Mutagenic DNA repair in Escherichia coli. VIII. Involvement of DNA polymerase III in constitutive and inducible mutagenic repair after ultraviolet and gamma irradiation.

Summary Mutagenic repair in Escherichia coli after ultraviolet (UV) irradiation has previously been shown to require a function of DNA polymerase III. In contrast, no effect of incubating a polC temperature-sensitive strain at 42° has been found after gamma irradiation. Thus at present there is no direct evidence for the involvement of polymerase III in gamma ray mutagenesis. This could, however, merely reflect the stability of the premutational lesion during the period of polymerase III insufficiency such that mutagenic repair is resumed on the plate during subsequent incubation at permissive temperature.It was previously suggested that an inducible factor might interact with polymerase III to enable it to polymerise in an error-prone way in daughter strand gaps opposite non-coding lesions in the template strand. A temperature-resistant revertant (CM 792) of a temperature-sensitive polC strain (CM 731) has been isolated which has properties expected of a strain in which the polymerase III complex is no longer susceptible to the inducible co-factor. Its UV sensitivity, spontaneous mutation rate and mutagenic response to ethyl methanesulphonate are all normal or near normal, also the rates of mutation to prototrophy after gamma irradiation and to streptomycin resistance after UV. These latter mutations are believed to arise through constitutive mutagenic repair at sites in pre-existing DNA. In contrast, the rate of UV-induced mutation to prototrophy due to changes at ochre suppressor loci is greatly depressed and no Weigle-reactivation of bacteriophage T3 is observable; both these effects are believed to result from the action of inducible mutagenic repair in newly-replicated DNA. It is suggested that the 3 to 5 exnnuclease activity of the polymerase III complex in CM 792 may not be susceptible to inhibition by an inducible factor and so continues to remove mismatched bases inserted in newly-replicated DNA opposite damage template sites thus preventing the fixation of errors as mutations.     

Mutagenic DNA repair in escherichia coli. V. Mutation frequency decline and error-free post-replication repair in an excision-proficient strain.

Mutation frequency decline (MFD) is an irreversible loss of newly-induced suppressor mutations occurring in excision-proficient Escherichia coli during a short period of incubation in minimal medium before plating on broth- or Casamino acids-enriched selective agar. It is known that MFD of UV-induced mutations may occur before DNA containing pre-mutagenic lesions is replicated, but we conclude that MFD can also occur after the damaged DNA has been replicated on the basis of the following evidence. (1) Mutation fixation in rich medium (i.e., loss of susceptibility to mutation frequency decline) with ethyl methanesulphonate mutagenesis begins immediately, whereas with UV it is delayed for 20--30 min. (2) The delay in mutation fixation after UV can be explained neither by inhibition of DNA replication nor by a delay in the appearance of error-prone repair activity in the irradiated population. (3) MFD at later times after UV irradiation is more rapid and is less strongly inhibited by caffeine than is MFD immediately after irradiation. (4) Excision is virtually complete 20 min after 3 J m-2 UV but at that time virtually all mutations are still susceptible to MFD. We have presented evidence elsewhere that in bacteria there is an alternative error-free excision-dependent type of post-replication repair of potentially mutagenic daughter strand gaps. We suggest that this process is inhibited at tRNA loci in the presence of nutrient broth or Casamino acids, possibly because of a broth-dependent change in the structure of the single-stranded region including the tRNA locus.     

Mutation induction by thymine deprivation in Escherichia coli B/R : II. Mutation in the repressed and derpressed tryptophan operon

True Trp⁺ reversions are induced by thymine deprivation in cells with repressed trp operons as efficiently as in derepressed cells. At least part of the mutations are fixed during thymine starvation, i.e. in the absence of net DNA synthesis. The hypothesis is put forward that thymineless mutagenesis is due to repair-replication under limited concentrations of 5′-dTTP, performed by an inducible error-prone “DNA-polymerizing activity” on single-strand gaps.     

Correlation between survival,ability to rejoin DNA and stability of DNA after preirradiation inhibition of protein synthesis in arec - mutant ofEscherichia coli K12

A 90 min inhibition of protein synthesis induced by starvation for amino acids (AA^-) or by treatment with chloramphenicol (CAP) prior to UV irradiation (2.5 J m^-2) increased the resistance of the strainEscherichia coli K12 SR19 to UV radiation more than ten-fold. Under these conditions, cultures in which protein synthesis was inhibited before the UV irradiation rejoin short regions of DNA synthesized after the irradiation to a normal-size molecule, whereas an exponentially growing culture does not rejoin DNA synthesized after UV irradiation to a molecule of a normal size. In the exponentially growing culture both the parental and the newly synthesized DNA are unstable after the irradiation. In cultures with inhibited protein synthesis only the parental DNA is somewhat unstable. InEscherichia coli K12 SR19 where protein synthesis was inhibited before the irradiation, a correlation between the survival of cells, the ability to rejoin short regions of DNA synthesized after UV irradiation and a higher stability of both parental and newly synthesized DNAs could be demonstrated.     

DNA repair in Proteus mirabilis. IV. Post-irradiation DNA degradation as influenced by a function inducible in rec+ cells.

Summary Post-irradiation DNA degradation in P. mirabilis rec ⁺ strains after UV irradiation is found to be more extensive in starvation buffer than in growth medium. In growth medium restriction of protein synthesis, but not DNA synthesis, largely prevents the expression of breakdown limitation. By the addition of chloramphenicol during post-irradiation incubation in growth medium the expression of break-down limitation was followed and found to occur 20 to 40 min after UV irradiation. Pre-irradiation by a low dose of UV leads after a corresponding time of post-irradiation incubation to breakdown limitation even in starvation buffer after a second UV exposure.Post-irradiation DNA degradation is presumed to be initiated at the sites of DNA lesions which arise at replication points damaged by UV. While pre-starvation restricts the efficiency of postirradiation DNA degradation by the reduction of the number of replication points active at the time of irradiation, caffeine as well as 2,4-dinitrophenol inhibit DNA degradation even in rec ^- cells probably by the interference with nicking or exonucleoltytic events initiated at those sites in the absence of breakdown limitation.Breakdown limitation is postulated to be due to inducible derepression of REC-functions which lead to the protection and, probably, repair of DNA lesions arising at the replication points following UV exposure.     

Influence of streptomycin on UV-induced TRP+ reversions in a streptomycin-resistant strain of E. Coli B/R

UV mutagenesis has been compared in the E. coli B/r trp^? WWP-2 Hcr⁺ and in a mutant (Stm^R 28) resistant to 100 μg streptomycin/ml. In the Stm^R strain it is possible to score UV-induced Trp⁺ reversions, and survival, on media supplemented with, or lacking, streptomycin. Such experiments revealed a marked enhancement of mutagenic effect of UV by streptomycin. On analysis it was shown that in the Stm^R strain a fraction of UV-induced Trp⁺ reversions, dut to ochre suppressor, achieve full phenotypic expression only in the presence of streptomycin. Thus an inhibitory effect of the Stm^R 28 mutation on the activity of some ochre suppressors is relieved by streptomycin.     

UV-inducible repair: influence on survival, dimer excision, DNA replication and breakdown in Escherichia coli B/r Her+ cells.

Summary Using a model of double-UV-irradiation with inducing¹ (non-lethal) and lethal fluences² we have studied involvement of UV-inducible functions in post-UV-irradiation restoration processes and survival of Escherichia coli B/r thy ^- thy ^- Hcr⁺. Cells irradiated with both inducing and lethal fluences differed from cells irradiated with lethal fluence in the following respects: They were more UV resistant; they did not die during postincubation with chloramphenicol³; they exhibited a significant reduction in dimer excision; they were able to resume DNA replication and produce normal-sized DNA molecules in the presence of chloramphenicol. Since induction was provoked in cell prestarved for amino acids it was not associated with damage to points active in replication. However, the inducible product was more important for repair of replicating than non-replicating cells. The data indicate that protein necessary for resumption of DNA synthesis after UV is not constitutive but inducible.Abbreviations ¹IF inducing fluence - ²IF lethal fluence - ³CAP chloramphenicol     

Increase in the fraction of non-repaired mutations in Escherichia coli cells, incubated in the absence of glucose after UV-irradiation

In E. coli WP2 trpE65 cells irradiated with UV-dose of 11 J/m2, the additional small portion of induced Trp+ mutations became resistant to photoreactivation or "dark" (excision) repair after a short-termed (10-30 min) postirradiation incubation of bacteria in a minimal medium deprived of glucose and tryptophan. Since protein synthesis could not proceed in those cells because of the lack of energy and tryptophan, the data indicate that an unknown mechanism exists which imparts some mutations with the resistance to antimutagenic repair in the absence of the inducible mutagenic system. In the light of this result, one could suggest that the normal process of mutation fixation (that is the loss of sensitivity of mutations to photoreactivation or to excision repair in cells incubated in growth medium after irradiation) should not necessarily be a direct consequence of manifestation of the activity of an inducible mutagenic system.     

Induction of protein X in Escherichia coli.

Summary Certain treatments that damage DNA and/or inhibit replication in E. coli have been reported to induce synthesis of a new protein, termed protein X, in recA ⁺ lexA ⁺ strains. We have examined some of the treatments that might induce protein X and we have, in particular, tested the hypothesis of Gudas and Pardee (1975) that DNA degradation products play an essential role in the induction process.We confirmed that UV irradiation, nalidixic acid treatment, or thymine starvation result in protein X synthesis in wild type strains. However, we found that UV irradiation, unlike nalidixic acid, also induced protein X in recB strains, in which little DNA degradation occurs. Furthermore, we found that the presence of DNA fragments resulting from host-controlled restriction of phage DNA did not affect protein X synthesis. We conclude that no causal relationship exists between the production of DNA fragments and induction of protein X.The presence of the plasmid R46, which confers enhanced mutagenesis and UV resistance on its host, did not affect protein X synthesis. Growth in the presence of 5-bromouracil, which does not result in production of degradation fragments, resulted eventually in a low rate of protein X synthesis. In dnaA mutants, deficient in the initiation of new rounds of replication, UV irradiation induced protein X, again unlike nalidixic acid. Thus, the inhibition of active replication forks is not an essential requirement for protein X induction.     

Replication of Damaged DNA and the Molecular Mechanism of Ultraviolet Light Mutagenesis

Abstract

On UV irradiation of Escherichia coli cells, DNA replication is transiently arrested to allow removal of DNA damage by DNA repair mechanisms. This is followed by a resumption of DNA replication, a major recovery function whose mechanism is poorly understood. During the post-UV irradiation period the SOS stress response is induced, giving rise to a multiplicity of phenomena, including UV mutagenesis. The prevailing model is that UV mutagenesis occurs by the filling in of single-stranded DNA gaps present opposite UV lesions in the irradiated chromosome. These gaps can be formed by the activity of DNA replication or repair on the damaged DNA. The gap filling involves polymerization through UV lesions (also termed bypass synthesis or error-prone repair) by DNA polymerase III. The primary source of mutations is the incorporation of incorrect nucleotides opposite lesions. UV mutagenesis is a genetically regulated process, and it requires the SOS-inducible proteins RecA, UmuD, and UmuC. It may represent a minor repair pathway or a genetic program to accelerate evolution of cells under environmental stress conditions.     

Recombined molecules of mitochondrial DNA obtained from crosses between cytoplasmic petite mutants of Saccharomyces cerevisiae: The stoichiometry of parental DNA repeats within the recombined molecule

Summary Ultraviolet mutagenesis of phage is produced by host functions which are inducible by ultraviolet irradiation of the host cell. Induction kinetics and the half life of the inducible mutagenic DNA repair (SOS-repair) in E. coli have been determined using phage assays. At 37°C, both mutagenic and repair activities are maximal approximately 30 min following irradiation and decay with a half life of approximately 30 min. The presence of 100 g/ml chloramphenicol during the first 40 min after irradiation completely abolishes induction of repair and mutagenesis. The ultraviolet induction pattern of SOS repair very much resembles that of prophage in lysogenic induction (Monk and Kinross, 1975).     

Repair promoted by plasmid pKM101 is different from SOS repair.

In E. coli K12 bacteria carrying plasmid pKM101, prophage lambda was induced at UV doses higher than in plasmid-less parental bacteria. UV-induced reactivation per se was less effective. Bacteria with pKM101 showed no alteration in their division cycle. Plasmid pKM101 coded for a constitutive error-prone repair different from the inducible error-prone repair called SOS repair. Plasmid pKM101 protected E. coli bacteria from UV damage but slightly sensitized them to X-ray lesions. Protection against UV damage was effective in mutant bacteria deficient in DNA excision-repair provided that the recA, lexA and uvrE genes were functional. Survival of phages lambda and S13 after UV irradiation was enhanced in bacteria carrying plasmid pKM101; phage lambda mutagenesis was also increased. Plasmid pKM101 repaired potentially lethal DNA lesions, although wild-type DNA sequences may not necessarily be restored; hence the mutations observed are the traces of the original DNA lesions.     

Indirect and intragenic suppression of the lexA102 mutation in E. coli B/r.

Summary In Escherichia coli B/r the expression of UV inducible (SOS) functions is under the control of the recA and lexA genes. In this study we have characterized mutants which are altered in their ability to express SOS functions. These mutants were isolated as UV resistant UV nonmutable (Rnm) derivatives of the lexA102 uvrA155 mutant strain WP51. The UV resistance of these Rnm strains is a result of the suppression of lexA102 mediated UV sensitivity. Genetic mapping of rnm mutations shows that the two predominant classes, rnmA and rnmB, map in or very near the lexA and recA genes respectively. rnmA mutations differ from rnmB with respectively recA protein synthesis. rnmA mutations do not restore the ability to express high levels of recA protein after UV treatment whereas rnmB mutations result in constitutive expression of high levels of recA protein. However, both rnmA and rnmB mutant strains inhibit postirradiation DNA degradation. This shows that in rnmA strains, high levels of recA protein are not needed to inhibit postirradiation DNA degradation.The genetic map location and constitutive expression of recA protein synthesis resulting from rnmB mutations suggests that they are operator constitutive mutations of the recA gene. The result that the lexA ⁺ gene is required for the expression of UV mutagenesis in rnmB mutants shows that high levels of recA protein do not circumvent the need for the lexA ⁺ gene product in this process. Thus, while the lexA gene product is required for the induction of recA protein synthesis, lexA must have an additional role in UV induced mutagenesis.     

Kinetics of induction of error-prone repair of bacteriophage lambda by temperature shift in an Escherichia coli dnaB mutant.

Summary Preincubation at 42^o, before infection at permissive temperature by phage , of an Escherichia coli dnaB mutant, provokes a significant increase in survival and mutagenesis of ultraviolet irradiated phage as well as mutagenesis of untreated phage. Similarly to UV irradiation and many chemical mutagens, the inhibition of DNA synthesis by temperature shift of this dnaB mutant induces SOS repair. This work shows that replication blockage in bacterial DNA is not only mutagenic for bacterial DNA itself (Witkin, 1975) but also for normally replicating DNA, probably due to induction of diffusible products.     

Repair and plasmid R46 mediated mutation requires inducible functions in Proteus mirabilis

Summary In Proteus mirabilis nalidixic acid or a predose of UV induce Rec protein formation, a portion of post-UV replication repair and post-UV replication enhancement. These inducible functions are not significantly affected by the plasmid R46, which renders P. mirabilis efficiently UV-mutable. The R46-mediated UV induction of rif ^r mutations requires additional inducible functions, as existing after malidixic acid treatment in rec ⁺ strains. After a nalidixic acid pretreatment UV efficient induction of rif ^r mutations occurs without an otherwise obligatory period of post-UV incubation prior to plating on rifampicin agar. The inducible character of this qualification of plasmid R46-mediated UV mutagenesis in P. mirabilis is evident from the inhibitory effects of chloramphenicol and starvation. Constitutive high-level synthesis of Rec protein in cells harboring the recombinant (multi-copy) rec ⁺ plasmid pPM1 reduced plasmid R46-mediated UV mutagenesis, probably by preventing (inducible?) functions required by the plasmid R46 repair-mutator.     

Strand specificity for UV-induced DNA repair and mutations in the Chinese hamster HPRT gene.

DNA excision repair modulates the mutagenic effect of many genotoxic agents. The recently observed strand specificity for removal of UV-induced cyclobutane dimers from actively transcribed genes in mammalian cells could influence the nature and distribution of mutations in a particular gene. To investigate this, we have analyzed UV-induced DNA repair and mutagenesis in the same gene, i.e. the hypoxanthine phosphoribosyl-transferase (hprt) gene. In 23 hprt mutants from V79 Chinese hamster cells induced by 2 J/m2 UV we found a strong strand bias for mutation induction: assuming that pre-mutagenic lesions occur at dipyrimidine sequences, 85% of the mutations could be attributed to lesions in the nontranscribed strand. Analysis of DNA repair in the hprt gene revealed that more than 90% of the cyclobutane dimers were removed from the transcribed strand within 8 hours after irradiation with 10 J/m2 UV, whereas virtually no dimer removal could be detected from the nontranscribed strand even up to 24 hr after UV. These data present the first proof that strand specific repair of DNA lesions in an expressed mammalian gene is associated with a strand specificity for mutation induction.     

Dependence of DNA dark repair on protein synthesis in Escherichia coli.

Summary We investigated the influence of aminoacidless treatments applied prior and after UV irradiation on survival, dimer excision, postirradiation DNA degradation, DNA synthesis and sedimentation profiles of parental DNA ofE. coli B/r Hcr⁺ cells. In dependence on the treatment applied, the fluence 50 J/m² yielded distinctly different fractions of survivors within 0,03–85%. In all cases dimers were completely excised. The rate of DNA degradation was similar during a 30–40 min period after UV during which the bulk of dimers was excised. Degradation ceased, however, earlier in the prestarved cells than in exponentially growing ones; it was prolonged by aminoacidless postincubation. Sedimentation profiles of parental DNA did not differ during the whole period of dimer excision. In cells DNA synthesis was not restored for several hours after addition of amino acids. In cells addition of amino acids resulted in a fast resumption of DNA synthesis. We conclude that removal of dimers and repair of gaps were similar in all cases. We believe that aminoacidless treatments influence production and repair of damage to the sites of DNA replication. The treatment appears to prevent this damage when applied before UV irradiation, but interferes with its restoration when applied after UV irradiation. Consequently, the former treatment increases survival of cells while the latter produces an opposite effects.     

DNA repair properties of Escherichia coli tif-1, recAo281 and lexA1 strains deficient in single-strand DNA binding protein

Summary Mutations affecting single-strand DNA binding protein (SSB) impair induction of mutagenic (SOS) repair. To further investigate the role of SSB in SOS induction and DNA repair, isogenic strains were constructed combining the ssb ⁺, ssb-1 or ssb-113 alleles with one or more mutations known to alter regulation of damage inducible functions. As is true in ssb ⁺ strains tif-1 (recA441) was found to allow thermal induction of prophage ⁺ and Weigle reactivation in ssb-1 and ssb-113 strains. Furthermore, tif-1 decreased the UV sensitivity of the ssb-113 strain slightly and permitted UV induction of prophage ⁺ at 30°C. Strains carrying the recAo281 allele were also constructed. This mutation causes high constitutive levels of RecA protein synthesis and relieves much of the UV sensitivity conferred by lexA ^– alleles without restoring SOS (error-prone) repair. In contrast, the recAo281 allele failed to alleviate the UV sensitivity associated with either ssb ^– mutation. In a lexA1 recAo281 background the ssb-1 mutation increased the extent of postirradiation DNA degradation and concommitantly increased UV sensitivity 20-fold to the level exhibited by a recA1 strain. The ssb-113 mutation also increased UV sensitivity markedly in this background but did so without greatly increasing postirradiation DNA degradation. These results suggest a direct role for SSB in recombinational repair apart from and in addition to its role in facilitating induction of the recA-lexA regulon.     

Mutagenic DNA repair in Escherichia coli

Summary The photoreversibility of UV-induced mutations to Trp⁺ in strain Escherichia coli WP2 uvrA trp (unable to excise pyrimidine dimers) was lost at different rates during incubation in different media. In Casamino acids medium after a short initial lag, photoreversibility was lost over about one generation time; in minimal medium with tryptophan, photoreversibility persisted for more than two generations; in Casamino acids medium with pantoyl lactone photoreversibility was lost extremely slowly. The rate of loss of photoreversibility was unaffected by UV dose in either Casamino acids medium or in minimal medium. The same eventual number of induced mutants was obtained when cells were incubated for two generations in any of the three media before being transferred to selective plates supplemented with Casamino acids. Thus in each the proportion of cells capable of giving rise to a mutant was the same and only the rate at which these cells did so during post-irradiation growth varied, suggesting that there might be a specific fraction of pyrimidine dimers at a given site capable of initiating a mutagenic repair event, and that the size of this fraction is dose dependent. Segregation experiments have shown that error-prone repair appears to occur once only and is not repeated in subsequent replication cycles, in contrast to (presumed error-free) recombination repair.The results are discussed in the light of current models of UV mutagenesis.     

Photoreactivation reverses ultraviolet radiation induced premutagenic lesions leading to frameshift mutations in Escherichia coli

Summary The effect of photoreactivation of the ultraviolet radiation induced reversion of a trpE9777 frameshift mutation was studied in a uvrA6 derivative of Escherichia coli K12. Two different photoreactivation treatments were used, one providing a single flash of photoreactivating light and another providing 10 min of light from fluorescent lamps. The reversion frequency of the trpE9777 frameshift mutation was strongly reduced when subsequently exposed to visible light. The dose modification factor (the ratio of equally effective doses), for cells challenged with single-flash photoreactivation, for survival and induction of reversion to Trp⁺ was 3.6 and 3.4, respectively. UV induction of RecA protein synthesis was not reversed by a single flash of photoreactivation. The dose modification factor for 10 min of fluorescent lamp photoreactivation for survival and for induction of reversion to Trp⁺ was 6.5 and 6.3, respectively. The dose modification factor for 10 min of photoreactivation for induction of RecA protein was 1.7–2.5. Photoreactivation decreased the reversion of trpE9777 and increased survival to the same extent. We concluded that cyclobutyl pyrimidine dimers are the premutagenic lesions of UV mutagenesis of the trpE9777 allele in a uvrA6 background.