Uvm mutants of Escherichia coli K12 deficient in UV mutagenesis

Summary Uvm mutants of Escherichia coli K12 selected for defective UV reversion induction have previously been reported to differ considerably from the UV-reversion-less recA and lexA mutants with regard to survival or mutagenic response to UV, X-rays and alkylating agents. In the present study, the phenotypic characterization of uvm mutants was extended to investigate several cellular processes which also may be related to or involved in UV mutagenesis. Like recA and lexA mutations, the uvm mutations exhibit highly reduced Weigle reactivation and normal host cell reactivation of UV irradiated phage . But unlike recA and lexA, the uvm mutations do not impair genetic recombination, UV induction of prophage or R plasmid-mediated UV resistance and mutagenesis. These phenotypical characteristics and preliminary results of genetic mapping lend further support to the assumption that the uvm site may be a novel locus affecting, apart from the recA and lexA loci, the error-prone repair pathway in E. coli.     

Repair mechanisms involved in prophage reactivation and UV reactivation of UV-irradiated phage lambda

Survival of UV-irradiated phage λ is increased when the host is lysogenic for a homologous heteroimmune prophage such as λimm434 (prophage reactivation). Survival can also be increased by UV-irradiating slightly the non-lysogenic host (UV reactivation).Experiments on prophage reactivation were aimed at evaluating, in this recombination process, the respective roles of phage and bacterial genes as well as that of the extent of homology between phage and prophage.To test whether UV reactivation was dependent upon recombination between the UV-damaged phage and cellular DNAs, lysogenic host cells were employed. Such hosts had thus as much DNA homologous to the infecting phage as can be attained. Therefore, if recombination between phage and host DNAs was involved in this repair process, it could clearly be evidenced.By using unexposed or UV-exposed host cells of the same type, prophage reactivation and UV reactivation could be compared in the same genetic background.The following results were obtained: (1) Prophage reactivation is strongly decreased in a host carrying recA mutations but quite unaffected by mutation lex-I known to prevent UV reactivation; (2) In the absence of the recA⁺ function, the red⁺ but not the int⁺ function can substitute for recA⁺ to produce prophage reactivation, although less efficiently; (3) Prophage reactivation is dependent upon the number of prophages in the cell and upon their degree of homology to the infecting phage. The presence in a recA host of two prophages either in cis (on the chromosome) or in trans (on the chromosome and on an episome) increases the efficiency of prophage reactivation; (4) Upon prophage reactivation there is a high rate of recombination between phage and prophage but no phage mutagenesis; (5) The rate of recombination between phage and prophage decreases if the host has been UV-irradiated whereas the overall efficiency of repair is increased. Under these conditions UV reactivation of the phage occurs as in a non-lysogen, as attested by the high rate of mutagenesis of the restored phage.These results demonstrate that UV reactivation is certainty not dependent upon recombination between two pre-existing DNA duplexes. The hypothesis is offered that UV reactivation involves a repair mechanism different from excision and recombination repair processes.     

Plasmid (pKM101)-mediated enhancement of repair and mutagenesis: Dependence on chromosomal genes in Escherichia coli K-12

Summary The drug resistance plasmid pKM101 plays a major role in the Ames Salmonella/microsome carcinogen detecting system by enhancing chemical mutagenesis. It is shown that in Escherichia coli K-12 the plasmid pKM101 enhances both spontaneous and methyl methanesulfonate-caused reversion of an ochre mutation, bacterial survival after ultraviolet irradiation, and reactivation of ultraviolet-irradiated in unirradiated cells. All these effects are shown to be dependent on the recA ⁺ lexA⁺ genotype but not on the recB ⁺ recC⁺ or recF ⁺ genotypes. The recA lexA-dependence of the plasmid-mediated repair and mutagenesis suggests an interaction with the cell's inducible error-prone repair system. The presence of pKM101 is shown to cause an additional increase in methyl methanesulfonate mutagenesis in a tif mutant beyond that caused by growth at 42°. The presence of the plasmid raises the level of the Weigle-reactivation curve for the reactivation of ultraviolet-irradiated in E. coli and causes a shift of the maximum to a higher UV fluence. These observations suggest that pKM101 does not exert its effects by altering the regulation of the cell's error-prone repair system but rather by supplying a mechanistic component or components.     

Ultraviolet reactivation of the single stranded DNA phage phiX 174.

Summary When UV-irradiated X174 was grown in pre-irradiated host cells of various strains, ultraviolet reactivation (UVR) was observed only in recombination proficient strains such as E. coli C (uvrA ⁺ recA ⁺) and HF4704 (uvrA ^- recA ⁺), but not in the recombination deficient strain HF4712 (uvrA ⁺ recA ^-). By increasing the multiplicity of infection, no rise in the amount of such reactivation was observed. From the study of the neutral and alkaline sucrose gradient sedimentation patterns of DNA samples extracted from unirradiated cells infected with unirradiated phage, it appears that after the conversion of the viral single stranded (SS) DNA to the double stranded form (DS), nicks or scissions were produced on it within all three strains, which were ultimately sealed up in the recA ⁺ but persisted within the recA ^- host cells. When UV-irradiated phage infected unirradiated host cells, such nicking of the DS DNA appeared to be much more extensive in uvrA ⁺ recA ⁺, but slightly reduced in uvrA ⁺ recA ^- and severely suppressed in uvrA ^- recA ⁺ strains. When the host cells were also UV-irradiated, the conversion of the infecting viral SS DNA to DS DNA as well as its subsequent nicking were reduced in all the three strains to a much greater extent. Although nicking of the DS DNA molecule is an essential step even in the normal intracellular replication of X DNA, the production and the sealing up of such nicks appear not to have any positive correlation with UVR of these phages. A drastic reduction in nicking due te pre-irradiation of the host cells might, however, mean slowing down of the replication of the damaged parental RF molecules which would facilitate their repair perhaps through recombination with the homologous parts of the host genome.     

Influence of plasmids carrying the lexA gene on DNA repair and related processes in Escherichia coli K-12

Summary Plasmid pLC44-14 from the Clarke and Carbon collection has been shown to carry the lexA gene. The presence of lexA was demonstrated by complementation of tsl mutants which lie close to lexA on the E. coli K-12 linkage map and are probably in the lexA gene, and by crossing the dominant lexA mutation on to pLC44-14 to produce a recombinant plasmid, pSEl, which gave the host cell the properties of a lexA mutant. The lexA gene has been cloned on to pBR322 (Little, 1980). pJL21, which carries the lexA ⁺ gene, rendered the host cell moderately sensitive to UV light, greatly reduced the extent of Weigle reactivation and mutagenesis of UV-irradiated phage , and inhibited induction of protein X by either UV light or nalidixic acid. A similar plasmid carrying a mutant lexA3 allele produced extreme sensitivity to UV light, reduced recombinant production 10 to 50-fold following Hfr x F^– conjugation crosses, and otherwise mimicked the effects of pJL21. Introduction of an amber mutation into the lexA gene carried by the plasmid greatly reduced the UV-sensitivity of the host, thereby indicating that the extreme sensitivity was due to the mutant lexA gene product. These properties of strains with lexA plasmids are thought to originate from high levels of the lexA protein in the cell due to a large plasmid copy number. This protein, which appears from other studies to regulate negatively the recA gene, may inhibit expression of recA or other DNA repair genes when present in excess amounts in the cell.     

Interstrain crosses enhance excision of Tc1 transposable elements in Caenorhabditis elegans

Summary We have studied spontaneous and UV mutagenesis of the glyU gene in Escherichia coli trpA461 (GAG) strains carrying the pIP11 plasmid, in which the dnaQ gene encoding the 3–5 exonuclease subunit (epsilon) of DNA polymerase III is fused to the tac(trp-lac) promoter. We have used a pair of M13glyU phage in which the gene encoding the glycyl-tRNA is cloned in opposite orientations, consequently the phage present either GGG or CCC anticodon triplets for mutagenesis. The presence of IPTG, the inducer of the tac-dnaQ fusion, results in about 100-fold decrease in frequency of spontaneous Su⁺ (GAG) mutations arising in the CCC phage. The enhanced expression of tac-dnaQ reduces 10-fold the frequency of UV-induced Su⁺ (GAG) mutations in the CCC phage and nearly completely prevents generation by UV of Su⁺ (GAG) mutations in the GGG phage, in which UV-induced pyrimidine photoproducts can be formed only in the vicinity of the target triplet. These results suggest that both locally and regionally targeted mutagenesis is affected by overproduction of the epsilon subunit. By delayed photoreversal mutagenesis we have shown that UV-induced chromosomal mutagenesis of the umuC36 trpA461 strain harboring pIP11 is completely abolished in the presence of IPTG. This result seems to indicate that the misinocorporation step of DNA translesion synthesis is affected by excess of the epsilon subunit. Finally, we have introduced the pIP13 plasmid carrying the dnaQ gene into the recA1207 strain, which is deficient in the recombinase activity of RecA but constitutive in the protease activity. We demonstrate that the transformant shows much higher UV sensitivity than recA1207 carrying the vector plasmid pBR325, indicating that translesion synthesis significantly contributes to DNA repair capacity of cells deficient in recombination.     

Cloned truncated recA genes in E. coli

Summary Plasmids pMH1 and pDR1461, possessing the control region and 22% or 73% of the E. coli recA gene, conferred UV sensitivity to wild-type uvrA, and umuC bacteria. Sensitization was less in recA441 (tif-1) mutants and absent in lexA cells. Radiosensitization correlated with inhibition of recombinational repair, even through induced recA protein synthesis and recombination in Hfr matings were normal. Plasmids pMH1 and pDR1461 also prevented induction of some, but not all, SOS functions. Mutagenic reversion to tryptophan prototrophy and induced reactivation of UV-irradiated phage were eliminated, and the efficiency of lysogenic induction reduced. However, naladixic acid induced filamentous growth, mitomycin-C induced uvrA gene expression and post UV-irradiation DNA degradation control were little changed. Explanations of these effects are discussed which involve the presence of either truncated recA protein or multiple copies of the recA gene control sequence.A preliminary account of this work is presented in Chromosome Damage and Repair, edited by E. Seeberg and K. Klepper, to be published by Plenum Press     

Pathways for repair of DNA damaged by alkylating agent in Escherichia coli.

Summary A strain with both the polA12 and the alk-1 mutation is only slightly more sensitive to methyl methane sulfonate (MMS) than isogenic strains with only one of the mutations. On the other hand, alk-1 recA1 double mutant is much more sensitive to MMS than are strains carrying either one of alk or recA mutation. It was suggested that the alk and the polA gene products are involved in the same DNA repair process whereas the recA function is independent from the process. The yield of MMS-induced mutation (Arg^- (argE) to Arg⁺ reversion) in alk mutant is considerably higher than that in wild type strain. Thus, the repair process in which the alk gene product is involved is relatively accurate. When MMS-treated phages were plated on MMS-treated bacteria, there were considerable increases in survival of treated phage even in recA alk double mutant. It seems that a new repair pathway, which is specific for alkylating agent-induced damages and is not dependent on the RecA function, may be induced on exposure of bacteria to the alkylating agent.     

Nucleotide excision repair and recombination are engaged in repair of trans-4-hydroxy-2-nonenal adducts to DNA bases in Escherichia coli

One of the major products of lipid peroxidation is trans-4-hydroxy-2-nonenal (HNE). HNE forms highly mutagenic and genotoxic adducts to all DNA bases. Using M13 phage lacZ system, we studied the mutagenesis and repair of HNE treated phage DNA in E. coli wild-type or uvrA, recA, and mutL mutants. These studies revealed that: (i) nucleotide excision and recombination, but not mismatch repair, are engaged in repair of HNE adducts when present in phage DNA replicating in E. coli strains; (ii) in the single uvrA mutant, phage survival was drastically decreased while mutation frequency increased, and recombination events constituted 48 % of all mutations; (iii) in the single recA mutant, the survival and mutation frequency of HNE-modified M13 phage was slightly elevated in comparison to that in the wild-type bacteria. The majority of mutations in recA^- strain were G:C → T:A transversions, occurring within the sequence which in recA⁺ strains underwent RecA-mediated recombination, and the entire sequence was deleted; (iv) in the double uvrA recA mutant, phage survival was the same as in the wild-type although the mutation frequency was higher than in the wild-type and recA single mutant, but lower than in the single uvrA mutant. The majority of mutations found in the latter strain were base substitutions, with G:C → A:T transitions prevailing. These transitions could have resulted from high reactivity of HNE with G and C, and induction of SOS-independent mutations.     

Expression of the plasmid pKM101-determined DNA repair system in recA? and lex? strains of Escherichia coli

Summary pKM101, a plasmid R factor of the N compatibility group increases methylmethane sulfonate mutagenesis and diminishes UV-killing in recA ⁺ lex ⁺ and recA ⁺ lex ^– strains, but not in recA ^– lex ⁺ strains. The induction of a reclex dependent colicin is not present in lex ^– strains carrying the pKM101 factor. These facts indicate that pKM101 acts through an error-prone DNA repair system, which is recA ⁺ dependent, but not lex ⁺ dependent.This paper is published on the occasion of Dr. C. Callerio's seventy-fifth birthday     

Molecular cloning and nucleotide sequence of the Rhizobium phaseoli recA gene

Summary A recombinant phage carrying the recA gene of Rhizobium phaseoli was isolated from a R. phaseoli genomic library by complementation of the Fec^– phenotype of the recombinant phage in Escherichia coli. When expressed in E. coli, the cloned recA gene was shown to restore resistance to both UV irradiation and the DNA alkylating agent methyl methanesulphonate (MMS). The R. phaseoli recA gene also promoted homologous recombination in E. coli. The cloned recA gene was only weakly inducible in E. coli recA strains by DNA damaging agents. The DNA sequence of the R. phaseoli recA gene was determined and compared with published recA sequences. No LexA-binding site was detected in the R. phaseoli recA upstream region.     

Prophage induction and cell division in E. coli. II. Linked (recA, zab) and unlinked (lex) suppressors of tif-1-mediated induction and filamentation

Summary The pleiotropy of tif-1, a mutation in E. coli K12 causing, among other effects, cellular filamentation at 42° and thermal induction of lysogenic derivatives, can be explained by the participation of the tif ⁺ gene product in more than one reaction pathway. Pathways that involve the tif ⁺ product may be analyzed by selection of secondary mutations that reverse both tif-1-mediated prophage induction and cell filamentation. Among revertants of a tif-1 lysogen among 20% are recombination deficient. These appear to carry a recA mutation. In addition to this class is a rarer (7%) phenotypically distinguishable class of revertants, called zab, first described here. Markers tif-1, recA and zab are closely linked. Mutations lex which are dominant and located near malB also appear (3%) among tif-1 revertants. The lex ⁺ function is needed for normal UV, X-ray and mitomycin C induction of prophage .The zab mutation resembles recA in causing (1) high sensitivity to UV, X-rays and mitomycin C, (2) drastic DNA degradation following UV irradiation but normal capacity to repair UV-damaged infecting phage (Hcr⁺), (3) failure to carry out UV reactivation and UV mutagenesis of UV-irradiated bacteriophage , (4) a markedly reduced level of spontaneous induction of . In contrast, other capacities, strikingly diminished by recA, are affected less, if at all by zab. Thus zab (1) permits 30–60% normal recombination proficiency, (2) shows real, although very low inducibility of by UV or mitomycin C, (3) permits 100% efficiency of plating of red gam, and (4) does not degrade DNA spontaneously.The hypothesis is proposed that the tif-1 mutation is a regulatory mutation controlling the activity, or more likely the synthesis of repair enzyme(s). The level of these repair enzyme(s), rather than DNA lesions, may govern the stability of the prophage repressor and the capacity of the bacteria to form septa.     

Genetic evidence that the elevated levels of Escherichia coli helicase II antagonize recombinational DNA repair

Some phages survive irradiation much better upon multiple than upon single infection, a phenomenon known as multiplicity reactivation (MR). Long ago MR of UV-irradiated λ red phage in E. coli cells was shown to be a manifestation of recA-dependent recombinational DNA repair. We used this experimental model to assess the influence of helicase II on the type of recombinational repair responsible for MR. Since helicase II is encoded by the SOS-inducible uvrD gene, SOS-inducing treatments such as irradiating recA⁺ or heating recA441 cells were used. We found: i) that MR was abolished by the SOS-inducing treatments; ii) that in uvrD background these treatments did not affect MR; and iii) that the presence of a high-copy plasmid vector carrying the uvrD⁺ allele together with its natural promoter region was sufficient to block MR. From these results we infer that helicase II is able to antagonize the type of recA-dependent recombinational repair acting on multiple copies of UV-damaged λ DNA and that its antirecombinogenic activity is operative at elevated levels only.     

Complementation studies with the repair-deficient uvrD3, uvrE156, and recL152 mutations in Escherichia coli

Summary Previous work showed that the mutations uvrD3, uvrE156, and recL152 were closely linked and increased UV-sensitivity. They were phenotypically distinguishable in that only the uvrD3 mutation significantly decreases host cell reactivation of UV-irradiated phage (Hcr^-) and repair of methylmethane sulfonate (MMS)-induced damage, and only the uvrE156 mutation increased mutation rates (Mut^-). MMS-resistant revertants of a uvrD3 mutant were still UV-sensitive and fell into two phenotypic classes, Hcr^- Mut⁺ (non-mutator) and Hcr⁺ Mut^-. In this work complementation tests were done by examining UV-and MMS-sensitivity and host cell reactivation in heterogenotes containing combinations of uvrD3, uvrE156, recL152, and the MMS-resistant mutations derived from uvrD3. The mutations could not complement each other in the repair of UV-damage, the one trait all had in common, indicating that they were in one gene. For the most part, the different mutations were able to complement each other in respect to traits in which one was deficient and the other had wild type activity.     

DNA repair in E. coli strains deficient in single-strand DNA binding protein

Summary Weigle reactivation and mutagenesis have been found to be defective in strains of E. coli deficient in single-strand DNA binding protein (SSB). These defects parallel those previously found in prophage induction and amplification of recA protein synthesis in ssb strains. Together, these results demonstrate a role for SSB in the induction of SOS responses. UV survival studies of ssb ^- recA^- and ssb ^- uvr^- strains are presented which also suggest a role for SSB in recombinational repair processes but not in excision repair. Studies of host cell reactivation support this latter conclusion.     

Construction in Escherichia coli K12 of derivatives of sex factor F143 carrying lexB and recA mutations

Summary Various F sex factors have been derived from F143, an episome extending from lysA to pheA. F143 derivatives carrying recA and lexB alleles and also mutations in genes thyA, argA, cysC were constructed as follows. Recombination was used as a means to generate genetically labelled F-primes. Using trimethoprim as agent of counterselection of Thy⁺ cells in thyA ^–/F-thyA ⁺bacteria, it was possible to collect, after transfer, F-primes modified by deletion of the thyA region or recombination between chromosome and episome. F-primes which had spontaneously recombined with the chromosome and integrated chromosomal markers, were also selected by transfer to proper F^– recipients. P1 transduction of a dominant marker allele into a strain homodiploid for a recessive allele was used to construct F-primes carrying mutations introduced by cotransduction. These F-primes have been useful to establish the dominance and complementation pattern of recA and lexB mutations (Morand, Goze and Devoret, accompanying paper; Glickman, Guijt and Morand, accompanying paper).     

Investigation of a phage resistantSerratia entomophila strain (BC4B), establishment of generalised transduction and construction ofS. entomophila RecA mutants

ArecA clone was isolated from a cosmid library ofSerratia entomophila constructed in theEscherichia coli strain HB101. Subcloning and transposon mutagenesis were used to identify a 1.36 kb fragment containing therecA gene. A clonedrecA mutation, generated by transposon mutagenesis and the replacement of a portion of therecA gene with an antibiotic resistance cassette, was introduced into the chromosome via a marker exchange technique. TherecA strains created were deficient in DNA repair, homologous recombination and both the spontaneous and UV induction of prophages.S. entomophila recA strains showed continued pathogenicity towards the New Zealand grass grub,Costelytra zealandica. Simple procedures for further construction ofS. entomophila recA strains have been demonstrated.     

Evidence that pretreatment of Escherichia coli cells with N-methyl-N''-nitro-N-nitrosoguanidine enhances mutability of subsequently infecting phage lambda

Summary The frequency of mutations in is significantly higher for host cells of a rec ⁺ or recA ^- strain pretreated with N-methyl-N-nitro-N-nitrosoguanidine (NG) than for untreated control cells. Direct NG treatment of DNA-injected host cells results in about tenfold higher mutation frequency in than NG treatment of host cells alone. Since mutability of is enhanced by UV preirradiation of host cells in the rec ⁺ strain but not in the recA ^- strain, indirect NG mutagenesis is different in mechanism from indirect UV mutagenesis. It is concluded that NG mutagenesis in consists of at least two processes: induction of rec ⁺-independent mutagenic activity in the host bacterium and production of rec ⁺-independent mutational damage to intracellular DNA.     

Nature of the SOS mutator activity: genetic characterization of untargeted mutagenesis in Escherichia coli

Summary In Escherichia coli, induction of the SOS functions by UV irradiation or by mutation in the recA gene promotes an SOS mutator activity which generates mutations in undamaged DNA. Activation of RecA protein by the recA730 mutation increases the level of spontaneous mutation in the bacterial DNA. The number of recA730-induced mutations is greatly increased in mismatch repair deficient strains in which replication errors are not corrected. This suggests that the majority of recA730-induced mutations (90%) arise through correctable, i.e. non-targeted, replication errors. This recA730 mutator effect is suppressed by a mutation in the umuC gene. We also found that dam recA730 double mutants are unstable, segregating clones that have lost the dam or the recA mutations or that have acquired a new mutation, probably in one of the genes involved in mismatch repair. We suggest that the genetic instability of the dam recA730 mutants is provoked by the high level of replication errors induced by the recA730 mutation, generating killing by coincident mismatch repair on the two unmethylated DNA strands. The recA730 mutation increases spontaneous mutagenesis of phage poorly. UV irradiation of recA730 host bacteria increases phage untargeted mutagenesis to the level observed in UV-irradiated recA ⁺ strains. This UV-induced mutator effect in recA730 mutants is not suppressed by a umuC mutation. Therefore UV and the recA730 mutation seem to induce different SOS mutator activities, both generating untargeted mutations.