Frameshift mutagenesis by ultraviolet and gamma radiations in Salmonella: SOS inducibility and effects of DNA polymerase I

Ultraviolet (UV) and gamma-induced mutagenesis have been studied using a doubly auxotrophic strain of Salmonella typhimurium carrying the amber leuA150 mutation (which reverts by base-pair substitution) and the frameshift hisC3076 marker (which reverts by compensating frameshifts). In the initially constructed LT2 background, both markers were poorly revertible by UV and essentially non-revertible by gamma-radiation. A derivative of this strain carrying the mutation-enhancing plasmid pKM101 was however readily reverted by both UV and gamma, with either Leu+ (base substitution) or His+ (frameshift) revertants being observed on appropriate selective media. Photoreactivation experiments suggested that the lesions leading to formation of the two types of mutagenic event were similar if not identical. Support for this suggestion was obtained when it was found that yields of both types of UV-induced revertant were significantly increased in an excision-deficient background, while no revertants of either type were found in a recA background. Yields of gamma-induced revertants were not greatly altered in a uvrB background, but were also reduced to zero (for both markers) in the recA background. These results are consistent with what has previously been well-documented for UV and gamma-induced base-pair substitution mutagenesis, and serve to emphasize the similarities between base-pair substitution mutagenesis and frameshift mutagenesis by these agents. There are differences, however, since although UV-induced reversion of the leuA150 marker was little affected and gamma-induced reversion of leuA150 was somewhat reduced in the presence of a polA mutation (polA3), the yields of His+ frameshift revertants were significantly increased in the polA3 background following treatment with either UV or gamma. Thus while inducible DNA repair (SOS repair) appears to be involved in generating both types of mutational event following either UV- or gamma-irradiation, at some stage in the processing of premutational lesions the level (or type) of DNA polymerase I activity in the cell seems to have an important role in determining whether or not frameshifts or base-pair substitutions will be produced at a particular frequency.     

Induction of genetic duplications and frameshift mutations in Salmonella typhimurium by acridines and acridine mustards: dependence on covalent binding of the mutagen to DNA

Summary The aroC321 allele permits positive selection for the detection of a large genetic duplication that arises in the Salmonella typhimurium chromosome by homologous recombination. Strains that contain both aroC321 and the hisC3076 allele were constructed so that the induction of genetic duplications and frameshift mutations in a run of GC base pairs could be studied simultaneously by selecting for tryptophan and histidine prototrophy, respectively. Using these strains, we examined the ability of 9-aminoacridine, quinacrine, four acridine mustards (ICR-170, ICR-191, ICR-372, and quinacrine mustard) and the nitroacridine Entozon to induce genetic duplications and frameshift mutations. Although all these compounds induce reversion of hisC3076, only the four mustards and Entozon are effective as inducers of genetic duplications under identical treatment conditions. The induction of genetic duplications by acridine mustards, like the toxic and mutagenic effects of these compounds, is enhanced by a deficiency for excision repair caused by a deletion through the uvrB gene. The ineffectiveness of 9-aminoacridine and quinacrine in the test for genetic duplications indicates that simple intercalation is sufficient for the mutagenic effect measured with the hisC3076 allele but that the induction of duplications by the acridine mustards and Entozon requires covalent binding of the chemical to DNA.     

Variation in the mutagenic potential of acridines in Salmonella typhimurium, with stage of the culture

The mutagenic potential of proflavine differed by up to two orders of magnitude in three strains of Salmonella typhimurium carrying the frameshift marker hisC3076, depending upon the stage of the culture. In cultures at a specific stage, proflavine showed decreased mutagenesis in a recA as compared with a wildtupe or uvrB derivative. The related compound 9-aminoacridine also showed marked variation in mutagenic potential with the stage of the bacterial culture, but was equally mutagenic in all three strains, at least in log phase cells. Within the literature, there are conflicting data regarding the mutagenicity of acridines, and the contribution of repair enzymes to this event. We suggest that many of the reported differences can be related to the variations in bacterial growth conditions in different laboratories, and urge authors to supply more details of these.     

Spontaneous and induced mutability or frameshift strains of Salmonella typhimurium carrying uvrB and polA mutations

Three strains Salmonella typhimurium carrying frameshift mutations affecting the histidine genes (hisC3076, hisD3052 and hisC207) showed increased sensitivity to mutagenesis by ICR-191 (as judged by measuring back mutation to prototrophy), if they were made deficient in excision repair by deleting the uvrB gene. One frameshift strain, hisC3076, also showed increased sensitivity to mutagenesis by ICR-191 when it carried either of two different polA alleles, whereas the hidD305 and hisD207 frameshifts reduced sensitivity to mutagenesis in the presence of these alleles. Studies of spontaneous back mutation to prototrophy revealed siginificant mutator effects of the polA1 mutation on reversion of the hisD3052 frameshift and of the polA3 mutation on reversion of the hisC3076 frameshift. Other smaller mutator effects of the polA alleles on reversion of the his mutations may also be present. In an attempt to explain the complex interactions between different polA alleles and different frameshift mutations, it is tentatively suggested that deletion frameshift may arise mainly during DNA replication, while addition frameshifts may arise mainly during post-replication repair.     

P22 antirepressor protein prevents in vivo recA-dependent proteolysis of P22 repressor

A method was developed to demonstrate recA-dependent P22-repressor breakdown in vivo by SDS-polyacrylamide electrophoresis of unfractionated extracts of phage-infected, lysogenic Salmonella typhimurium strains TA1530 rec+ and TA1530 recA1-. The antirepressor of P22 is not cleaved by recA protein. Under conditions of unregulated ant-overproduction (Harvey et al. 1981) antirepressor protects c2-repressor in vivo against proteolytic cleavage by recA protein.     

Mutagenic activity of nitracrine derivatives in Salmonella typhimurium: relationship to drug physicochemical parameters, and to bacterial uvrB and recA genes and plasmid pKM101

To determine whether it is possible to separate antitumour and mutagenic properties in the nitracrine series, a number of 4-substituted derivatives of the hypoxia-selective drug nitracrine have been evaluated for their mutagenic effects at three loci in several strains of Salmonella typhimurium differing in DNA-repair capacity (uvrB, recA, plasmid pKM101). The drugs divided into two series in terms of their biological effects. Group A compounds (nitracrine and its Cl, F, Me and OMe derivatives) were very toxic to bacteria, and uvrB and recA deletions enhanced toxicity by 10-80-fold. Mutagenic potency was high, being slightly enhanced by uvrB and reduced by recA deletions. In contrast the toxicities and mutagenic potentials of Group B compounds (COOMe, NMe2, and two other bulky amine derivatives) were reduced by at least an order of magnitude, with uvrB and recA deletions showing lesser influence. The COOMe derivative was the only compound showing greater effects at the hisC3076 locus than the hisD3052 or hisG46 loci. The data suggest that all the compounds cause mutations through intercalation and/or monoadduct formation, but only for the COOMe derivative is intercalation the dominant mode of action. Group A compounds appear to have the additional ability to cross-link DNA, a property which amounts for their high potency but which is not compatible with bulky 4-substituents. Apart from these generalizations, there was considerable variation in mutagenic efficiency (as measured by the maximum numbers of revertant colonies) within each series. Of the compounds studied, the 4-OMe derivative appears to best retain the desirable antitumour properties of nitracrine while showing greatly-reduced mutagenic potential, and is an interesting lead for further development.     

Frameshift mutagenesis by nitracrine analogues in wild-type, uvrB, polA and recA strains of Salmonella typhimurium, with and without plasmid pKM101

The mutagenic potential of 9-[(3-dimethylaminopropyl)amino]-acridine and its 1-, 2-, 3- and 4-nitro derivatives was studied in several strains of Salmonella typhimurium carrying the frameshift marker hisC3076. The strains all carried deep rough (rfa) mutations, and were either wild-type with respect to DNA repair capacity or carried recA, uvrB, polA1 or polA3 (amber) mutations. Derivatives with and without plasmid pKM101 were also studied. The des-nitro compound resembled 9 aminoacridine and other simple intercalating compounds. Both toxicity and mutagenesis were apparently unaffected by the uvrB and recA mutations or by the presence of plasmid pKM101. However, mutagenicity was reduced by the polA1 mutation, and virtually eliminated by the polA3 mutation. The drug was substantially more toxic in the latter, slightly more toxic in the former, of these polA- strains. Plasmid pKM101 enhanced mutagenesis and protected from toxicity in both polA1- and polA3- strains, although it did not restore either of these parameters to the level in the wild-type strain. The 2-nitro compound was generally similar to the des-nitro compound, except that it was considerably more toxic and apparently non-mutagenic in the recA-bearing strain. By contrast, mutagenicity of the 3- and 4-nitro compounds was enhanced by the uvrB mutation and by the presence of the plasmid. These compounds were highly toxic but non-mutagenic in the recA- strain, and showed some increased toxicity in polA1- and polA3- strains. The 1-nitro compound has been previously found to cross-link DNA. Unlike well-characterised cross-linkers such as mitomycin C it was highly mutagenic in the uvrB- strain, and this mutagenesis was enhanced by plasmid pKM101, but eliminated by the recA mutation. At high doses, where the drug was completely toxic towards uvrB- or recA-carrying strains, it became mutagenic in the DNA-repair-proficient strains. This 'high-dose' mutagenesis was enhanced by plasmid pKM101, but reduced by the polA1 mutation and almost eliminated by the polA3 mutation. Although there are several possible interpretations of these data, they are compatible with the suggestion that the lesion induced by high doses (but not by low doses) of nitracrine is a cross-link, but that this is not the major mutagenic lesion.     

recA-dependent and recA-independent N-ethyl-N-nitrosourea mutagenesis at a plasmid-encoded herpes simplex virus thymidine kinase gene in Escherichia coli

We have compared isogenic recA13/recA+ Escherichia coli K-12 strains for the induction by N-ethyl-N-nitrosourea (ENU) of forward mutations at a plasmid-encoded herpes simplex virus type 1 thymidine kinase (HSV-tk) gene. Treatment of plasmid-bearing bacteria with ENU resulted in a dose-dependent increase in the mutant frequencies of the chromosomal udk locus and of the plasmid HSV-tk locus in both recA13 and recA+ strains. Although the recA13 strain was considerably more sensitive to the cytotoxic effects of ENU treatment than was the recA+ strain, the ENU-induced mutation frequency at both loci was greater for the recA+ strain than for the recA13 strain. When plasmid DNA modified by in vitro reaction with ENU was used to transform recA13, recA+, and UV pre-irradiated recA+ strains, an increase in the HSV-tk mutant frequency was observed in all 3 cases. The induction of mutations in recA13 and recA+ strains followed a similar dose-response, while the ENU-induced HSV-tk mutant frequency was significantly greater for UV pre-irradiated recA+ bacteria. These results indicate that fixation of ENU-induced premutagenic lesions can occur by both recA-dependent and recA-independent pathways.     

Mutational spectrum analysis of umuC-independent and umuC-dependent gamma-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.     

Mutagenesis of anaerobic cultures of Salmonella typhimurium by nitrosoguanidine, diethyl sulfate and 9-aminoacridine

Despite a previous report to the contrary, anaerobic cultures of Salmonella typhimurium strain LT2 hisG46 are revertible (although to a slightly reduced extent) by both N-methyl-N'-nitro-N-nitrosoguanidine and diethyl sulfate, while anaerobic cultures of a strain carrying the frameshift hisC3076 marker are fully revertible by 9-aminoacridine.     

Mutagenicity of coolwhite fluorescent light for Salmonella

The most common fluorescent lamps in use today in homes and businesses in the United States, 'coolwhite' fluorescent lamps, emit light that is mutagenic for Salmonella. Strains that carry both a uvrB mutation and plasmid pKM101 are extremely susceptible to this light-induced mutation. Both base substitution and frameshift mutations can be induced without substantial lethal effects on the bacteria. Induced mutations accumulate essentially as a linear function of the time bacteria are exposed to illumination. Of Salmonella histidine-requiring strains with known nucleotide target sequences (Hartman et al., 1986; Cebula and Koch, 1989, 1990), strains either carrying one of the base substitution mutations, hisG428 and hisG46, or one of the frameshifts, hisC3076 and hisD6610, are most highly mutagenized whereas frameshift strains with hisD6580 and hisD3052 exhibit lower rates of mutagenesis. Mutagenicity does not appear to require the presence of oxygen. A filter blocking wavelengths below 370 nm eliminates mutagenesis. Polystyrene, cellulose acetate and, especially, mylar and glass filters reduce mutagenesis, indicating that at least some of the mutagenic effects can be attributed to leakage of radiations below 290 nm (far-ultraviolet light) from 'coolwhite' lamps. The more recently introduced fluorescent 'softwhite' lamps are roughly 10-fold less mutagenic at approximately equal light intensity. Incandescent light bulbs are much less mutagenic than are these fluorescent lamps. Our mutational data correlate closely with previous results in eukaryotic cells (Jacobson and Krell, 1982). A uvrB recA Salmonella double mutant is hypersensitive to the lethal effects of coolwhite fluorescent light, even when illuminated through the lids of glass Petri dishes. Thus, appropriate Salmonella strains would appear to be simple and useful screens for both the mutagenic and the lethal activities of fluorescent lamps. These systems are amenable to classroom laboratory use as relatively safe and effective means of demonstrating environmental mutagenesis.     

The mutA mistranslator tRNA-induced mutator phenotype requires recA and recB genes, but not the derepression of lexA-regulated functions

The mapping of mutA and mutC mutator alleles to the glyV and glyW glycine tRNA genes, respectively, and the subsequent discovery that the mutA phenotype is abolished in a DeltarecA strain raise the possibility that asp --> gly misinsertion may induce a novel mutagenic pathway. The recA requirement suggests three possibilities: (i) the SOS mutagenesis pathway is activated in mutA cells; (ii) loss of recA function interferes with mutA-promoted asp --> gly misinsertion; or (iii) a hitherto unrecognized recA-dependent mutagenic pathway is activated by translational stress. By assaying the expression levels of a reporter plasmid bearing a umuC :lacZ fusion, we show that the SOS regulon is not in a derepressed state in mutA cells. Neither overexpression of the lexA gene through a multicopy plasmid nor replacement of the wild-type lexA allele with the lexA1[Ind-] allele interferes with the expression of the mutA phenotype. The mutA phenotype is unaffected in cells defective for dinB, as shown here, and is unaffected in cells defective for umuD and umuC genes, as shown previously. We show that mutA-promoted asp --> gly misinsertion occurs in recA- cells and, therefore, the requirement for recA is 'downstream' of mistranslation. Finally, we show that the mutA phenotype is abolished in cells deficient for recB, suggesting that cellular recombination functions may be required for the expression of the mutator phenotype. We propose that translational stress induces a previously unrecognized mutagenic pathway in Escherichia coli.     

An alternative pathway of recombination of chromosomal fragments precedes recA-dependent recombination in the radioresistant bacterium Deinococcus radiodurans.

Deinococcus radiodurans R1 and other members of this genus are able to repair and survive extreme DNA damage induced by ionizing radiation and many other DNA-damaging agents. The ability of R1 to repair completely > 100 double-strand breaks in its chromosome without lethality or mutagenesis is recA dependent. However, during the first 1.5 h after irradiation, recA+ and recA cells show similar increases in the average size of chromosomal fragments. In recA+ cells, DNA continues to enlarge to wild-type size within 29 h. However, in recA cells, no DNA repair is observed following the first 1.5 h postirradiation. This recA-independent effect was studied further, using two slightly different Escherichia coli plasmids forming adjacent duplication insertions in the chromosome, providing repetitive sequences suitable for circularization by non-recA-dependent pathways following irradiation. After exposure to 1.75 Mrad (17,500 Gy), circular derivatives of the integration units were detected in both recA+ and recA cells. These DNA circles were formed in the first 1.5 h postirradiation, several hours before the onset of detectable recA-dependent homologous recombination. By comparison, D. radiodurans strains containing the same E. coli plasmids as nonrepetitive direct insertions did not form circular derivatives of the integration units before or after irradiation in recA+ or recA cells. The circular derivatives of the tandemly integrated plasmids were formed before the onset of recA-dependent repair and have structures consistent with the hypothesis that DNA repair occurring immediately postirradiation is by a recA-independent single-strand annealing reaction and may be a preparatory step for further DNA repair in wild-type D. radiodurans.     

Dark Recovery Processes in Escherichia coli Irradiated with Ultraviolet Light I. Effect of rec− Mutations on Liquid Holding Recovery

We have examined various derivatives of Escherichia coli K-12 for liquid holding recovery, a type of recovery originally observed in E. coli B irradiated with ultraviolet light. Although most of the K-12 derivatives tested showed relatively little or no recovery under our conditions, four of the six independent rec(-) mutants examined, those carrying recA1, rec-12, recA13, and rec-56, respectively, displayed marked recovery. These mutants are distinguished from rec(+) strains by their increased sensitivity to ultraviolet radiation and decreased ability to undergo genetic recombination. Two of them have also been reported to release large amounts of their deoxyribonucleic acid as acid-soluble material, especially after irradiation. None of the three uvr(-) mutants examined, containing uvrA6, uvrB5, or uvrC34, showed comparable liquid holding recovery. The one rec(-) uvr(-) derivative tested, carrying recA13 and uvrA6, did not appear to undergo liquid holding recovery, although recA13 uvr(+) strains did. Genetic analysis of one strain, a recA13 mutant, indicated that all the rec(+) derivatives obtained from it by conjugation, transduction and reversion, had lost the property of showing liquid holding recovery. From these results, we conclude that in E. coli K-12 the expression of liquid holding recovery depends upon certain rec(-) mutations.     

Comparative mutagenesis and interaction between near-ultraviolet (313- to 405-nm) and far-ultraviolet (254-nm) radiation in Escherichia coli strains with differing repair capabilities.

Comparative mutagenesis and possible synergistic interaction between broad-spectrum (313- to 405-nm) near-ultraviolet (black light bulb [BLB]) radiation and 254-nm radiation were studied in Escherichia coli strains WP2 (wild type), WP2s (uvrA), WP10 (recA), WP6 (polA), WP6s (polA uvrA), WP100 (uvrA recA), and WP5 (lexA). With BLB radiation, strains WP2s and WP6s demonstrated a high level of mutagenesis, whereas strains WP2, WP5, WP6, WP10, and WP100 did not demonstrate significant mutagenesis. In contrast, 254-nm radiation was mutagenic in strains WP2, WP2s, WP6, and WP6s, but strains WP5, WP10, and WP100 were not significantly mutated. The absence of mutagenesis by BLB radiation in lexA and recA strains WP10, WP5, and WP100 suggests that lex+ rec+ repair may play a major role in mutagenesis by both BLB and 254-nm radiation. The hypothesis that BLB radiation selectively inhibits rec+ lex+ repair was tested by sequential BLB-254-nm radiation. With strain WP2, a fluence of 30 J/m2 at 254 nm induced trp+ revertants at a frequency of 15 X 10(-6). However, when 10(5) J/m2 or more of BLB radiation preceded the 254-nm exposure, no trp+ revertants could be detected. A similar inhibition of 254-nm mutagenesis was observed with strain WP6 (polA). However, strains WP2s (uvrA) and wP6s (polA uvrA) showed enhanced 254-nm mutagenesis when a prior exposure to BLB radiation was given.     

Genetic control of heat resistance and thermotolerance by recA and uvrA in E. coli K12

Several recA and uvrA derivatives of E. coli K12 AB1157 develop a transient increase in heat resistance, i.e. induced thermotolerance after a brief exposure to 43.5 degrees C (less than 1 h). Thermotolerance was identified from the appearance of an inflection in the survival curve or from the loss of heat resistance in the presence of chloramphenicol (CAM) or rifampicin. Heat resistance and induced thermotolerance were enhanced by recA and uvrA gene functions and their contribution was roughly as follows: AB1157 (recA+ uvrA+) greater than AB2463 (recA- uvrA+) greater than AB1886 (recA+ uvrA-) greater than AB2480 (recA- uvrA-). In heat resistance, uvrA and recA contributed approximately equally and their effects were additive. Induced thermotolerance developed sooner and was maintained at a higher level in the presence of uvrA as compared with recA. Since uvrA-dependent excision repair is scheduled prior to recA-dependent (postreplication) repair, induction of thermotolerance may be linked to DNA repair. Although recA and uvrA play a distinct role, they are not essential, and thermotolerance can develop in the absence of either one or both of these gene functions. Furthermore, since thermotolerance can be induced in recA mutants (AB2463 and AB2480), its biochemical pathway must be different from that of the recA-dependent SOS system.     

Molecular analysis of the Deinococcus radiodurans recA locus and identification of a mutation site in a DNA repair-deficient mutant, rec30

Deinococcus radiodurans strain rec30, which is a DNA damage repair-deficient mutant, has been estimated to be defective in the deinococcal recA gene. To identify the mutation site of strain rec30 and obtain information about the region flanking the gene, a 4.4-kb fragment carrying the wild-type recA gene was sequenced. It was revealed that the recA locus forms a polycistronic operon with the preceding cistrons (orf105a and orf105b). Predicted amino acid sequences of orf105a and orf105b showed substantial similarity to the competence-damage inducible protein (cinA gene product) from Streptococcus pneumoniae and the 2'-5' RNA ligase from Escherichia coli, respectively. By analyzing polymerase chain reaction (PCR) fragments derived from the genomic DNA of strain rec30, the mutation site in the strain was identified as a single G:C to A:T transition which causes an amino acid substitution at position 224 (Gly to Ser) of the deinococcal RecA protein. Furthermore, we succeeded in expressing both the wild-type and mutant recA genes of D. radiodurans in E. coli without any obvious toxicity or death. The gamma-ray resistance of an E. coli recA1 strain was fully restored by the expression of the wild-type recA gene of D. radiodurans that was cloned in an E. coli vector plasmid. This result is consistent with evidence that RecA proteins from many bacterial species can functionally complement E. coli recA mutants. In contrast with the wild-type gene, the mutant recA gene derived from strain rec30 did not complement E. coli recA1, suggesting that the mutant RecA protein lacks functional activity for recombinational repair.     

Influence of a uvrD mutation on survival and repair of X-irradiated Escherichia coli K-12 cells.

The presence of a uvrD mutation increased the X-ray sensitivities of E. coli wild-type and polA strains, but had no effect on the sensitivities of recA and recB strains, and little effect on a lexA strain. Incubation of irradiated cells in medium containing 2,4-dinitrophenol or chloramphenicol decreased the survival of wild-type and uvrD cells, but had no effect on the survival of recA, recB and lexA strains. Alkaline sucrose gradient sedimentation studies indicated that the uvrD strain is deficient in the growth-medium-dependent (Type III) repair of DNA single-strand breaks. These results indicate that the uvrD mutation inhibits certain rec+lex+-dependent repair processes, including the growth-medium-dependent (Type III) repair of X-ray-induced DNA single-strand breaks, but does not inhibit other rec+lex+-dependent processes that are sensitive to 2,4-dinitrophenol and chloramphenicol.     

Characterization of RecA424 and RecA670 proteins from Deinococcus radiodurans.

RecA protein is considered to be the most important participant in the radiation resistance of Deinococcus radiodurans. However, it is still unclear how RecA contributes to the resistance. In this study, we identified a new recA mutation (recA424) in the DNA-repair deficient mutant strain KI696, the phenotype of which is remarkably different from mutant strain rec30 carrying recA670. The properties of the gene products from the recA mutants were compared. recA424 could not complement the deficiency in Escherichia coli RecA, as found for recA670. In vitro, neither RecA424 nor RecA670 could promote DNA strand exchange under conditions in which wild-type RecA promoted the reaction, indicating that both RecA424 and Rec670 are defective in recombination activity. RecA424 promoted the autocleavage reaction of LexA in vitro, whereas RecA670 did not. The intracellular LexA level in KI696 was decreased following gamma-irradiation. However, the LexA level in strain rec30 was constant irrespective of irradiation. These results indicate that RecA424 retains co-protease activity, whereas RecA670 does not. While strain rec30 is extremely radiation sensitive, strain KI696 is only slightly sensitive. Together, these observations suggest that the co-protease activity rather than the recombination activity of RecA contributes to radiation resistance in D. radiodurans.     

5-Azacytidine: survival and induction of the SOS response in Escherichia coli K-12

Survival and induction of the SOS system by 5-azacytidine, an analog of cytidine, were studied in Escherichia coli K-12. This compound did not produce any effect on the viability of dcm and dam dcm mutants. Furthermore, recA430 and lexA1 strains (both mutations interfere with LexA repressor cleavage but not recombination proficiency) were more resistant than the wild-type strain of E. coli K-12. In contrast, recBC and recA13 mutants were more sensitive to 5-azacytidine than the wild type. Transient exposure of E. coli to 5-azacytidine for 60 min induced both recA-dependent inhibition of cell division and induction of lambda prophage in Dcm+ strains but not in Dcm- mutants. Expression of both functions was dependent on recBC exonuclease. On the other hand, 5-azacytidine was unable to trigger the induction of umuCD and mucB genes and no amplification of RecA protein synthesis in either Dcm+ or Dcm- strains was observed. These last results are in agreement with previously reported data suggesting that there is a discrimination in the expression of the several SOS functions and that some SOS genes may be induced without amplification of RecA protein synthesis.