Reduction In Mutation Frequency Research Articles

Implication of uracil in spontaneous mutagenesis on a single-stranded shuttle vector replicated in mammalian cells

Almost all spontaneous point mutations found on a single-stranded shuttle vector after its transfection and replication in monkey cells were located at cytosine residues. In order to understand this very specific type of targeting we have studied the possible implication of uracil residues in the induction of these spontaneous mutations. The single-stranded shuttle vector pCF3A carrying the supF tRNA gene as a mutagenesis target has been allowed to replicate in mammalian COS7 cells, mutations being screened in bacteria using the beta-galactosidase assay. Progenies from untreated DNA and DNA treated with the uracil-DNA glycosylase prior to transfection were analyzed to determine the amount and classes of mutations. While spontaneous mutation frequency was 9.7 x 10(-4) for control DNA, single-stranded vector treated with the E. coli uracil-DNA glycosylase exhibited a reduced mutation frequency of about 30%. The abolished mutations were mainly confined to the cytosine to thymine transitions for which a decrease by a factor of 5 was indeed observed. This finding fits well with the fact that it is usually admitted that uracil pairs with adenine, indicating therefore that approximately 30% of spontaneous mutations observed in our experimental conditions and 80% of C to T transitions may be due to the presence of uracil instead of cytosine.

Inducibility of the DNA repair gene encoding O6-methylguanine-DNA methyltransferase in mammalian cells by DNA-damaging treatments.

The inducibility of the mammalian O6-methylguanine-DNA methyltransferase (MGMT) gene encoding the MGMT protein (EC 2.1.1.63) responsible for removal of the procarcinogenic and promutagenic lesion O6-alkylguanine from DNA was examined by an analysis of transcription of the MGMT gene following exposure of repair-competent (Mex+) and repair-deficient (Mex-) cells to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). While human and rodent Mex- cells (CHO-9, V79, HeLa MR) showed no detectable MGMT mRNA despite the presence of the gene in their genome, the amount of it in several Mex+ lines (NIH 3T3, HeLa S3, HepG2) paralleled their MGMT activity. However, none of these cell lines showed an increase in the MGMT mRNA level after treatment with various concentrations of MNNG. In contrast, MNNG-treated rat hepatoma cells, H4IIE and FTO-2B, both Mex+, had three- to fivefold more MGMT mRNA than the corresponding untreated controls as measured 12 to 72 h after alkylation. N-Methyl-N-nitrosourea, methyl methanesulfonate, N-hydroxyethyl-N-chloroethylnitrosourea, UV light, and X rays caused a similar accumulation of MGMT mRNA in rat hepatoma cells. Studies with inhibitors of RNA and protein synthesis indicate that the induced increase in the amount of MGMT mRNA was due to enhanced transcription of the gene. Furthermore, they revealed the turnover of the MGMT mRNA to be relatively low (half-life, greater than 7 h). Mutagen-induced increase of transcription of MGMT mRNA in H4IIE cells was accompanied by elevation of MGMT repair activity and resulted in reduction of mutation frequency after a challenge dose of MNNG. Although induction of MGMT mRNA transcription has been observed in two rodent hepatoma cell lines so far, this appears to be the first demonstration of inducibility of a mammalian gene encoding a clearly define DNA repair function. The transcription activation of the MGMT gene protects cells from the mutagenic effects of methylating agents.

Inducibility of the DNA Repair Gene Encoding O6-Methylguanine- DNA Methyltransferase in Mammalian Cells by DNA-Damaging Treatments

The inducibility of the mammalian O6-methylguanine-DNA methyltransferase (MGMT) gene encoding the MGMT protein (EC 2.1.1.63) responsible for removal of the procarcinogenic and promutagenic lesion O6-alkylguanine from DNA was examined by an analysis of transcription of the MGMT gene following exposure of repair-competent (Mex+) and repair-deficient (Mex-) cells to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). While human and rodent Mex- cells (CHO-9, V79, HeLa MR) showed no detectable MGMT mRNA despite the presence of the gene in their genome, the amount of it in several Mex+ lines (NIH 3T3, HeLa S3, HepG2) paralleled their MGMT activity. However, none of these cell lines showed an increase in the MGMT mRNA level after treatment with various concentrations of MNNG. In contrast, MNNG-treated rat hepatoma cells, H4IIE and FTO-2B, both Mex+, had three- to fivefold more MGMT mRNA than the corresponding untreated controls as measured 12 to 72 h after alkylation. N-Methyl-N-nitrosourea, methyl methanesulfonate, N-hydroxyethyl-N-chloroethylnitrosourea, UV light, and X rays caused a similar accumulation of MGMT mRNA in rat hepatoma cells. Studies with inhibitors of RNA and protein synthesis indicate that the induced increase in the amount of MGMT mRNA was due to enhanced transcription of the gene. Furthermore, they revealed the turnover of the MGMT mRNA to be relatively low (half-life, greater than 7 h). Mutagen-induced increase of transcription of MGMT mRNA in H4IIE cells was accompanied by elevation of MGMT repair activity and resulted in reduction of mutation frequency after a challenge dose of MNNG. Although induction of MGMT mRNA transcription has been observed in two rodent hepatoma cell lines so far, this appears to be the first demonstration of inducibility of a mammalian gene encoding a clearly define DNA repair function. The transcription activation of the MGMT gene protects cells from the mutagenic effects of methylating agents.

Accumulation of CHO Cells in G 2 Phase Following Exposure to WR-1065

The radioprotector WR-1065 (2-[(aminopropyl)amino]ethanethiol) is known to protect mammalian cells from the cytotoxic and mutagenic effects of radio- and chemotherapeutic agents, but the exact mechanisms involved in this protection are not fully known. To help determine the effects of WR-1065 alone on cells, we examined its effect on a variety of cellular processes. Incubation of AA8 cells in 4 mM WR-1065 did not significantly affect the rate of DNA synthesis. Autoradiographic analysis of heavily labeled (S-phase population) nuclei of AA8 cells showed no significant difference in the S-phase population of WR-1065-treated versus control cells for up to 3 h. An examination of the effect of WR-1065 on repair synthesis, as measured by unscheduled DNA synthesis (UDS) in cells exposed to 15 Gy, showed no difference between treated and sham-treated cells for up to 2 h exposure. A significant reduction in the amount of UDS was seen in cells treated with the protector for 2.5 and 3 h. Incubation of cells in WR-1065 did alter the cell cycle distributions. An increase in the G2-phase population with a corresponding decrease in the G1-phase population was observed in cells incubated up to 3 h in the presence of 4 mM WR-1065. After the removal of WR-1065 at 3 h, a redistribution of the cells throughout the cell cycle occurred as has been observed in cells treated with other synchronization agents. These data suggest that perturbations in cell cycle progression, rather than direct effects on the rate of DNA synthesis, could play a role in the increased survival and reduced mutation frequencies observed in the presence of WR-1065.

Transfection and expression of human O6-methylguanine-DNA methyltransferase (MGMT) cDNA in Chinese hamster cells: the role of MGMT in protection against the genotoxic effects of alkylating agents

O6-Methylguanine-DNA methyltransferase (MGMT) is responsible for removal of O6-alkylguanine from DNA induced by alkylating mutagens/carcinogens. To analyze the involvement of O6-alkylguanine in the generation and MGMT in avoidance of various genotoxic effects of alkylating agents, we transfected Chinese hamster ovary (CHO) cells that lack MGMT activity with human MGMT cDNA cloned into a mammalian expression vector (pSV2MGMT). A high proportion (60-80%) of transfectants selected for a cotransfected neo gene survived treatment with high doses of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and N-hydroxyethyl-N-chloroethylnitrosourea (HeCNU). Parallel transfections with an expression vector containing the bacterial ada gene (pSV2ada) showed the human MGMT to be more effective than the ada expression vector in mediating alkylation resistance. Various clonal CHO cell lines have been established stably transfected with the human MGMT cDNA. The transfectants expressed human MGMT at levels ranging from 8600 to 210,000 molecules per cell. The high MGMT expressors became strongly resistant to the killing effects of MNNG, HeCNU, N-methyl-N-nitrosourea (MNU) and, to a significant lesser degree, methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS). No killing resistance was observed to N-ethyl-N-nitrosourea (ENU), though the MGMT and ada transfectants showed reduction in mutation frequency induced by this agent. Protection from mutation induction by MGMT (and ada) expression was also demonstrated for MNNG. The transfectants were also protected from the sister chromatid exchange (SCE) inducing and, to a lesser degree, clastogenic effect of MNNG and MNU, and slightly to EMS and MMS. Again no protection was observed towards ENU. Correlations between MGMT activity and resistance to a given end point suggest that, for MNNG, O6-methylguanine is the preponderant toxic, mutagenic and SCE inducing lesion. About 90% of MNNG (and MNU) induced SCEs and nearly all of the MNNG-induced gene mutations seem to be due to this adduct. For alkylation-induced chromosomal aberrations, however, and for cell killing and SCEs induced by MMS, EMS and ENU, other lesions than O6-alkylguanine appear to be of major importance. The data strongly support the view that O6-methylguanine is a genotoxic lesion and MGMT a function decisively involved in avoidance of genotoxic effects in cells exposed to MNNG and related compounds. They indicate also that it is important to take into account the property and mode of action of any given alkylating agent in assessing the protective role of MGMT against alkylation-induced genotoxicity.

The radioprotector WR1065 reduces radiation-induced mutations at the hypoxanthine-guanine phosphoribosyl transferase locus in V79 cells.

N-(2-mercaptoethyl)-1,3-diaminopropane (WR1065) protects against radiation-induced cell killing and mutagenesis at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in V79 Chinese hamster lung fibroblast cells. At a concentration of 4 mM, WR1065 was found to be effective in protecting against radiation-induced cell lethality only if present during irradiation, e.g., a dose modification factor (DMF) of 1.9. No protective effect was observed if the protector was added within 5 min after irradiation or 3 h later, e.g., DMFs of 1.0 and 1.1, respectively. The effect of WR1065 on radiation-induced mutation, expressed as resistance to the cytotoxic purine analogue 6-thioguanine (HGPRT), was also investigated. In contrast to the treatment-schedule dependence for protection by WR1065 against cell killing, this agent was effective in reducing radiation-induced mutations regardless of when it was administered. Following a dose of 10 Gy of 60Co gamma-rays, the mutation frequencies observed per 10(6) survivors were 77 +/- 8, 27 +/- 6, 42 +/- 7, and 42 +/- 7 for radiation only, and WR1065 present during, immediately after, or 3 h after irradiation. These data suggest that although a segment of radiation-induced damage leading to reproductive death cannot be modulated through the postirradiation action of WR1065, processes leading to the fixation of gross genetic damage and mutation induction in surviving cells can be effectively altered and interfered with leading to a marked reduction in mutation frequency.

Adaptive response to simple alkylating agents in Bacillus subtilis cells

When the cells of Bacillus subtilis Marburg strains were grown in the presence of N-methyl- N′-nitro- N-nitrosoguanidine (MNNG) at a non-toxic concentration, they acquired resistance with regard to both lethality (enhancement of survival) and mutagenicity (reduction of mutation frequency) to subsequent challenge with MNNG at higher concentrations. Thus, like Escherichia coli cells (Samson and Cairns, 1977), B. subtilis cells respond adaptively to MNNG. Protein synthesis was necessary for the adaptation, and an activity to destroy specifically O 6-methylguanine residues in methylated DNA was present in the extracts of the adapted cells. Therefore, this response is attributable to an error-free DNA-repair system induced by the exposure to MNNG. The adaptive response was observed with the cells carrying uvrA, polA or recA mutation, indicating this to be independent of the excision repair or recA-dependent repair. For the acquisition of the resistance to MNNG, methylating or ethylating agents were effective, but neither N-butyl- N-nitrosorea (BNU) nor N-propyl- N′-nitro- N-nitrosoguanidine (PNNG) was effective. The cells pre-treated with MNNG became resistant to the challenge with alkynitrosoureas or N-alkyl- N′-nitro- N-nitrosoguanidine, but not to the challenge with methyl methanesulphonate (MMS) or dimethyl sulphate (DMS). Thus, the target of the induced DNA-repair activity is likely to be O-alkylated bases in DNA. Though the phenomenology of the adaptive response seems similar to that in E. coli, the following two lines of evidence indicate that the underlying mechanisms may not be identical: (1) in B. subtilis, the cells of polA strains are adapted both with regard to lethality and to mutagenicity; (2) in B. subtilis, MMS or DMS acts only as the inducer of the response.

Mechanism for the differential induction of mutation by S9 activated benzo[ a]pyrene employing either a glucose-6-phosphate-dependent NADPH-regenerating system or an isocitrate-dependent system

A significant difference in mutation frequency has been observed in CHO cells exposed to benzo[ a]pyrene with alternative activation systems. Each system employed rat-liver S9 homogenate with one using isocitrate dehydrogenase to provide reduced NADP, while the other method uses glucose-6-phosphate dehydrogenase. Total aryl hydrocarbon hydroxylase (AHH) activity was greater for the isocitrate dependent system, however, this yielded a lower level of HGPRT mutants. It was ascertained that this reduced mutation frequency may result from sequestering of B[ a]P substrate by crystals in the medium, possibly calcium phosphate, which decreased the effective substrate concentration. This sequestration enhances B[ a]P internalization, which would explain the dichotomy between the AHH values and the mutation frequency data. The production of specific B[ a]P metabolites was also examined by reverse phase HPLC quantitation of extracts of solutions in which the two activation systems were used. The levels of 7,8 dihydroxybenzo[ a]pyrene produced by the glucose-6-phosphate protocol were consistently greater than with isocitrate. This may also be a contributing mechanism for elevating the mutation frequency with this procedure. These results demonstrate several interactions between test compound, cells, and metabolizing system which must be considered with in vitro activation systems.

ANALYSIS OF THE ALBINO-LOCUS REGION OF THE MOUSE. I. ORIGIN AND VIABILITY

Numerous specific-locus experiments designed to test the mutagenic effect of external radiation have yielded, in over 3,600,000 animals observed, altogether 119 presumed mutations involving the c locus. Of these, 55 were viable and albino (cav), 13 were viable and of various intermediate pigment types (cxv), four were subvital (cas and cxs), seven were neonatally lethal albinos (cal), 28 prenatally lethal albinos (cal); 12 died untested. All of the prenatally lethal and at least one of the neonatally lethal c-locus mutations (cal classes) are probably deficiencies that we have analyzed extensively in other experiments. Since absence of the locus mimics albino in phenotype, the intermediates (cxv and cxs groups) probably resulted from intragenic changes. The class of viable albino mutants (cav) might include, in addition to intragenic changes, some extremely small deficiencies. --The effects on viability of c-locus lethals (cal's) in heterozygous condition are not drastic enough to be perceived in stocks of mixed genetic background except in the case of the two longest known deficiencies and a few others. --Analysis of the relation between radiation treatment and type of c-locus mutants obtained shows that the relative frequency of viable mutations, for each germ-cell type, is greater for low-LET than for neutron irradiation; however, the difference for any individual cell type is not significant. The majority (66.7%) of mutations derived from X- or gamma-ray irradiated spermatogonia are viable, and the proportion of "intermediates" among these viables is similar to that among presumed spontaneous c-locus mutations. No significant dose-rate effect on the proportion of lethals could be demonstrated within the set of mutants induced by low-LET irradiation of spermatogonia. Although sets from other germ-cell stages are too small for statistical tests, the results for oocytes are similar, as far as they go. Furthermore, most of the c-locus mutations induced in spermatogonia, even by high-dose-rate X-ray or gamma irradiation, are of a type most likely to result from single-tract events (62% cxv, cxs, and cav; plus 16% presumed deficiencies not involving the closest marker). These results support the view that most of the reduction in mutation frequency at low dose rates is not due to a change in relative proportion of two-track and one-track ionizing events.

Influence of X-ray dose fractionation on the frequency of somatic mutations induced in tradescantia stamen hairs

Abstract X-rays were used to investigate the influence of dose fractionation on the induction of pink and colorless somatic mutations in stamen hair cells of Tradescantia clone 02. Inflorescences were exposed to a single acute dose of 60 rad, two acute doses of 30 rad, or three acute doses of 20 rad. The dose rate in all cases was 30 rad/min. Intervals between dose fractions were varied from 35 sec to 48 h and the mutation frequency was compared with that resulting after the single dose of 60 rad. The data show a reduction in mutation frequency for fractionation intervals longer than 15 and 6 min for pink and colorless mutations, respectively, but not for shorter intervals. One interpretation of the data predicts that pink mutation frequencies are reduced by 11% for fraction intervals of from 30 min to 6 h, and that colorless mutation frequencies are reduced by 24% for intervals of from 15 min to 6 h. The corresponding sparing effect of dose fractionation is equal to 6 rad for pink mutations and 9 rad at the colorless mutation endpoint. A calculation has been made which indicates that the percentages of the total repairable (presumably two-hit) damage that is repaired during fraction intervals up to 6 h, are 16 and 35% for pink and colorless mutations respectively.

The induction of 8-azaguanine-resistant mutants in cultured Chinese hamster cells by ultraviolet light the effect of chenges in post-irradiation conditions

The influence of caffeine (100 μg/ml) and incubation in salts medium (“liquid holding”) was examined on the induction by UV light of mutants resistant to 8-azaguanine. Liquid-holding conditions enhanced survival and gave a significant but small reduction in mutation frequency. The influence of caffeine was seen to depend upon the length of time it was contact with the cells. In a procedure where caffeine was present only during the period allowed for the expression of mutants, the induced mutant freqency was enhanced. Where caffeine was present for the complete post-irradiation incubation period, the frequency was reduced. The first of these experimental procedures is the more rigorous in that it reduces the opportunity for any possible interaction between the effects of the two purine analogues, caffeince and 8-azaguanine. The results are interpreted in terms of an error-prone component of the repair processes.

Mutable genes in the light of Callan's hypothesis of serially repeated gene copies.

MUTABLE genes have been intensively studied in maize by McLintock1–3 and Brink4–6 and they occur widely in higher plants7. Among many features of mutable genes, all difficult to explain by conventional mutation theory, the following are especially relevant to the present discussion, (a) One original labile gene often gives rise with high frequency to a whole range of more or less stable alleles, quantitatively graded in their expression. (b) In addition, a labile gene can frequently mutate to other unstable alleles, differing from the original in the timing or frequency of mutation or in the kinds of stable derivatives most often produced, (c) The frequency of mutation is frequently drastically affected by changes of temperature (in several examples higher temperature causes reduced mutation frequency), or by genes elsewhere in the genotype; such variables affect the mutable gene specifically in the sense that they have no evident effect on mutation in general. All these features are well shown in the pallida-recurrens mutable system of Antirrhinum majus8,9. In the following discussion I shall refer to the Antirrhinum mutants because I am familiar with them, although McLintock has documented most of the same effects much more thoroughly in maize.

Post-radiation recovery in early spermatids sampled from Drosophila pupae.

Abstract When 24- and 48-h old Drosophila pupae were subjected to 6-h pre-treatment with nitrogen, irradiated under anoxia, then post-treated for a further 2 h with either oxygen or nitrogen, it was found that in the first one-day brood from the 24-h pupae there was a consistent and highly significant decrease in the mutation frequency following oxygen post-treatment, as compared to that with nitrogen. This result confirms earlier findings of Sobels , and demonstrates the existence of an oxygen-dependent repair process operating in early spermatids of Drosophila . It was observed that more repair occurred at low than at high doses ( i.e. the relative reduction in mutation frequency was greater), and the suggestion is put forward that the repair system can handle effectively only a limited amount of damage, but becomes saturated when this amount is exceeded at higher doses. The fact that translocation frequencies are also lowered by oxygen-post-treatment is taken as an indication that potential breaks, rather than chromosome breaks, are involved in the repair process. Dominant lethal studies showed that it is most unlikely that the repair process could be due to an artefact caused by oxygen selectively eliminating cells carrying mutations.

Quantitative and qualitative aspects of photoreactivation of premutational ultraviolet damage at the ad-3 loci of Neurospora crassa

Experiments have been carried out to determine whether photoreactivation is specific for particular fraction of the UV-induced premutational damage leading to ad-3 B mutants in Neurospora. Populations of mutants were compared which had been induced by UV in the presence and absence of photoreactivation. In spite of the reduction in mutation frequency brought about by photoreactivation, no evidence was found to suggest that any part of the mutant population was repaired preferentially. The simplest interpretation of this result is that there is but one type of premutational lesion induced by UV in this system. Repair of a fraction of this damage should affect all types of mutants similarly. Alternatively, if more than one type of premutational lesion exist, some of which are nonphotoreactivable, it is necessary to assume that, in terms of the mutants they are capable of producing, the photoreactivable and nonphotoreactivable lesions are equivalent.

Factors modifying mutation frequency patterns detected after irradiation of Drosophila melanogaster males

The frequency of sex-linked recessive lethal mutations detected in matings made immediately after irradiation of 3-day-old males which have mated twice just prior to irradiation is significantly lower than the mutation frequency observed when virgin 3-day-old males are irradiated with the same dose. A similar reduction in mutation frequency is also observed when 7-day-old premated males are irradiated, but the reduction is not so pronounced as in the case of 3-day-old males. The effect of mating prior to irradiation is simply to reduce the number of fully mature sperm present in the male in comparison with the number found in a virgin male of the same age; however, the number of immature sperm is not affected. Therefore, premating decreases the proportion of fully mature to immature sperm present in the male at the time of irradiation. The reduction in mutation frequency that is associated with premating irradiated males can be explained by the higher intrinsic mutability of fully mature as compared with less mature sperm; it cannot be explained by recovery from genetic effects of irradiation. This conclusion is supported by the fact that withholding irradiated virgin 3-and 7-day-old males from mating for 24 or 48 h also reduces the mutation frequency, but only to a level intermediate between that observed in the first mating immediately after irradiation of virgin males of comparable age, and that observed from matings made 24 to 48 h later, if in the meantime the males have been allowed to mate repeatedly. The change in frequency of mutations detected at successive intervals after irradiation is a dynamic reflection of the progress of spermatogenesis, as germ cells having different mutabilities because they were at different stages of development at the time of irradiation are sampled one after another.

Enhancement of ultraviolet-induced mutation in bacteria by caffeine

The addition of caffeine or theophylline to the growth medium of irradiatedE. coli B/rtry − resulted in a 10-fold or greater increase in the frequency oftry + mutants. These observations extend those ofWitkin (1958). Caffeine produced a slight reduction in the rate of RNA and protein synthesis, and a somewhat greater but temporary reduction in the rate of DNA synthesis. The analogue must be added immediately after UV-irradiation to produce its optimal effect, and the ability of an irradiated culture to respond to caffeine was lost completely after 20 min incubation in broth. Normal purine ribosides did not compete with caffeine. The optimal exposure time to caffeine was correlated with the time of DNA doubling, but marked increases of mutation frequency resulted when caffeine was present for 30 min in the absence of DNA synthesis. Incubation in caffeine before irradiation had no effect. Caffeine also reduced mutation frequency decline caused by incubation of irradiated bacteria in chloramphenicol. It is suggested that caffeine interfers with a “dark repair” enzyme system which removes a UV photoproduct (s) whose presence during DNA synthesis leads to mutation.

SOME BIOCHEMICAL ASPECTS OF THE POSTIRRADIATION MODIFICATION OF ULTRAVIOLET-INDUCED MUTATION FREQUENCY IN BACTERIA

Postirradiation treatments blocking RNA or protein syntesis lower markedly the ultraviolet-induced mutation frequency (reversion of tryptophan requirement) in Escherichia coli strain WP2. Treatments of less than 20 min reduce the mutation frequency to a minimal level without significant modification of the time or rate of subsequent synthesis of RNA, DNA, and protein. Therefore the mutation frequency decline process apparently is an active process, probably enzymatically mediated, and not dependent on delay in postirradiation macromolecular synthesis. The progressive loss of susceptibility of the potential mutation to these treatments is closely correlated with the progression of ribonucleic acid snythesis in the culture. A relation exists between the amoumt of RNA synthesized at the time of chloramphenicol addition, the relative rate of DNA synthesis in the presence of chloramphenicol, and the induced mutation frequency. Apparently only when the RNA and protein necessary for genetic replication are synthesized prior to chloramphenicol addition will DNA be formed. When the RNA and protein involved have not been formed, the potentiality for mutation is lost through the active decline process. Decline in induced mutation frequency is promoted by 5-hydroxyuridine after a considerable lag. This decline is correlated with the doubling of RNA in the culture, suggesting thatmore » the analogue causes the decline through incorporation into the RNA. However, loss of susceptibility to the 5-hydroxyuridine-promoted reduction in mutation frequency occurs prior to measurable RNA synthesis and is correlated with the mutation stabilization process. Icubation with 5hydroxyuridine of sufficient duration to prevent all sensitive mutations does not delay DNA synthesis. Several lines of evidence indicate that DNA synthesis is the terminal event in ultraviolet-induced mutation. However, delay of DNA synthesis does not modify ultraviolet-induced mutation frequency in the absence of the active decline process unless drastic treatments are employed which grossly alter cellular metabolism. Loss of mutation photoreversibility is correlated with DNA synthesis in the culture. Dinitrophenol (but not chloramphenicol) blocks the loss of mutation photoreversibility. (auth)« less