Role of constitutive and inducible repair in radiation resistance of Escherichia coli

Radiation resistance of Escherichia coil cells depends on how efficiently DNA is recovered after damage, which is determined by the function of constitutive and inducible repair branches. The effects of additional mutations of the key genes of constitutive and inducible repair (recA, lexA, recB, polA, lig, gyr, recE, recO, recR, recJ, recQ, uvrD, helD, recN, and ruv) on radiation resistance were studied in E. coli K-12 strain AB 1157 and highly radiation-resistant isogenic strain Gam(r)444. An optimal balance ensuring a high gamma resistance of the Gam(r)444 radiation-resistant E. coli mutant was due to expression of the key SOS repair genes (recA, lexA, recN, and ruv) and activation of the presynaptic functions of the RecF homologous recombination pathway as a result of a possible mutation of the uvrD gene, which codes for repair helicase II.     

Effect of an Insertional Mutation in the cspA Gene Encoding the Major Cold-Shock Protein on Radiation Resistance of Escherichia coli

Plasmid pCspA::Km carrying a cloned mutant allele of the cspA gene for the major Escherichia coli cold-shock protein CspA with an insertion of the kanamycin resistance gene cassette from transposon Tn903 into the core region of the coding sequence causes a 2.3-fold increase in radioresistance of wild-type E. coli cells (cspA ⁺). The radiation protective effect of this plasmid is abolished or drastically reduced in mutants recA13and rpoH15defective in RecA protein and in induction of the heat-shock protein regulon, respectively. Plasmid pCspA::Km causes a 1.3-fold elevation in the resistance to -irradiation of E. coli mutants with an intermediate level of radiation resistance (Gam^r445 and KS0160) but slightly diminishes resistance of a highly radiation-resistant Gam^r444 mutant. In the chromosome of E. coli strain with normal DNA repair systems, the cspA::Km mutation in the homozygous state enhances resistance to the lethal effect of -rays and UV light 2.9 and 1.4 times, respectively. These data suggest that the system of cold-shock proteins can modulate resistance of E. colicells to the lethal effect of -rays and UV light.     

Escherichia coli K-12 mutants with enhanced resistance to ionizing radiation. III. The effect of rec and lexA mutations on radioresistance

Lethal action of gamma-rays on derivatives of the wild-type strain AB1157 and of two radiation-resistant mutants (Gamr444 and Gamr445) containing additional mutations dnaA46, recB21, recF143, recA56, recA430, lexA3, lexA102 or lexA3 recAo98, was studied. When the mean number of genomes per cell was reduced by means of pre-incubation at 43 degrees C, radioresistance of the strains AB1157 dnaA46 and Gamr445 dnaA46 was not changed, and that of the strain Gamr444 dnaA46 was reduced to the level of the Gamr445 dnaA46 strain. Introduction of additional mutations recB21, recA56 or lexA3 (lexA102) into the genome of the strains Gamr444 or Gamr445 made them as radiosensitive as the corresponding variants of AB1157. Additional mutations recF143 or recA430 (lexB30) significantly decreased the radioresistance of Gamr444 and Gamr445 mutants, although did not level them to corresponding derivatives of AB1157. Operator-constitutive mutation recAo98 enhanced radioresistance of all lexA3 derivatives tested but not to the level of the corresponding lexA+ strains. The role of recombinational repair and the inducible SOS system in enhanced radioresistance of Gamr mutants is discussed. The data of post-irradiation DNA degradation in various derivatives of the strains AB1157 and Gamr suggest that Gamr mutants have a constitutive inhibitor of degradation which does coincide with RecA protein.     

Effect of ruv mutations on recombination and DNA repair in Escherichia coli K12

Summary Mutation of the ruv gene of E. coli is associated with sensitivity to radiation, and filamentous growth after transient inhibition of DNA synthesis. The filamentation of ruv strains is abolished by mutations in sfiA or sfiB that prevent SOS induced inhibition of cell division, but this does not restore resistance to UV radiation. Double mutants carrying both ruv and uvr mutations are considerably more sensitive to UV radiation than the single mutants, but there is no additive effect of ruv with recA, recF, recB, or recC mutations. ruv mutations have little effect on conjugal recombination in wild-type strains but confer recombination deficiency and extreme sensitivity to ionizing radiation in recBC sbcB strain. These results, together with the fact that ruv strains are excision proficient and mutable by UV light, are interpreted to suggest that the ruv ⁺ product is involved in recombinational repair of damaged DNA rather than in cell division as suggested by Otsuji et al. (1974).     

Involvement in DNA repair of the ruvA gene of Escherichia coli

Summary The ruv operon of Escherichia coli consists of two genes, orfl1 and ruv, which encode 22 and 37 kilodalton proteins, respectively, and are regulated by the SOS system. Although the distal gene, ruv, is known to be involved in DNA repair, the function of orf1 has not been studied. To examine whether orf1 is also involved in DNA repair, we constructed a strain with a deletion of the entire ruv operon. The strain was sensitive to UV even after introduction of low copy number plasmids carrying either orf1 or ruv, but UV resistance was restored by introduction of a plasmid carrying both orfl and ruv. These results suggest that orf1 as well as ruv is involved in DNA repair. Therefore, orf1 and ruv should be renamed ruvA and ruvB, respectively.     

Genetic control of damage-inducible restriction alleviation in Escherichia coli K12: an SOS function not repressed by lexA

Summary The alleviation of K-specific DNA restriction after treatment of cells by UV or nalidixic acid has been studied in mutants with various alleles of recA and lexA and combinations of these alleles and with recB and recF mutations. The studies show that induction of restriction alleviation by UV or nalidixic acid is abolished in mutants in which the recA protein is defective (recA13, recA56), its protease activity is altered (recA430) or in which it cannot be efficiently activated (recA142). Thermoinduction of restriction alleviation was observed in tif mutant (recA441). In lexA amber mutants restriction alleviation is not constitutive but is still inducible. In a lexA3 mutant restriction alleviation is inducible by nalidixic acid provided that recA protein is overproduced as a result of a recA operator mutation. Induction by UV depends on the recF function and an unidentified function (Y) which is controlled by the lexA protein. The recBC enzyme is necessary for induction by UV or by nalidixic acid. Temperature shift experiments with a thermosensitive recB mutant indicate that the recBC enzyme functions in an early step during UV-induction. It is concluded that the damage-inducible function which alleviates restriction is similar to other damage inducible repair (SOS) functions in the dependence on activated recA protease for induction, but that it differs from these functions by the absence of a direct control through the lexA repressor.     

The recN locus of Escherichia coli K12: molecular analysis and identification of the gene product

Summary The recN gene which is necessary for inducible DNA repair and recombination in Escherichia coli has been cloned into the low copy plasmid vector pHSG415. Analysis of the recombinant plasmid, pSP100, revealed a 5.6 Kb HindIII insert of chromosomal DNA. Transposon inactivation of recN function and analysis of a recN::Mu(Ap lac) fusion located the coding region to a 1.4 Kb region within a 2.1 Kb BglII-AvaI DNA fragment transcribed in a clockwise direction with respect to the chromosome map. The gene product was identified in maxicells as a 60,000 dalton protein. Synthesis of this protein was increased in cells lacking LexA activity or in strains carrying recN cloned into the multicopy vector pBR322. Multiple copies of recN increase resistance to ionizing radiation in recN mutants but reduce the survival of a wild-type strain.     

Effects of a recA operator mutation on mutant phenotypes conferred by lexA and recF mutations

Depression of recA by an operator mutation (recAo281) produces effects opposite to those obtained from its derepression following DNA damage. Inducible reactivation of Λvir and S13 phages is decreased and inducible UV mutahenesis of a φX174 amber mutant is lessened in a recAo281 strain compared to a recAo⁺ strain. The decreases could not be accounted for by increases in constitutive levels of these processes. Consistent with these results the UV resistance of a recAo281 strain is less than that of a recAo⁺ strain. This may indicate that too much recA protein immediately after irradiation interferes with derepression of the lexA regulon or functioning of its products. Effects of increasing the recAo⁺ and recA⁺ copy number on a ColE1 plasmid are compared with the effects of recAo281.recAo281 partially suppresses UV sensitivity due to lexA102 and lexA3 in E. coli K-12. This increase in resistance is not correlated with an increase in constitutive or inducible reactivation of UV-irradiated Λvir or S13. This is consistent with the previous suggestion that the UV resistance stems from a decrease in DNA degradation allowing an increase in DNA repair. lexA3 blocks UV mutagenesis of φX174 as measured by reversion of amber mutations and this was not suppressed by recAo281.recF143 blocks UV mutagenesis of φX174. recAo281 suppresses neither this effect nor the decrease in bacterial UV resistance caused by recF143.     

Genetic analysis and molecular cloning of the Escherichia coli ruv gene

Summary The genetic organisation of the ruv gene, a component of the SOS system for DNA repair and recombination in Escherichia coli, was investigated. New point mutations as well as insertions and deletions were generated using transposon Tn10 inserted in eda as a linked marker for site specific mutagenesis, or directly as a mutagen. The mutations were ordered with respect to one another and previously isolated ruv alleles by means of transductional crosses. The direction of chromosome mobilization from ruv:: Mud(Ap^R lac)strains carrying F42lac ⁺ established that ruv is transcribed in a counterclockwise direction. Recombinant phages able to restore UV resistance to ruv mutants were identified, and the ruv ⁺ region was subcloned into a low copy number plasmid. The ruv ⁺ plasmid was able to correct the extreme radiation sensitivity and recombination deficiency of ruv recBC sbcB strains.     

Viability of Escherichia coli K-12 DNA adenine methylase (dam) mutants requires increased expression of specific genes in the SOS regulon

Summary We have examined the level of expression of the SOS regulon in cells lacking DNA adenine methylase activity (dam ^-). Mud (Ap, lac) fusions to several SOS operons (recA, lexA, uvrA, uvrB, uvrD, sulA, dinD and dinF) were found to express higher levels of -galactosidase in dam ^- strains than in isogenic dam ⁺ strains. The attempted construction of dam ^- strains that were also mutant in one of several SOS genes indicated that the viability of methylase-deficient strains correlates with the inactivation of the SOS repressor (LexA protein). Consistent with this, the wild-type functions of two LexA-repressed genes (recA and ruv) appear to be required for dam ^- strain viability.     

TheisfA mutation inhibits mutator activity and processing of UmuD protein inEscherichia coli recA730 strains

Further studies on theisfA mutation responsible for anti-SOS and antimutagenic activities inEscherichia coli are described. We have previously shown that theisfA mutation inhibits mutagenesis and other SOS-dependent phenomena, possibly by interfering with RecA coprotease activity. TheisfA mutation has now been demonstrated also to suppress mutator activity inE. coli recA730 andrecA730 lexA51(Def) strains that constitutively express RecA coprotease activity. We further show that the antimutator activity of theisfA mutation is related to inhibition of RecA coprotease-dependent processing of UmuD. Expression of UmuD' from plasmid pGW2122 efficiently restores UV-induced mutagenesis in therecA730 isfA strain and partially restores its mutator activity. On the other hand, overproduction of UmuD'C proteins from pGW2123 plasmid markedly enhances UV sensitivity with no restoration of mutability.     

Repair of DNA double-strand breaks in Escherichia coli K12 requires a functional recN product

Summary Mutation of the recN gene of Escherichia coli in a recBC sbcB genetic background blocks conjugational recombination and confers increased sensitivity to UV light and mitomycin C. The basis for this phenotype was investigated by monitoring the properties associated with recN mutations in otherwise wild-type strains. It was established that recN single mutants are almost fully resistant to UV irradiation, and that there is no detectable defect in repair of UV lesions by excision, error-prone, or recombinational mechanisms. However, recN mutations confer sensitivity to mitomycin C and ionizing radiation both in wild-type and recB sbcB strains. The sensitivity to ionizing radiation is correlated with a deficiency in the capacity to repair DNA double-strand breaks by a UV inducible mechanism. Recombinant phages that complement the recombination and repair defects of recN recBC sbcB mutants have been identified, and the recN gene has been cloned from these phages into a low copy-number plasmid.     

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.     

Damage to DNA induces expression of the ruv gene of Escherichia coli

Summary Regulation of the ruv gene of E. coli was studied using phage Mud (Ap lac) to obtain a fusion of the lac genes to the ruv promoter. -galactosidase synthesis in the ruv-lac fusion strain was induced by mitomycin C and other agents that damage DNA. The induction of -galactosidase could be altered by mutations either in lexA or recA from which it is concluded that ruv is regulated by lexA repressor. A possible function of ruv in promoting cell recovery following damage to DNA is discussed.     

Amplified UvrA protein can ameliorate the ultraviolet sensitivity of an Escherichia coli recA mutant.

When a recA strain of Escherichia coli was transformed with the multicopy plasmid pSF11 carrying the uvrA gene of E. coli, its extreme ultraviolet (UV) sensitivity was decreased. The sensitivity of the lexA1 (Ind(-)) strain to UV was also decreased by pSF11. The recA cells expressing Neurospora crassa UV damage endonuclease (UVDE), encoding UV-endonuclease, show UV resistance. On the other hand, only partial amelioration of UV sensitivity of the recA strain was observed in the presence of the plasmid pNP10 carrying the uvrB gene. Host cell reactivation of UV-irradiated lambda phage in recA cells with pSF11 was as efficient as that in wild-type cells. Using an antibody to detect cyclobutane pyrimidine dimers, we found that UV-irradiated recA cells removed dimers from their DNA more rapidly if they carried pSF11 than if they carried a vacant control plasmid. Using anti-UvrA antibody, we observed that the expression level of UvrA protein was about 20-fold higher in the recA strain with pSF11 than in the recA strain without pSF11. Our results were consistent with the idea that constitutive level of UvrA protein in the recA cells results in constitutive levels of active UvrABC nuclease which is not enough to operate full nucleotide excision repair (NER), thus leading to extreme UV sensitivity.     

UV resistance of E. coli K-12 deficient in cAMP/CRP regulation.

Deletion of genes for adenylate cyclase (delta cya) or cAMP receptor protein (delta crp) in E. coli K-12 confers a phenotype that includes resistance to UV radiation (254 nm). Such mutations lead to UV resistance of uvr+, uvrA, lexA and recA strains which could partly be abolished by the addition of cAMP to delta cya but not to delta crp strain culture medium. This effect was not related to either inducibility of major DNA repair genes or growth rate of the bacteria. Enhanced survival was also observed for UV-irradiated lambda bacteriophage indicating that a repair mechanism of UV lesions was involved in this phenomenon.     

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.     

Evidence of abortive recombination in ruv mutants of Escherichia coli K12

Summary Genetic recombination in Escherichia coli was investigated by measuring the effect of mutations in ruv and rec genes on F-prime transfer and mobilization of nonconjugative plasmids. Mutation of ruv was found to reduce the recovery of F-prime transconjugants in crosses with recB recC sbcA strains by about 30-fold and with recB recC sbcB sbcC strains by more than 300-fold. Conjugative plasmids lacking any significant homology with the chromosome were transferred normally to these ruv mutants. Mobilization of the plasmid cloning vectors pHSG415, pBR322, pACYC184 and pUC18 were reduced by 20- to 100-fold in crosses with ruv rec ⁺ sbc ⁺ strains, depending on the plasmid used. Recombinant plasmids carrying ruv ⁺ were transferred efficiently. With both F-prime transfer and F-prime cointegrate mobilization, the effect of ruv was suppressed by inactivating recA. It is proposed that the failure to recover transconjugants in ruv recA ⁺strains is due to abortive recombination and that the ruv genes define activities which function late in recombination to help convert recombination intermediates into viable products.     

Mutation in the <Emphasis Type="Italic">cspH-cspG</Emphasis> gene cluster enhances expression of heat-shock proteins and SOS repair system of <Emphasis Type="Italic">Escherichia coli</Emphasis>

The recessive radioresistance allele gam12 cloned in plasmid pBC4042-gam12 slightly increases the radiation resistance of Escherichia coli wild-type cells. Meanwhile, irradiation by γ-rays induces transition of gam ^r 12 mutation to the homozygous state and causes a 3.37-fold increase in radiation resistance of these cells. The mutation gam ^r 12 was located at 22.68 min of the chromosomal map in the region of cspH-cspG gene cluster of cold-shock proteins. Sequence analysis of gam12 allele revealed the nucleotide sequence of cold-shock gene cspG and insertions in the C-terminal part of the gene. Translation of mutant cspG gene can lead to synthesis of a truncated product that represents the N-terminal protein fragment with motifs governing binding with DNA and RNA. Analysis of the Escherichia coli genome revealed motifs recognized by proteins of the cspA family in genes of cold shock, heat shock, SOS regulon, and other systems. These data suggest the possibility of involvement of mutant RNA-chaperones of type CspA′ and CspG′ in the expression of key genes in systems of SOS repair and recombination or auxiliary stress systems, including heat-shock proteins, in radiation resistant mutants of E. coli.