Neil J. Sargentini

Quantitation of the Involvement of the recA, recB, recC, recF, recJ, recN, lexA, radA, radB, uvrD, and umuC Genes in the Repair of X-Ray-Induced DNA Double-Strand Breaks in Escherichia coli

Isogenic Escherichia coli strains carrying single DNA-repair mutations were compared for their capacity for (i) the repair of X-ray-induced DNA double-strand breaks (DSB) as measured using neutral sucrose gradients; (ii) medium-dependent resistance, i.e., a recA-dependent X-ray survival phenomenon that correlates closely with the capacity for repairing DSB; and (iii) the growth medium-dependent, recA-dependent repair of X-ray-induced DNA single-strand breaks (SSB) as measured using alkaline sucrose gradients (about 80% of these SSB are actually parts of DSB). These three capacities were measured to quantitate more accurately the involvement of the various genes in the repair of DSB over a wide dose range. The mutations tested were grouped into five classes according to their effect on the repair of X-ray-induced DSB: (I) the recA, recB, recC, and lexA mutants were completely deficient; (II) the radB and recN mutants were about 90% deficient; (III) the recF and recJ mutants were about 70% deficient; (IV) the radA and uvrD mutants were about 30% deficient; and (V) the umuC mutant resembled the wild-type strains in its capacity for the repair of DSB.

Spontaneous mutagenesis: the roles of DNA repair, replication, and recombination

There appears to be no dearth of mechanisms to explain spontaneous mutagenesis. In the case of base substitutions, data for bacteriophage T4 and especially for E. coli and S. cerevisiae suggest important roles in spontaneous mutagenesis for the error-prone repair of DNA damage (to produce mutations) and for error-free repair of DNA damage (to avoid mutagenesis). Data from the very limited number of studies on the subject suggest that about 50% of the spontaneous base substitutions in E. coli, and perhaps 90% in S. cerevisiae are due to error-prone DNA repair. On the other hand, spontaneous frameshifts and deletions seem to result from mechanisms involving recombination and replication. Spontaneous insertions have been shown to be important in the strongly polar inactivation of certain loci, but it is less important at other loci. Perhaps with continued study, the term spontaneous mutagenesis will be replaced by more specific terms such as 5-methylcytosine deamination mutagenesis, fatty acid oxidation mutagenesis, phenylalanine mutagenesis, and imprecise-recombination mutagenesis. While most studies have concentrated on mutator mutations, the most conclusive data for the actual source of spontaneous mutations have come from the study of antimutator mutations. Further study in this area, perhaps along with an understanding of chemical antimutagens, should be invaluable in clarifying the bases of spontaneous mutagenesis.

umuC-dependent and umuC-independent γ- and UV-radiation mutagenesis in Escherichia coli

Abstract The effects of the umuC 36 and umuC 122::Tn5 mutations on γ- and UV radiation mutagenesis (nonsense, missense, and frameshift mutation assays) in Escherichia coli K12 were studied. Although both mutations reduced radiation mutagenesis, the umuC 36 mutation appeared to be leaky since considerably more UV radiation mutagenesis could be detected in the umuC 36 strain than in the umuC 122::Tn5 strain. In general, the umuC strains showed much larger deficiencies in UV radiation mutagenesis than they did for γ-radiation mutagenesis. The mutability of the umuC 122::Tn5 strain varied depending on the radiation dose, and the mutation assay used. For γ-radiation mutagenesis, the deficiency varied from no deficiency to a 50-fold deficiency; for UV radiation mutagenesis, the deficiency varied from 100-fold to at least 5000-fold. We concluded that both umuC -dependent and umuC -independent modes function for γ-radiation m mutagenesis, while UV radiation mutagenesis seems to depend almost exclusively on the umuC -dependent mode.

DNA sequence analysis of γ-radiation (anoxic)-induced and spontaneous lacId mutations in Escherichia coli K-12

Abstract An extensive spectrum of ionizing radiation mutagenesis was determined by sequencing 318 137 Cs γ-radiation (anoxic)-induced episomal lacI d mutations in Escherichia coli strain NR9102. The most commonly found radiation-induced mutations were based substitutions (44% transversions and 41% transitions). The radiation-induced spectrum consisted of: 23% G · C → A · T, 18% A · T → G · C, 17% G · C → T · A, 14% G · C → C · G, 8% A · T → T · A, 6% A · T → C · G, 8% single-based deletions, 5% multiple mutations, 3% multi-base deletions, and essentially no single-or multi-base additions. This spectrum compared better with spectra for other systems obtained by in vivo irradiation than with one obtained by in vitro irradiation. Multiple mutations, which were unique to the radiation-induced spectrum, generally consisted of one active and one closely linked silent mutation and are suggested to result from an altered replication complex of reudced fidelity. Mutation rates were 4.1 × 10 −8 lac-constitutive mutations/gene/Gy and 1.2 × 10 −10 base substitutions/base pair/Gy. Thirty-two percent more radiation-induced mutations occurred at G · C vs. A · T base pairs. A strand asymmetry was noted for G · C → C · G and A · T → T · A transversions. A nearest-neighbor analysis showed that C (vs. A, G. or T), on either side of the mutation site, substantially enhanced most types of base substitutions. Similarly, G and C flanked both sides of single-base deletion sites twice as frequently as would be expected from the base composition of the mutation target. For comparative purpose, we sequenced 411 spontaneous lac-constitutive mutants of which 269 were lacI d mutants, and there was good agreement between these and previously published mutational spectra. The spontaneous and radiation-induced mutational spectra differed substantially for virtually every class of mutation. For example, the set of spontaneous dominant lac-constitutive mutations contained many more mutations that did not map in the normal region for lacI d mutations (i.e., 35% vs. 3%) and were presumed to be lacO-constitutive mutations. A sampling of these presumptive lacO c mutations was also sequenced: 17 22 (spontaneous) and 1 9 (radiation) were found to be lacO c long deletions, one from each set were base substitutions, and the remaining mutations showed the wild-type lacO sequence. Like the radiation-induced spectrum, the spontaneous spectrum showed enhanced mutagenesis at G · C sites, strand asymmetry, and enhanced mutagenesis when G or C were the nearest neighbors.

Characterization of an Escherichia coli mutant (radB101) sensitive to. gamma. and uv radiation, and methyl methanesulfonate

After N-methyl-N-nitro-N-nitrosoguanidine mutagenesis of Escherichia coli K-12 (xthA14), and X-ray-sensitive mutant was isolated. This sensitivity is due to a mutation, radB101, which is located at 56.5 min on the E. coli K-12 linkage map. The radB101 mutation sensitized wildtype cells to gamma and uv radiation, and to methyl methanesulfonate. When known DNA repair-deficient mutants were ranked for their gamma-radiation sensitivity relative to their uv-radiation sensitivity, their order was (starting with the most selectively gamma-radiation-sensitive strain): recB21, radB101, wild type, polA1, recF143, lexA101, recA56, uvrD3, and uvrA6. The radB mutant was normal for gamma- and uv-radiation mutagenesis, it showed only a slight enhancement of gamma- and uv-radiation-induced DNA degradation, and it was approximately 60% deficient in recombination ability. The radB gene is suggested to play a role in the recA gene-dependent (Type III) repair of DNA single-strand breaks after gamma irradiation and in postreplication repair after uv irradiation for the following reasons; the radB strain was normal for the host-cell reactivation of gamma- and uv-irradiated bacteriophage lambda; the radB mutation did not sensitize a recA strain, but did sensitize a polA strain to gamma and uv radiation; the radB mutation sensitized a uvrB strain to uv radiation.

Involvement of RecB-mediated (but not RecF-mediated) repair of DNA double-strand breaks in the γ-radiation production of long deletions in Escherichia coli

Experiments were designed to determine the association between the repair of gamma-radiation-induced DNA double-strand breaks (DSB) and the induction of 700-1000 bp long deletions (Lac(-)----Lac+), base substitutions (leuB19----Leu+), and frameshifts (trpE9777----Trp+) in Escherichia coli K-12. Over the range of 2.5-20 krad, deletions were induced with linear kinetics, as has been shown for the induction of DSB, while the induction kinetics of base substitutions and frameshifts were curvilinear. Like the repair of DSB, deletion induction showed an absolute requirement for an intact recB gene as well as a dependency on the type of preirradiation growth medium; these requirements were not seen for base substitutions or frameshifts. In addition, about 80% of the spontaneous deletions were absent in the recB21 strain. A recC1001 mutation, which confers a hyper-Rec phenotype, increased the rate of gamma-radiation-induced deletions as well as the low-dose production of base substitutions and frameshifts. A recF143 mutation increased the yield of gamma-radiation-induced deletions without increasing base substitutions or frameshifts. A mutS mutation markedly enhanced the gamma-radiation induction of frameshifts, and had a slight effect on base substitutions, but did not affect the induction of deletions. Resistance to gamma-irradiation and the capacity to repair DSB (albeit at about half the normal rate) were restored to the radiosensitive recB21 strain by the addition of the sbcB21 and sbcC201 mutations. However, the radioresistant recB sbcBC strain, which is recombination proficient via the RecF pathway, was still grossly deficient in the ability to produce deletions. A model for deletion induction as a by-product of the recB-dependent (Chi-dependent) repair of gamma-radiation-induced DSB is discussed, as is the inability to detect deletions in cells that use only the recF-dependent (Chi-independent) mechanism to repair DSB.

Mutational spectrum analysis of umuC-independent and umuC-dependent γ-radiation mutagenesis in Escherichia coli

gamma-Radiation mutagenesis (oxic versus anoxic) was examined in wild-type, umuC and recA strains of Escherichia coli K-12. Mutagenesis [argE3(Oc)----Arg+] was blocked in a delta (recA-srlR)306 strain at the same doses that induced mutations in umuC122::Tn5 and wild-type strains, indicating that both umuC-independent and umuC-dependent mechanisms function within recA-dependent misrepair. Analyses of various suppressor and back mutations that result in argE3 and hisG4 ochre reversion and an analysis of trpE9777 (+1 frameshift) reversion were performed on umuC and wild-type cells irradiated in the presence and absence of oxygen. While the umuC strain showed the gamma-radiation induction of base substitution and frameshifts when irradiated in the absence of oxygen, the umuC mutation blocked all oxygen-dependent base-substitution mutagenesis, but not all oxygen-dependent frameshift mutagenesis. For anoxically irradiated cells, the yields of GC----AT [i.e., at the supB and supE (Oc) loci] and AT----GC transitions (i.e., at the argE3 and hisG4 loci) were essentially umuC independent, while the yields of (AT or GC)----TA transversions (i.e., at the supC, supL, supM, supN and supX loci) were heavily umuC dependent. These data suggest new concepts about the nature of the DNA lesions and the mutagenic mechanisms that lead to gamma-radiation mutagenesis.

Characterization of a New Radiation-Sensitive Mutant, Escherichia coli K-12 radC102

A new radiation-sensitive mutant, radC , has been isolated. The radC gene is located at 81.0 min on the Escherichia coli K-12 linkage map. The radC mutation sensitized cells to uv radiation, but unlike most DNA repair mutations, sensitization to X rays was observed only for rich medium-grown cells. For cells grown in rich medium, the radC mutant was normal for gamma-radiation mutagenesis, but showed less uv-radiation mutagenesis than the wild-type strain; it showed normal amounts of X- and uv-radiation-induced DNA degradation, and it was approximately 60% deficient in recombination ability. The radC strain was normal for host cell reactivation of gamma-and uv-irradiated bacteriophage lambda; the radC mutation did not sensitize a recA strain, but did sensitize a radA and a polA strain to X and uv radiation and a uvrA strain to uv radiation. Therefore, we suggest that the radC gene product plays a role in the growth medium-dependent, recA gene-dependent repair of DNA single-strand breaks after X irradiation, and in postreplication repair after uv irradiation.

Role of ruvAB genes in UV- and γ-radiation and chemical mutagenesis in Escherichia coli

Abstract Escherichia coli umuC122 :: Tn5 was mutagenized with N -methyl- N ′-nitro- N -nitrosoguanidine to isolate mutations that block the residual γ-radiation mutagenesis observed in umuC strains. Two of these mutations were shown by transductional mapping and plasmid complementation to map in the ruvA and ruvB genes (i.e., ruvA200 and ruvB201 ). Whereas ruvA200 was complemented by ruvA + plasmids, the only other known ruvA mutation, ruvA59 :: Tn 10 required both the ruvA + and ruvB + genes to show complementation. The ruvA200 , ruvB201 , ruvA59 :: Tn 10 and ruvB60 :: Tn 10 mutations all reduced γ-radiation-induced ochre reversion [ argE3 (Oc) → Arg + ]] to about 30% of the wild-type level, and they all reduced UV-radiation-induced ochre reversion to about 15% of the wild-type level. The ruvA200 and ruvB201 mutants also showed reduced γ- and UV-radiation mutagenesis with two other assays [ hisG4 (Oc) → His + and Rif s → Rif r ]. Streptozotocin mutagenesis (Rif r was reduced to about half of the wild-type level in ruv strains, but ethyl methanesulfonate mutagenesis was normal. While the umuC strain did not show the oxygen enhancement of γ-radiation mutagenesis, the ruvA200 strain showed an oxygen effect that was similar to that shown by the wild-type strain. When the ruvA200 mutation was combined with the umuC mutation, γ-radiation mutagenesis was further reduced to 5% of the wild-type level and cells showed a synergistic sensitization to UV- and γ-radiation-induced killing. A mutational spectrum analysis indicates a general depression of both umuC -dependent and umuC -independent γ-radiation mutagenesis in the ruvA strain, which is in contrast with the site-specific reduction in γ-radiation mutagenesis that is observed in the umuC mutant. The reduced radiation mutagenesis in the ruvA strain could not be correlated with a reduction in transcription of the recA or umuC genes.

The Effect of Growth Conditions on Inducible, recA-Dependent Resistance to X Rays in Escherichia coli

Escherichia coli cells grown to logarithmic phase in, and plated on, rich medium (yeast extract-nutrient broth) were more resistant to X rays, ultraviolet (uv) radiation, and methyl methanesulfonate (MMS) than cells grown in, and plated on, minimal medium. We have called this enhanced survival capability medium-dependent resistance (MDR). The magnitude of MDR observed after oxic X irradiation was greater than that observed after anoxic X irradiation, uv irradiation, or MMS treatment. MDR was not observed in stationary-phase cells with X or uv radiation. MDR was associated with an increased ability to repair X-ray-induced DNA single-strand breaks, and with reduced X-ray-induced DNA degradation and protein synthesis retardation. Postirradiation protein synthesis was concluded to be critical in allowing the high X-ray survival associated with MDR, because of the large radiosensitization caused by a postirradiation growth medium shift down or treatment with rifampicin (RIF), recA protein must be at least one of the proteins whose synthesis is critical to MDR, as judged by the absence of MDR or a RIF effect in X-irradiated recA and lexA mutants. The results with X-irradiated temperature-conditional recA cells suggest that it is only after cells have been damaged that the recA gene plays a role in MDR.