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The multispecific transcription factor and tumor suppressor FOXO3 is an important mediator of apoptosis, but the mechanisms that control its proapoptotic function are poorly understood. There has long been evidence that acetylation promotes FOXO3-driven apoptosis and recently a specific JNK (c-Jun N-terminal kinase)-dependent S574 phosphorylated form (p-FOXO3) has been shown to be specifically apoptotic. This study examined whether acetylation and S574 phosphorylation act independently or in concert to regulate the apoptotic function of FOXO3. We observed that both sirtuins 1 and 7 (SIRT1 and SIRT7) are able to deacetylate FOXO3 in vitro and in vivo, and that lipopolysaccharide (LPS) treatment of THP-1 monocytes induced a rapid increase of FOXO3 acetylation, partly by suppression of SIRT1 and SIRT7. Acetylation was required for S574 phosphorylation and cellular apoptosis. Deacetylation of FOXO3 by SIRT activation or SIRT1 or SIRT7 overexpression prevented its S574 phosphorylation and blocked apoptosis in response to LPS. We also found that acetylated FOXO3 preferentially bound JNK1, and a mutant FOXO3 lacking four known acetylation sites (K242, 259, 290 and 569R) abolished JNK1 binding and failed to induce apoptosis. This interplay of acetylation and phosphorylation also regulated cell death in primary human peripheral blood monocytes (PBMs). PBMs isolated from alcoholic hepatitis patients had high expression of SIRT1 and SIRT7 and failed to induce p-FOXO3 and apoptosis in response to LPS. PBMs from healthy controls had lower SIRT1 and SIRT7 and readily formed p-FOXO3 and underwent apoptosis when similarly treated. These results reveal that acetylation is permissive for generation of the apoptotic form of FOXO3 and the activity of SIRT1 and particularly SIRT7 regulate this process in vivo, allowing control of monocyte apoptosis in response to LPS.

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This review assesses recent data on mutational risk to the germline after radiation exposure obtained by molecular analysis of tandemly repeated DNA loci (TRDLs): minisatellites in humans and expanded simple tandem repeats in mice. Some studies, particularly those including exposure to internal emitters, indicate that TRDL mutation can be used as a marker of human radiation exposure; most human studies, however, are negative. Although mouse studies have suggested that TRDL mutation analysis may be more widely applicable in biomonitoring, there are important differences between the structure of mouse and human TRDLs. Mutational mechanisms probably differ between the two species, and so care should be taken in predicting effects in humans from mouse data. In mice and humans, TRDL mutations are largely untargeted with only limited evidence of dose dependence. Transgenerational mutation has been observed in mice but not in humans, but the mechanisms driving such mutation transmission are unknown. Some minisatellite variants are associated with human diseases and may affect gene transcription, but causal relationships have not yet been established. It is concluded that at present the TRDL mutation data do not warrant a dramatic revision of germline or cancer risk estimates for radiation.

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Two assumptions are commonly made in the estimation of genetic risk: (1) that the seven specific loci in the mouse constitute a suitable basis for extrapolation to genetic disease in humans, and (2) that mutations are induced by radiation damage (energy-loss events leading to double-stranded damage) occurring within the gene and are induced linearly with dose, at least at low doses. Recent evidence on the mutability of repeat sequences is reviewed that suggests that neither of these assumptions is as well founded as we like to think. Repeat sequences are common in the human genome, and alterations in them may have health consequences. Many of them are unstable, both spontaneously and after irradiation. The fact that changes in DNA repeat sequences can clearly arise as a result of radiation damage outside the sequence concerned and the likely involvement of some sort of signal transduction process mean that the nature of the radiation dose response cannot be assumed. While the time has not come to abandon the current paradigms, it would seem sensible to invest more effort in exploring the induction of changes in repeat sequences after irradiation and the consequences of such changes for health.

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A summary is given of a meeting held at Sussex University, UK, in October 2000, which allowed the exchange of ideas on methods of assessment of dose to the public arising from potential authorised radioactive discharges from nuclear sites in the UK. Representatives of groups with an interest in dose assessments were invited, and hence the meeting was called the Consultative Exercise on Dose Assessments (CEDA). Although initiated and funded by the Food Standards Agency, its organisation, and the writing of the report, were overseen by an independent Chairman and Steering Group. The report contains recommendations for improvement in co-ordination between different agencies involved in assessments, on method development and on the presentation of data on assessments. These have been prepared by the Steering Group, and will be taken forward by the Food Standards Agency and other agencies in the UK. The recommendations are included in this memorandum.

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The formation of base substitution mutations following exposure of bacteria to ultraviolet light and many other mutagens occurs during translesion synthesis opposite a photoproduct or other lesion in the template strand of DNA. This process requires the UmuD(2)' UmuC complex, only formed to a significant extent in SOS-induced cells. The "two-step" model proposed that there were two steps, insertion of a wrong base (misincorporation) and use of the misincorporated base as a primer for further chain extension (bypass). The original evidence suggested that UmuD(2)' UmuC was needed only for the second step and that in its absence other polymerases such as DNA polymerase III could make misincorporations. Now we know that the UmuD(2)' UmuC complex is DNA polymerase V and that it can carry out both steps in vitro and probably does both in vivo in wild-type cells. Even so, DNA polymerase III clearly has an important accessory role in vitro and a possibly essential role in vivo, the precise nature of which is not clear. DNA polymerases II and IV are also up-regulated in SOS-induced cells and their involvement in the broader picture of translesion synthesis is only now beginning to emerge. It is suggested that we need to think of the chromosomal replication factory as a structure through which the DNA passes and within which as many as five DNA polymerases may need to act. Protein-protein interactions may result in a cassette system in which the most appropriate polymerase can be engaged with the DNA at any given time. The original two-step model was very specific, and thus an oversimplification. As a general concept, however, it reflects reality and has been demonstrated in experiments with eukaryotic DNA polymerases in vitro.

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A temporary state of hypermutation can in principle arise through an increase in the rate of polymerase errors (which may or may not be triggered by template damage) and/or through abrogation of fidelity mechanisms such as proofreading and mismatch correction. In bacteria there are numerous examples of transient mutator states, often occurring as a consequence of stress. They may be targeted to certain regions of the DNA, for example by transcription or by recombination. The initial errors are made by various DNA polymerases which vary in their error-proneness: several are inducible and are under the control of the SOS system. There are several structurally related polymerases in mammals that have recently come to light and that have unusual properties, such as the ability to carry out 'accurate' translesion synthesis opposite sites of template damage or the possession of exceedingly high misincorporation rates. In bacteria the initial errors may be genuinely spontaneous polymerase errors or they may be triggered by damage to the template strand, for example as a result of attack by active oxidative species such as singlet oxygen. In mammalian cells, hypermutable states persisting for many generations have been shown to be induced by various agents, not all of them DNA damaging agents. A hypermutable state induced by ionizing radiation in male germ cells in the mouse results in a high rate of sequence errors in certain unstable minisatellite loci; the mechanism is unclear but believed to be associated with recombination events.

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The appearance over many days of Lac(+) frameshift mutations in Escherichia coli strain FC40 incubated on lactose selection plates is a classic example of apparent "adaptive" mutation in an episomal gene. We show that endogenously overproduced carotenoids reduce adaptive mutation under selective conditions by a factor of around two. Carotenoids are known to scavenge singlet oxygen suggesting that the accumulation of oxidative base damage may be an integral part of the adaptive mutation phenomenon. If so, the lesion cannot be 7,8-dihydro-8-oxoguanine since adaptive mutation in FC40 is unaffected by mutM and mutY mutations. If active oxygen species such as singlet oxygen are involved in adaptive mutation then they should also induce frameshift mutations in FC40 under non-selective conditions. We show that such mutations can be induced under non-selective conditions by protoporphyrin photosensitisation and that this photodynamic induction is reduced by a factor of just over two when endogenous carotenoids are present. We argue that the involvement of oxidative damage would in no way be inconsistent with current understanding of the mechanism of adaptive mutation and the role of DNA polymerases.

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Until recently, it had been concluded from genetic evidence that DNA polymerase III (Pol III, the main replicative polymerase in E. coli) was also responsible for mutagenic translesion synthesis on damaged templates, albeit under the influence of inducible proteins UmuD' and UmuC. Now it appears that these proteins themselves have polymerase activity (and are now known as Pol V) and can carry out translesion synthesis in vitro in the absence of Pol III. Here I discuss the apparent contradictions between genetics and biochemistry with regard to the role of Pol III in translesion synthesis. Does Pol V interact with Pol III and constitute an alternative component of the replication factory (replisome)? Where do the other three known polymerases fit in? What devices does the cell have to ensure that the "right" polymerase is used in a given situation? The debate about the role of Pol III in translesion synthesis reveals a deeper divide between models that interpret everything in terms of mass action effects and those that embrace a replisome held together by protein-protein interactions and located as a structural entity within the cell.

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In Escherichia coli trpA23 bacteria lacking the MutY glycosylase and incubated on plates in the absence of tryptophan, tryptophan-independent mutants continue to arise during incubation over many days. Their appearance is enhanced in umuD+,C+ strains in comparison with strains carrying a deletion through the umu operon and the umuD,C-dependent mutants were greater in number in uvrA bacteria (lacking nucleotide excision repair) than in uvr+ bacteria. Sequencing of mutations occurring in uvrA bacteria revealed the presence of G:C to C:G transversions but only in umuD+,C+ strains. There is thus a pathway in starved bacteria that generates G:C to C:G transversions and requires the inducible UmuD,C proteins. The data are consistent with the occurrence of a lesion, probably 8-oxoguanine, against which guanine may be incorporated during DNA synthesis by "dNTP stabilised" misalignment against the downstream template base. Upon realignment the configuration is substrate for MutY glycosylase which can remove the unmodified guanine. It is hypothesised that UmuD,C proteins are required for primer extension from the mismatch once formed.

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Replicative DNA polymerases generally cannot pass lesions in the template strand. Now there is accumulating evidence for the widespread existence of a separate class of DNA polymerases that can carry out translesion synthesis in both mutagenic and error-free ways.

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The paper considers the relationship between the quality of radiation and biological lesions produced by ionizing radiation. The paper provides a brief review of the modelling of induction of strand breakage, chromosome aberration, revertant mutation in bacteria and Drosophila melanogaster. Experimental data are presented for the relative biological effectiveness of helium ions and alpha-particles for mutation induction and genome lethality in Escherichia coli. The paper examines the relationship between the mutational events and LET. The RBE-LET values for T4 phage, E. coli WP2 and mwh (multiple wing hair) show dependency on LET while the wi (white-ivory) allele mutants show no dependency.

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Bacteria survive many types of synthesis-blocking DNA lesion by inducing a number of proteins that enable their polymerases to synthesize past a lesion, albeit at the cost of an increased mutation rate. This process has now been convincingly achieved in vitro, opening the way to a fuller understanding of the mechanism.

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Under starvation conditions, a variety of stationary phase genes are up-regulated under the control of the stationary phase sigma factor RpoS including at least two peroxidases and a protective DNA binding protein Dps. Previous work suggested that the reversion to prototrophy of certain amino acid auxotrophs of Escherichia coli that occurs when the bacteria are starved of a required amino acid results from the accumulation of oxidative damage to guanine residues in DNA. We report here that three strains lacking RpoS are indistinguishable from wild type in their ability to undergo this starvation-associated mutation, suggesting that basal levels of catalase activity are more than adequate in these strains, and that the induction of catalases and other proteins controlled by rpoS does not contribute to the protection of the DNA, at least in cells starved in early stationary phase. In comparison, the introduction of a plasmid specifying the production of singlet oxygen scavengers (carotenoids) in stationary phase cells led to a roughly twofold reduction in mutant yield. The results suggest that singlet oxygen may be an important endogenously produced mutagen in resting cells.

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Escherichia coli WP2 bacteria with an ochre amino acid auxotrophy show no evidence of growth during the first few days after plating at densities above 10(8) on plates lacking the required amino acid. They lose viability for some days, and then a subpopulation recovers and there is cell turnover. At very low plating densities (around 10(2) per plate), almost every cell will eventually form a small but visible colony. At intermediate plating densities (10(6) to 10(7) per plate), there is an immediate increase in the number of viable bacteria. The results are consistent with a model that assumes that growth is dependent on trace amounts of tryptophan or a tryptophan-complementing substance and that death is due to extracellular toxic species in the medium, including active oxygen species. Mutations in mutT bacteria under these conditions result from incorporation of 7,8-dihydro-8-oxo-dGTP into DNA and thus largely reflect DNA synthesis associated with the increase in the number of viable cells at the initial density used (10(7) per plate). We show that the increase in cell number and much of this DNA synthesis can be eliminated by the presence of 10(8) scavenger bacteria and by removal of early-arising mutant colonies that release the required amino acid. The synthesis that remains is equivalent to less than a quarter of a genome per day and is marginally reduced, if at all, in a polA derivative. We cannot exclude the possibility that this residual DNA synthesis is peculiar to mutT bacteria due to transcriptional leakiness, although there is no evidence that this is a major problem in this strain. If such DNA synthesis also occurs in wild-type bacteria, it may well be important for adaptive mutation since use of a more refined agar in selective plates both eliminated the initial increase in cell number seen at low density (10(7) per plate) and reduced the rate of appearance of mutants at plating densities above 10(8) per plate.

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