerance. DNA double strand breaks (DSBs)2 are major threats to the genomic integrity of cells. DSBs can be caused by exogenous and endogenous agents such as ionizing radiation and chemical mutagens and by endogenously produced radicals or as a result of replication arrest and/or collapse of the replication fork (1, 2). DSBs are lethal if unrepaired and if repaired improperly may result in genome instability such as mutations, genomic rearrangements, and chromosome loss. Therefore, the repair of DSBs is crucial for cell survival and for maintaining the integrity of the genome.In Escherichia coli, the RecBCD pathway of homologous recombination is responsible for the repair of DSBs. RecBCD initially processes broken ends into 3Ј single-stranded DNA tails by its helicase nuclease activities (2). These single-stranded DNA tails then invade homologous duplex DNA via a RecAmediated mechanism. However, the RecF pathway, which is another RecA-dependent recombination pathway, can also promote DSB repair when the RecBCD pathway is inactivated by mutations (e.g. recBC sbcBC). E. coli recN has two SOS boxes in the promoter region of recN that confer inducibility on recN in response to SOS signaling (3, 4). Mutation of recN reduces conjugational recombination in recBC sbcBC strains (3, 5), suggesting that RecN is part of the RecF pathway of recombination. However, in contrast to the other genes of the RecF pathway, mutations in recN do not restore resistance to thymineless death (6), and there is evidence suggesting that the RecN protein is also required for RecBCD-dependent repair of DSBs (6, 7). Recently, it was shown that RecN is required to repair chromosomal breaks at a specific location by I-SceI endonuclease (8). Furthermore, recN mutants are sensitive to ionizing radiation and mitomycin C but not to UV irradiation (5). Thus, RecN does not fall clearly ...

Introduction Cigarette smoking is associated with the risk of certain diseases, but non-combustible products may lower these risks. The potential long-term health effects of the next-generation non-combustible products (heat-not-burn tobacco products (HNBP) or electronic vapor products) have not been thoroughly studied. The present study aimed to investigate the impact of biomarkers of potential harm (BoPH) of one of HNBP (a novel vapor product: NTV), under the conditions of actual use. Methods This study was an observational, cross-sectional, three-group, multi-center study. Exclusive NTV users (NTV, n = 259), conventional cigarette smokers (CC, n = 100) and never-smokers (NS, n=100) were enrolled. Biomarkers of tobacco smoke exposure (cotinine and total NNAL) and BoPH including parameters of physical pulmonary functions relevant to smoking-related diseases were examined, and subjects answered a questionnaire on cough-related symptoms (J-LCQ) and health-related quality of life (SF-36v2®). Results Levels of cotinine, total NNAL and BoPH (HDL-cholesterol, triglyceride, sICAM-1, WBC count, 11-DHTXB2, 2,3-d-TXB2, 8-epi-PGF2α, FEV1, %FEV1 and FEF25-75) were significantly different in the NTV group as compared to levels in CC group (p<0.05). Significantly higher levels of cotinine, total NNAL, and 2,3-d-TXB2, and lower levels of FEV1 and %FEV1, were observed among NVT users compared to the NS group. Conclusion In a post-marketing study under actual use conditions, BoPH associated with smoking-related disease examined in exclusive NTV users were found to be favorably different from those of CC smokers, a finding attributable to a reduction in exposure to harmful substances of tobacco smoke. Implications Cigarette smoking is associated with increased risk of pulmonary diseases like COPD, cardiovascular diseases, and certain cancers. There is a growing body of evidence that HNBP reduces the exposure associated with smoking and that there is a favorable change in BoPH. However, long-term effects regarding the relative health risks to HNBP users compared to CC smokers have not been examined. This study provides post-marketing data under actual use conditions of the effects on biomarkers of potential harm in NTV, one of HNBP, exclusive users compared to CC smokers and never-smokers. The evidence suggests that exclusive NTV users have favorable levels of BoPH compared to CC smokers, and that is result from a sustained reduction in exposure to harmful substances of tobacco smoke.

Background: DNA double-strand breaks (DSBs) are induced by exogenous insults such as ionizing radiation and chemical exposure, and they can also arise as a consequence of stalled or collapsed DNA replication forks. Failure to repair DSBs can lead to genomic instability or cell death and cancer in higher eukaryotes. The Schizosaccharomyces pombe fbh1 gene encodes an F-box DNA helicase previously described to play a role in the Rhp51 (an orthologue of S. cerevisiae RAD51)-dependent recombinational repair of DSBs. Fbh1 fused to GFP localizes to discrete nuclear foci following DNA damage.