The combined effects of mixtures of ionizing radiations

Ionizing radiation is a special group of toxic agents whose general interaction can be calculated. This was demonstrated using a radiation interaction model previously published. In this paper, this model is refined and mathematically reformulated using a unified set of assumptions. It postulates the existence of a common intermediate lesion and the relative action of lesions before, at and after this common stage. General quantitative dose-effect relationships of mixed radiations can be derived from the dose-effect relationships of the components in the mixture.     

Inactivation and mutation of cultured mammalian cells by aluminium characteristic ultrasoft X-rays. II. Dose-responses of Chinese hamster and human diploid cells to aluminium X-rays and radiations of different LET.

The induction of inactivation and mutation to thioguanine-resistance of two types of cultured mammalian cells, V79 Chinese hamster and HF19 human diploid, was studied after irradiation with aluminium K characteristic ultrasoft X-rays, helium ion track intersections of different LET, 42 MeV d-Be neutrons, and hard X- or gamma-rays. The form of the dose-response curves was different for the two cell-types, and there was an overall difference in radiosensitivity, the human cells being the more sensitive to all radiations. However, for both inactivation and mutation-induction, the relative responses of both cell-types to these radiations was similar. Aluminium X-rays were considerably more effective than hard X- or gamma-rays and were at least as effective as helium ions of 20-28 keV micron-1, although aluminium X-rays produce tracks of very limited range (less than about 0.07 micron). Single track effects by aluminium X-rays cannot, therefore, extend beyond about 0.07 micron, and the subcellular sites involved in inactivation and mutation cannot be greater than this dimension or else the effectiveness of aluminium X-rays would be similar to that of low-LET radiations. This observation is in contradiction to models of radiation action which require relatively large sensitive sites; for example the 'theory of dual radiation action' requires a site diameter of about 0.4 micron to explain the shape of the dose-response curves for V79 hamster cells.     

The roles of specific glycosylases in determining the mutagenic consequences of clustered DNA base damage

The potential for genetic change arising from specific single types of DNA lesion has been thoroughly explored, but much less is known about the mutagenic effects of DNA lesions present in clustered damage sites. Localized clustering of damage is a hallmark of certain DNA-damaging agents, particularly ionizing radiation. We have investigated the potential of a non-mutagenic DNA base lesion, 5,6-dihydrothymine (DHT), to influence the mutagenicity of 8-oxo-7,8-dihydroguanine (8-oxoG) when the two lesions are closely opposed. Using a bacterial plasmid-based assay we present the first report of a significantly higher mutation frequency for the clustered DHT and 8-oxoG lesions than for single 8-oxoG in wild-type and in glycosylase-deficient strains. We propose that endonuclease III has an important role in the initial stages of processing DHT/8-oxoG clusters, removing DHT to give an intermediate with an abasic site or single-strand break opposing 8-oxoG. We suggest that this mutagenic intermediate is common to several different combinations of base lesions forming clustered DNA damage sites. The MutY glycosylase, acting post-replication, is most important for reducing mutation formation. Recovered plasmids commonly gave rise to both wild-type and mutant progeny, suggesting that there is differential replication of the two DNA strands carrying specific forms of base damage.     

African cichlid fish: a model system in adaptive radiation research

The African cichlid fish radiations are the most diverse extant animal radiations and provide a unique system to test predictions of speciation and adaptive radiation theory. The past few years have seen major advances in the phylogenetics, evolutionary biogeography and ecology of cichlid fish. Most of this work has concentrated on the most diverse radiations. Unfortunately, a large number of small radiations and 'non-radiations' have been overlooked, potentially limiting the contribution of the cichlid system to our understanding of speciation and adaptive radiation. I have reviewed the literature to identify 33 intralacustrine radiations and 76 failed radiations. For as many as possible I collected information on lake size, age and phylogenetic relationships. I use these data to address two questions: (i) whether the rate of speciation and the resulting species richness are related to temporal and spatial variation in ecological opportunity and (ii) whether the likelihood of undergoing adaptive radiation is similar for different African cichlid lineages. The former is a key prediction of the ecological theory of adaptive radiation that has been presumed true but remains untested for cichlid radiations. The second is based on the hypothesis that the propensity of cichlids to radiate is due to a key evolutionary innovation shared by all African cichlids. The evidence suggests that speciation rate declines through time as niches get filled up during adaptive radiation: young radiations and early stages of old radiations are characterized by high rates of speciation, whereas at least 0.5 Myr into a radiation speciation becomes a lot less frequent. The number of species in cichlid radiations increases with lake size, supporting the prediction that species diversity increases with habitat heterogeneity, but also with opportunity for isolation by distance. Finally, the data suggest that the propensity to radiate within lakes is a derived property that evolved during the evolutionary history of some African cichlids, and the appearance of which does not coincide with the appearance of proposed key innovations in morphology and life history.     

DNA core ionization and cell inactivation

Whether inner-shell ionizations of DNA atoms, called core ionizations, are critical events for cell inactivation by ionizing radiations such as 100 keV electrons and gamma rays has been investigated. The number of core ionizations in DNA atoms per gray of the two types of radiations is calculated from various Monte Carlo track simulations. The probability that a core ionization leads to cell inactivation is deduced from experimental values of the RBEs of ultrasoft X rays. The contribution to V79 cell inactivation solely due to the core ionizations in DNA is found to be 75 +/- 27% for energetic electrons and gamma rays. This surprisingly large contribution strongly suggests the presence of new mechanisms associated with critical lesions for cell inactivation.     

Effects of radiation quality and oxygen on clustered DNA lesions and cell death

Radiation quality and cellular oxygen concentration have a substantial impact on DNA damage, reproductive cell death and, ultimately, the potential efficacy of radiation therapy for the treatment of cancer. To better understand and quantify the effects of radiation quality and oxygen on the induction of clustered DNA lesions, we have now extended the Monte Carlo Damage Simulation (MCDS) to account for reductions in the initial lesion yield arising from enhanced chemical repair of DNA radicals under hypoxic conditions. The kinetic energy range and types of particles considered in the MCDS have also been expanded to include charged particles up to and including (56)Fe ions. The induction of individual and clustered DNA lesions for arbitrary mixtures of different types of radiation can now be directly simulated. For low-linear energy transfer (LET) radiations, cells irradiated under normoxic conditions sustain about 2.9 times as many double-strand breaks (DSBs) as cells irradiated under anoxic conditions. New experiments performed by us demonstrate similar trends in the yields of non-DSB (Fpg and Endo III) clusters in HeLa cells irradiated by γ rays under aerobic and hypoxic conditions. The good agreement among measured and predicted DSBs, Fpg and Endo III cluster yields suggests that, for the first time, it may be possible to determine nucleotide-level maps of the multitude of different types of clustered DNA lesions formed in cells under reduced oxygen conditions. As particle LET increases, the MCDS predicts that the ratio of DSBs formed under normoxic to hypoxic conditions by the same type of radiation decreases monotonically toward unity. However, the relative biological effectiveness (RBE) of higher-LET radiations compared to (60)Co γ rays (0.24 keV/μm) tends to increase with decreasing oxygen concentration. The predicted RBE of a 1 MeV proton (26.9 keV/μm) relative to (60)Co γ rays for DSB induction increases from 1.9 to 2.3 as oxygen concentration decreases from 100% to 0%. For a 12 MeV (12)C ion (681 keV/μm), the 'predicted RBE for DSB induction increases from 3.4 (100% O(2)) to 9.8 (0% O(2)). Estimates of linear-quadratic (LQ) cell survival model parameters (α and β) are closely correlated to the Monte Carlo-predicted trends in DSB induction for a wide range of particle types, energies and oxygen concentrations. The analysis suggests α is, as a first approximation, proportional to the initial number of DSBs per cell, and β is proportional to the square of the initial number of DSBs per cell. Although the reported studies provide some evidence supporting the hypothesis that DSBs are a biologically critical form of clustered DNA lesion, the induction of Fpg and Endo III clusters in HeLa cells irradiated by γ rays exhibits similar trends with oxygen concentration. Other types of non-DSB cluster may still play an important role in reproductive cell death. The MCDS captures many of the essential trends in the formation of clustered DNA lesions by ionizing radiation and provides useful information to probe the multiscale effects and interactions of ionizing radiation in cells and tissues. Information from Monte Carlo simulations of cluster induction may also prove useful for efforts to better exploit radiation quality and reduce the impact of tumor hypoxia in proton and carbon-ion radiation therapy.     

Interactions between uv radiation of different energies in the inactivation of bacteria.

A strong lethal interaction was observed between various monochromatic wavelengths (254, 334, 365, and 405 nm) in the repair-proficient E. coli K-12 strain AB 1157, except in the case of preexposure to 405-nm radiation which resulted in a protection against the inactivation resulting from subsequent exposure to 365-or 254-nm radiations. The results may be tentatively explained by assuming two classes of DNA lesions and two classes of damage to repair (reversible and inrreversible) whose proportions vary according to wavelength.     

Analysis of interaction for mixtures of agents using the linear isobole

The linear isobole that is commonly used as a reference for the study of interaction is derived from the interaction of an agent with itself. It is shown that the general use of the linear isobole in the study of the combined effects of mixtures of agents implies interaction between the agents whether the dose-effect curves of the agents are the same or not. It is difficult to generalize the interaction between two doses of the same agent to the interaction between two doses of different agents with different action mechanisms without the use of a mechanistic model. Predictions using non-interaction defined as independent action are generally different from those using linear isobole. A simple mechanistic framework based on the concept of common intermediate lesions is introduced in this paper to relate these two methods used for the analysis of synergism and antagonism. In this framework of lesion development, two agents that have no common intermediate lesion in their action will be non-interactive (referred to as independent action). When the two agents share a common intermediate, it is shown that the combined effect will follow the linear isobole (referred to as common action). This simple framework of analysis is applicable to the general study of interaction between two agents with different types of dose-effect curves.     

Relative biological effectiveness of 6 MeV neutrons with respect to cell inactivation and disturbances of the G1 phase

The relative biological effectiveness (RBE) of neutrons and other types of densely ionizing radiation appears to be close to 1.0 for the induction of strand breaks, but considerably higher RBEs have been found for cellular end points such as colony-forming ability. This may be due to differences in the processing of strand breaks or to the involvement of other lesions whose yields are more dependent on radiation quality. Because cell cycle delays may be of great importance in the processing of DNA damage, we determined the RBE for disturbances of the G1 phase in four different cell types (Be11 melanoma, 4197 squamous cell carcinoma, EA14 glioma, GM6419 fibroblasts) and compared them with the RBE for cell inactivation. The method we used to determine the progress from G1 into S was as follows: Cells were serum-deprived for a number of days and then stimulated to grow with culture medium containing normal amounts of serum. Immediately before the change of medium, cells were exposed to graded doses of either 240 kV X rays or 6 MeV neutrons. At different times afterward, cells were labeled with BrdU and the numbers of active S-phase cells were assessed using two-parameter flow cytometry. For all four cell types, cells started to progress from G1 into S after a few hours. Radiation suppressed this process in all cases, but there were some interesting differences. For Be11 and 4197 cells, the most obvious effect was a delay in G1; the labeling index increased a few hours later in irradiated samples than in controls, and there was no significant effect on the maximum labeling index. For EA14 and GM6419 cells, although smaller doses were used because of greater radiosensitivity, a delay of the entry into S phase was again noticeable, but the most significant effect was a reduction in the maximum percentage of active S-phase cells after stimulation, indicating a permanent or long-term arrest in G1. The RBE for the G1 delay was the same for all four cell types, about 2.8, while the RBE for the G1 arrest varied between 3.2 for the most resistant Be11 cells and 1.7 for the most sensitive GM6419 cells. This trend was similar to that observed for the RBE for cell inactivation. If, as described above, the same number of strand breaks per dose is induced by neutrons and by X rays, the signal transduction cascade translates them into a greater G1 delay in the case of higher LET. This appears to be independent of repair capacity, because it is similar in all cell types we investigated. We therefore assume that a higher lesion density or the presence of other types of lesions is important for this relatively early effect. A G1 arrest, however, is more closely related to the later events leading to cell inactivation, where strand break repair does play a major role, influencing X-ray sensitivity more strongly than sensitivity to neutrons because of a lower repairability of lesions induced by higher-LET radiation.     

Cancer risk from low dose radiation in Ptch1+/− mice with inactive DNA repair systems: Therapeutic implications for medulloblastoma

DSBs are harmful lesions produced through endogenous metabolism or by exogenous agents such as ionizing radiation, that can trigger genomic rearrangements. We have recently shown that exposure to 2 Gy of X-rays has opposite effects on the induction of Shh-dependent MB in NHEJ- and HR-deficient Ptch1^+/− mice. In the current study we provide a comprehensive link on the role of HR/NHEJ at low doses (0.042 and 0.25 Gy) from the early molecular changes through DNA damage processing, up to the late consequences of their inactivation on tumorigenesis. Our data indicate a prominent role for HR in genome stability, by preventing spontaneous and radiation-induced oncogenic damage in neural precursors of the cerebellum, the cell of origin of MB. Instead, loss of DNA-PKcs function increased DSBs and apoptosis in neural precursors of the developing cerebellum, leading to killing of tumor initiating cells, and suppression of MB tumorigenesis in DNA-PKcs^-/-/Ptch1^+/− mice. Pathway analysis demonstrates that DNA-PKcs genetic inactivation confers a remarkable radiation hypersensitivity, as even extremely low radiation doses may deregulate many DDR genes, also triggering p53 pathway activation and cell cycle arrest. Finally, by showing that DNA-PKcs inhibition by NU7441 radiosensitizes human MB cells, our in vitro findings suggest the inclusion of MB in the list of tumors beneficiating from the combination of radiotherapy and DNA-PKcs targeting, holding promise for clinical translation.     

Mutation and inactivation of cultured mammalian cells exposed to beams of accelerated heavy ions. III. Human diploid fibroblasts.

The induction of inactivation and mutation to thioguanine-resistance in cultured human diploid fibroblasts was studied after exposure to ionising radiations with LET's in the range 20--470 keV micrometer-1. Unique r.b.e. values were obtained for inactivation and mutation induction with nine different qualities of radiation. The plot of r.b.e. verus LET gave humped curves for both endpoints; r.b.e. maxima were in the LET range 90--200 keV micrometer-1 but the maximum r.b.e. value for mutation induction was almost twice that for inactivation. The accuracy of estimates of mutation induction are discussed with regard to possible selective effects against mutants during post-irradiation growth.     

The BIANCA model/code of radiation-induced cell death: application to human cells exposed to different radiation types

This paper presents a biophysical model of radiation-induced cell death, implemented as a Monte Carlo code called BIophysical ANalysis of Cell death and chromosome Aberrations (BIANCA), based on the assumption that some chromosome aberrations (dicentrics, rings, and large deletions, called “lethal aberrations”) lead to clonogenic inactivation. In turn, chromosome aberrations are assumed to derive from clustered, and thus severe, DNA lesions (called “cluster lesions,” or CL) interacting at the micrometer scale; the CL yield and the threshold distance governing CL interaction are the only model parameters. After a pilot study on V79 hamster cells exposed to protons and carbon ions, in the present work the model was extended and applied to AG1522 human cells exposed to photons, He ions, and heavier ions including carbon and neon. The agreement with experimental survival data taken from the literature supported the assumptions. In particular, the inactivation of AG1522 cells was explained by lethal aberrations not only for X-rays, as already reported by others, but also for the aforementioned radiation types. Furthermore, the results are consistent with the hypothesis that the critical initial lesions leading to cell death are DNA cluster lesions having yields in the order of ~2 CL Gy^?1 cell^?1 at low LET and ~20 CL Gy^?1 cell^?1 at high LET, and that the processing of these lesions is modulated by proximity effects at the micrometer scale related to interphase chromatin organization. The model was then applied to calculate the fraction of inactivated cells, as well as the yields of lethal aberrations and cluster lesions, as a function of LET; the results showed a maximum around 130 keV/μm, and such maximum was much higher for cluster lesions and lethal aberrations than for cell inactivation.     

Sexual dimorphism in an adaptive radiation: Does intersexual niche differentiation result in ecological character displacement?

Evolutionary radiations are one plausible explanation for the rich biodiversity on Earth. Adaptive radiations are the most studied form of evolutionary radiations, and ecological opportunity has been identified as one factor permitting them. Competition among individuals is supposedly highest in populations of conspecifics. Divergent modes of resource use might minimize trophic overlap, and thus intersexual competition, resulting in ecological character displacement between sexes. However, the role of intersexual differentiation in speciation processes is insufficiently studied. The few studies available suggest that intersexual niche differentiation exists in adaptive radiations, but their role within the radiation, and the extent of differentiation within the organism itself, remains largely unexplored. Here, we test the hypothesis that multiple morphological structures are affected by intersexual niche differentiation in “roundfin” Telmatherina, the first case where intersexual niche differentiation was demonstrated in an adaptive fish radiation. We show that sexes of two of the three morphospecies differ in several structural components of the head, all of these are likely adaptive. Sexual dimorphism is linked to the respective morphospecies‐specific ecology and affects several axes of variation. Trait variation translates into different feeding modes, processing types, and habitat usages that add to interspecific variation in all three morphospecies. Intrasexual selection, that is, male–male competition, may contribute to variation in some of the traits, but appears unlikely in internal structures, which are invisible to other individuals. We conclude that intersexual variation adds to the adaptive diversity of roundfins and might play a key role in minimizing intersexual competition in emerging radiations.     

Utilization of the inactivation rate of coenzyme A transferase by thiol reagents to determine properties of the enzyme-CoA intermediate.

The rate of inactivation of succinyl-CoA:3-ketoacid coenzyme A transferase by thiol reagents is increased 3 to 100 times by very low concentrations of acyl-CoA substrates. The same maximum inactivation rate is found with acetoacetyl-CoA and succinyl-CoA. The enhanced rate of inactivation is caused by the stoichiometric formation of the enzyme-CoA intermediate and an accompanying conformation change of the enzyme. The inactivation rate provides a simple assay for the amount of enzyme present as the enzyme-CoA intermediate, using only catalytic concentrations of enzyme. This technique has been utilized to measure (a) a rate constant for hydrolysis of the enzyme-CoA intermediate of 0.10 min-1 at pH 8.1; (b) a stoichiometry of two active sites per enzyme molecule; and (c) the equilibrium constants for formation of the enzyme-CoA intermediate from dilute solutions of substrates (and hence for the overall reaction) by determining the ratio of [enzyme-CoA]/[enzyme] in the presence of a series of substrate "buffers" at different ratios of [RCOO-]/[RCOSCoA]. As the total concentration of acyl-CoA and carbosylate substrates is increased, the inactivation rate is decreased. This indicates that the Michaelis complexes are protected against inactivation.     

Calmodulin-activated cyclic nucleotide phosphodiesterase from brain. Relationship of subunit structure to activity assessed by radiation inactivation

The apparent target sizes of the basal and calmodulin-dependent activities of calmodulin-activated phosphodiesterase from bovine brain were estimated using target theory analysis of data from radiation inactivation experiments. Whether crude or highly purified samples were irradiated, the following results were obtained. Low doses of radiation caused a 10 to 15% increase in basal activity, which, with further irradiation, decayed with an apparent target size of approximately 60,000 daltons. Calmodulin-dependent activity decayed with an apparent target size of approximately 105,000 daltons. The percentage stimulation of enzyme activity by calmodulin decreased markedly as a function of radiation dosage. These observations are consistent with results predicted by computer-assisted modeling based on the assumptions that: 1) the calmodulin-activated phosphodiesterase exists as a mixture of monomers which are fully active in the absence of calmodulin and dimers which are inactive in the absence of calmodulin; 2) in the presence of calmodulin, a dimer exhibits activity equal to that of two monomers; 3) on radiations destruction of a dimer, an active monomer is generated. This monomer-dimer hypothesis provides a plausible explanation for and definition of basal and calmodulin-dependent phosphodiesterase activity.     

A generalized formulation of dual radiation action

Dual radiation action is a process in which cellular lesions are produced as a result of the interaction of pairs of sublesions that are molecular alterations produced by ionizing radiation. Previous formulations of this process have employed a number of simplifying assumptions that limit the accuracy and the range of application of theoretical analysis. The formulation presented here removes some of these restrictions by introducing three functions that describe the geometry of the sensitive material in the cell, the geometry of the pattern of energy deposition, and the interaction probability of sublesions as a function of their separation. The relation derived is similar to that obtained previously, in that lesion production is found to depend on two terms that are proportional to the first and the second power of the absorbed dose. However, the coefficients of these terms are now derived on the basis of a more realistic treatment.     

Mechanisms of mutagenesis in mammalian cells. Application to human thyroid tumours

Mutations are defined as stable and irreversible modifications of the normal genetic message due to small changes in the number or type of bases, or to large modifications of the genome such as deletions, insertions or chromosome rearrangements. These lesions are due to either polymerase errors during normal DNA replication or unrepaired DNA lesions, which will give rise to mutations through a mutagenic pathway. The molecular process leading to mutagenesis depends largely on the type of DNA lesions. Base modifications, such as 8-oxo-guanine or thymine glycol, both induced by ionizing radiations (IR), are readily replicated leading to direct mutations, usually base-pair substitutions. The 8-oxo-G gives rise predominantly to G to T transversions, the type of mutations found in ras or p53 gene from IR-induced tumors. Bulky adducts produced by chemical carcinogens or UV-irradiation are usually repaired by the nucleotide excision repair (NER) pathway which is able to detect structural distortion in the normal double-strand DNA backbone. These lesions represent a blockage to DNA and RNA polymerases as well as some signal for p53 accumulation in the damaged cell. In the absence of repair, these lesions could be eventually replicated owing to the induction of specific proteins at least in bacteria during the SOS process. The precise nature of the error-prone replication across an unexcised DNA lesion in the template is not fully understood in detailed biochemical terms, in mammalian cells. IR basically produce a very large number of DNA lesions from unique base modifications to single- or double-strand breaks and even complex DNA lesions due to the passage of very high energy particles or to a local re-emission of numerous radicals. The breakage of the double-helix is a difficult lesion to repair. Either it will result in cell death or, after an incorrect recombinational pathway, it will induce frameshifts, large deletions or chromosomal rearrangements. Most of the IR-induced mutations are recessive ones, requiring therefore a second genetic event in order to exhibit any harmful effect and a long latency period before the development of a radiation-induced tumor. The fact that IR essentially induced deletions and chromosomal translocations renders very difficult the use of the p53 gene as a marker for mutation analysis. In agreement with the type of lesions induced by IR, it is interesting to point out that the presence has been observed, in a vast majority of radiation-induced papillary thyroid carcinomas (PTC), of an activated ret proto-oncogene originated by the fusion of the tyrosine kinase 3' domain of this gene with the 5' domain of four different genes. These ret chimeric genes which are due to intra- or inter-chromosomal translocations, were called RET/PTC1 to PTC5. The RET/PTC rearrangements were found in PTC from children contaminated by the Chernobyl fall-out as well as in tumours from patients with a history of therapeutic external radiation, with a frequency of 60-84%. This frequency was only 15% in 'spontaneous' PTC. The type of ret chimeric gene predominantly originated by the accidental or therapeutic IR was different. Indeed, PTC1 was present in 75% of the tumours linked to a therapeutic radiation and PTC3 in 75% of the Chernobyl ones. The other forms of RET/PTC were observed in only a minority of the post-Chernobyl PTC (< 20%). The difference in the frequency of PTC1 and PTC3 in both types of PTC, is statistically significant (P < 10(-5), Fischer's exact test). In two of the post-therapeutic radiation PTC, RET/PTC1 and PTC3 were simultaneously present. A PTC1 gene was also observed in 45% of the adenomas appearing after therapeutic radiation. The long-period of latency between exposure to IR and the appearance of thyroid tumours is probably due to the conversion of a heterozygote genotype of IR-induced mutations to a homozygote one. It will be interesting to use this time lag in accidental or therapeutic-irradiated p     

Argon laser micro-irradiation of mitochondria in rat myocardial cells in tissue culture

Three types of lesions produced by argon laser micro-irradiation of single mitochondria are described. A correlation between lesion severity and optical phase density and/or laser output power was observed. Cell survival was generally not affected by mitochondrial irradiation. As many as ten mitochondria were irradiated without cell death resulting. Absorption of the laser radiation was attributed to the natural chromophores, cytochromes c and c₁.     

C --> T mutagenesis and gamma-radiation sensitivity due to deficiency in the Smug1 and Ung DNA glycosylases

The most common genetic change in aerobic organisms is a C:G to T:A mutation. C --> T transitions can arise through spontaneous hydrolytic deamination of cytosine to give a miscoding uracil residue. This is also a frequent DNA lesion induced by oxidative damage, through exposure to agents such as ionizing radiation, or from endogenous sources that are implicated in the aetiology of degenerative diseases, ageing and cancer. The Ung and Smug1 enzymes excise uracil from DNA to effect repair in mammalian cells, and gene-targeted Ung(-/-) mice exhibit a moderate increase in genome-wide spontaneous mutagenesis. Here, we report that stable siRNA-mediated silencing of Smug1 in mouse embryo fibroblasts also generates a mutator phenotype. However, an additive 10-fold increase in spontaneous C:G to T:A transitions in cells deficient in both Smug1 and Ung demonstrates that these enzymes have distinct and nonredundant roles in suppressing C --> T mutability at non-CpG sites. Such cells are also hypersensitive to ionizing radiation, and reveal a role of Smug1 in the repair of lesions generated by oxidation of cytosine.     

Loss of heterozygosity in yeast can occur by ultraviolet irradiation during the S phase of the cell cycle

A CAN1/can1Δ heterozygous allele that determines loss of heterozygosity (LOH) was used to study recombination in Saccharomyces cerevisiae cells exposed to ultraviolet (UV) light at different points in the cell cycle. With this allele, recombination events can be detected as canavanine-resistant mutations after exposure of cells to UV radiation, since a significant fraction of LOH events appear to arise from recombination between homologous chromosomes. The radiation caused a higher level of LOH in cells that were in the S phase of the cell cycle relative to either cells at other points in the cell cycle or unsynchronized cells. In contrast, the inactivation of nucleotide excision repair abolished the cell cycle-specific induction by UV of LOH. We hypothesize that DNA lesions, if not repaired, were converted into double-strand breaks during stalled replication and these breaks could be repaired through recombination using a non-sister chromatid and probably also the sister chromatid. We argue that LOH may be an outcome used by yeast cells to recover from stalled replication at a lesion.