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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 DeltaclpX 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.

The effects of tobacco leaf types and the presence or absence of charcoal in the cigarette filters on gene expression were investigated using cigarette prototypes made of either flue-cured (FC) leaf or burley (BLY) leaf and Kentucky Reference 2R4F as a representative blend cigarette with cellulose acetate filters or charcoal filters. NCI-H292, human lung mucoepidermoid carcinoma cell line, was exposed to the total particulate matter (TPM) and gas/vapor phase (GVP) from each prototype for 8 h and then the changes in gene expression from microarray data were analyzed. A number of genes associated with oxidative stress, inflammation, DNA damage and xenobiotic response were modified by the two fractions, TPM and GVP, from the three prototypes with cellulose acetate filters. Both TPM and GVP fractions strongly enhanced the gene expression of HMOX1 , which is encoding the limiting enzyme in heme degradation and a key regulator of oxidative stress and inflammatory process. Comparing the effects of TPM and GVP fraction, TPM strongly activated Nrf2 pathway-mediated anti-oxidative stress reaction, whereas GVP caused notable DNA damage response. In comparison of FC and BLY, TPM from FC more strongly induced the expression of histone family proteins than that from BLY. GVP from FC markedly induced gene expression associated with HSP70-mediated inflammation relative to that from BLY. Charcoal included in the filter strongly reduced the effects of GVP from each cigarette on gene expression. However, charcoal did not modified the effects of TPM. As a whole, charcoal is a useful material for reducing the biological effects of GVP. [ABSTRACT FROM AUTHOR]

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. Results: To determine the functional roles of the highly conserved F-box and helicase domains, we have characterized fbh1 mutants carrying specific mutations in these domains. We show that the F-box mutation fbh1-fb disturbs the nuclear localization of Fbh1, conferring an fbh1 null-like phenotype. Moreover, nuclear foci do not form in fbh1-fb cells with DNA damage even if Fbh1-fb is targeted to the nucleus by fusion to a nuclear localization signal sequence. In contrast, the helicase mutation fbh1-hl causes the accumulation of Fbh1 foci irrespective of the presence of DNA damage and confers damage sensitivity greater than that conferred by the null allele. Additional mutation of the F-box alleviates the hypermorphic phenotype of the fbh1-hl mutant. Conclusion: These results suggest that the F-box and DNA helicase domains play indispensable but distinct roles in Fbh1 function. Assembly of the SCFFbh1 complex is required for both the nuclear localization and DNA damage-induced focus formation of Fbh1 and is therefore prerequisite for the Fbh1 recombination function. [ABSTRACT FROM AUTHOR]