Assessing radiation-associated mutational risk to the germline: repetitive DNA sequences as mutational targets and biomarkers.

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.

Radiation and germline mutation at repeat sequences: Are we in the middle of a paradigm shift?

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.

Consultative exercise on dose assessments.

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.

Hypermutation in bacteria and other cellular systems.

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.

Effect of endogenous carotenoids on "adaptive" mutation in Escherichia coli FC40.

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.

A UmuD,C-dependent pathway for spontaneous G:C to C:G transversions in stationary phase Escherichia coli mut Y.

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.

DNA repair: Polymerases for passing lesions.

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.

RBE-LET relationships in mutagenesis by ionizing radiation.

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.

DNA repair: getting past a lesion - at a cost.

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.

Effect of endogenous carotenoids and defective RpoS sigma factor on spontaneous mutation under starvation conditions in Escherichia coli: evidence for the possible involvement of singlet oxygen.

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.

DNA synthesis and viability of a mutT derivative of Escherichia coli WP2 under conditions of amino acid starvation and relation to stationary-phase (adaptive) mutation.

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.

Reversion of the tyrosine ochre strain Escherichia coli WU3610 under starvation conditions depends on a new gene tas.

When 3 x 10(8) bacteria of the Escherichia coli tyrA14(oc) leu308(am) strain WU3610 are plated on glucose salts agar supplemented with leucine only, colonies of slow-growing Tyr+ suppressor mutants begin to appear after about a week and increase in numbers roughly linearly with time thereafter (stationary phase or starvation-associated mutation). From a library constructed from two of these mutants, a clone was obtained that suppressed the tyrosine requirement of WU3610 when present on a multicopy plasmid. The activity was identified to an open reading frame we call tas, the sequence for which has homology with a variety of known genes with aldo-keto reductase activity. The activity of tas complements the prephenate dehydrogenase dysfunction of tyrA14 (the chorismate mutase activity of tyrA possibly being still functional). A strain deleted for tas showed no spontaneous mutation under starvation conditions. Whereas neither tas+ nor tas bacteria showed any increase in viable or total count when plated under conditions of tyrosine starvation at 3 x 10(8) cells per plate, at lower density (approximately 10(7) per plate) tas+ but not tas bacteria showed considerable residual growth. We suggest that the single copy of tas present in WU3610 allows cryptic cell or DNA turnover under conditions of tyrosine starvation and that this is an essential prerequisite for starvation-associated mutation in this system. The target gene for mutation is not tas, although an increase in the expression of this gene, for example, resulting from a suppressor mutation affecting supercoiling, could be responsible for the slow-growing Tyr+ phenotype.

UV-induced mutations and skin cancer: how important is the link?

Studies with patients having the inherited DNA repair disorders xeroderma pigmentosum (XP), Cockayne syndrome (CS), and sun-sensitive trichothiodystrophy (TTD) have shown that neither the defect in repair nor the consequent elevated frequency of sunlight-induced mutations in the skin is sufficient to account for the classically high incidence of skin cancer in XP patients. The possible ways in which different mutations in the XP(D) gene and deficiencies in the immune system may be involved are discussed.

Mutation in Escherichia coli under starvation conditions: a new pathway leading to small deletions in strains defective in mismatch correction.

Strains of Escherichia coli carrying the mutY mutation lack a mismatch correction glycosylase that removes adenines from various mismatch situations. In growing bacteria, 8-oxoguanine-adenine mispairs persist and can give rise to G-->T transversions during subsequent replication cycles. We now show that when trpA23 mutY bacteria are held under tryptophan starvation conditions the tryptophan-independent mutants that arise include small in-frame deletions in addition to transversions. The trpA23 reversion system appears to be unusual in that small in-frame deletions occurring in a particular region of the gene can lead to the production of a functional protein. We suggest that this is a consequence of the deletion causing the polar group on the arginine at the trpA23 site to be pulled away from the active site of the enzyme. Such deletions are also found with starved bacteria defective in methyl-directed mismatch correction activity (mutH, mutL or mutS), and deletion mutations are also found among the much lower number of mutants that arise in bacteria wild-type for mismatch correction. There is thus a pathway, hitherto undetected, leading to deletions probably from mismatches under conditions of growth restraint. RecA, UmuC, UvrA, MutH,L,S, SbcC and SbcD proteins are not required for the operation of the deletion pathway. A possible explanation is that the deletion pathway is not dependent upon further replication and that it fails to be discernible in growing cells because it is relatively slow acting and mismatches are likely to encounter a DNA replication fork before the initial step of the deletion pathway.

DNA turnover and mutation in resting cells.

There is growing evidence that mutations can arise in non-dividing cells (both bacterial and mammalian) in the absence of chromosomal replication. The processes that are involved are still largely unknown but may include two separate mechanisms. In the first, DNA lesions resulting from the action of endogenous mutagens may give rise to RNA transcripts with miscoded bases. If these confer the ability to initiate DNA replication, the DNA lesions may have an opportunity to miscode during replication and thus could give rise to apparently 'adaptive' mutations. A second mechanism is suggested by recent work in starved bacteria, showing that there is much more turnover of chromosomal DNA than has been previously thought. This could permit polymerase errors to lead to mutations in non-dividing cells. Such cryptic DNA synthesis, which may essentially replace existing DNA rather than duplicating it, could, in principle, act as an additional source of variability on which selection may act, initially in the absence of cell division. In mammals such processes would undoubtedly have implications for germ cell mutagenesis and carcinogenesis.