Role of constitutive and inducible repair in radiation resistance of <Emphasis Type="Italic">Escherichia coli</Emphasis>

Radiation resistance of Escherichia coli 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, recF, recO, recR, recJ, recQ, uvrD, helD, recN, and ruv) on radiation resistance were studied in E. coli K-12 strain AB1157 and highly radiation-resistant isogenic strain Gam^r444. An optimal balance ensuring a high γ resistance of the Gam^r444 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.     

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.     

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.     

Non-mutability by ultraviolet light in uvrD recB derivatives of Escherichia coli WP2 uvrA is due to inhibition of RecA protein activation

The deficiency in UV mutagenesis in uvrD3 recB21 strains of E. coli is almost completely overcome by constitutive activation of RecA protein and expression of the SOS system (by recA730 or 43 degrees C treated recA441 lexA71). When SOS was expressed but RecA protein not self-activated (recA441 lexA71 at 30 degrees C), uvrD3 recB21 still reduced UV mutagenesis at low doses. The uvrD3 recB21 combination is therefore inhibiting activation of RecA protein. It is suggested that the DNA unwinding activity of the products of the uvrD and recB genes may be involved in generating single-stranded DNA needed to activate RecA protein both for the cleavage of LexA repressor and for a further role in UV mutagenesis.     

The Genetic Dependence of Recombination in Recd Mutants of Escherichia Coli

RecBCD enzyme has multiple activities including helicase, exonuclease and endonuclease activities. Mutations in the genes recB or recC, encoding two subunits of the enzyme, reduce the frequency of many types of recombinational events. Mutations in recD, encoding the third subunit, do not reduce recombination even though most of the activities of the RecBCD enzyme are severely reduced. In this study, the genetic dependence of different types of recombination in recD mutants has been investigated. The effects of mutations in genes in the RecBCD pathway (recA and recC) as well as the genes specific for the RecF pathway (recF, recJ, recN, recO, recQ, ruv and lexA) were tested on conjugational, transductional and plasmid recombination, and on UV survival. recD mutants were hyper-recombinogenic for all the monitored recombination events, especially those involving plasmids, and all recombination events in recD strains required recA and recC. In addition, unlike recD+ strains, chromosomal recombination events and the repair of UV damage to DNA in recD strains were dependent on one RecF pathway gene, recJ. Only a subset of the tested recombination events were affected by ruv, recN, recQ, recO and lexA mutations.     

Genetic and Physical Analysis of Plasmid Recombination in Recb Recc Sbcb and Recb Recc Sbca Escherichia Coli K-12 Mutants

The effect of mutations in known recombination genes (recA, recB, recC, recE, recF, recJ, recN, recO, recQ and ruv) on intramolecular recombination of plasmids was studied in recB recC sbcB and recB recC sbcA Escherichia coli mutants. The rate of recombination of circular dimer plasmids was at least 1000-fold higher in recB recC sbcB or recB recC sbcA mutants as compared to wild-type cells. The rate was decreased by mutations in recA, recF, recJ, recO, ruv or mutS in recB recC sbcB mutants, and by mutations in recE, recN, recO, recQ, ruv or mutS in recB recC sbcA mutants. In addition to measuring the recombination rate of circular dimer plasmids, the recombination-mediated transformation of linear dimer plasmids was also studied. Linear dimer plasmids transformed recB recC sbcB and recB recC sbcA mutants 20- to 40-fold more efficiently than wild-type cells. The transformation efficiency of linear dimer plasmids in recB recC sbcB mutants was decreased by mutations in recA, recF, recJ, recO, recQ or lexA (lexA3). In recB recC sbcA mutants the transformation efficiency of linear dimers was decreased only by a recE mutation. Physical analysis of linear dimer- or circular dimer-transformed recB recC sbcB mutants revealed that all transformants contained recombinant monomer genotypes. This suggests that recombination in recB recC sbcB cells is very efficient.     

Genetic recombination of bacterial plasmid DNA: effect of RecF pathway mutations on plasmid recombination in Escherichia coli.

Tn5 insertion mutations in the recN gene, and in what appears to be a new RecF pathway gene designated recO and mapping at approximately 55.4 min on the standard genetic map, were isolated by screening Tn5 insertion mutations that cotransduced with tyrA. The recO1504::Tn5 mutation decreased the frequency of recombination during Hfr-mediated crosses and increased the susceptibility to killing by UV irradiation and mitomycin C when present in a recB recC sbcB background, but only increased the sensitivity to killing by UV irradiation when present in an otherwise Rec+ background. The effects of these and other RecF pathway mutations on plasmid recombination were tested. Mutations in the recJ, recO, and ssb genes, when present in otherwise Rec+ E. coli strains, decreased the frequency of plasmid recombination, whereas the lexA3, recAo281, recN, and ruv mutations had no effect on plasmid recombination. Tn5 insertion mutations in the lexA gene increased the frequency of plasmid recombination. These data indicate that plasmid recombination events in wild-type Escherichia coli strains are catalyzed by a recombination pathway that is related to the RecF recombination pathway and that some component of this pathway besides the recA gene product is regulated by the lexA gene product.     

Quantitation of the involvement of the recA, recB, recC, recF, recJ, recN, lexA, radA, radB, uvrD, and umuC genes in the repair of X-ray-induced DNA double-strand breaks in Escherichia coli

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

Genetic analysis of conjugational recombination in Escherichia coli K12 strains deficient in RecBCD enzyme

Conjugational recombination in Escherichia coli was investigated by comparing the effects of recN, recO, ruv and lexA mutations on the formation of recombinants in crosses with strains lacking RecBCD enzyme. The results presented reveal that recN and ruv mutations do not abolish residual recombination in a recB mutant, and have only a rather modest effect on recombination in recBC sbcA strains; in these respects they are quite different from recF, recJ and recO mutations. The differences between these two groups of genes are discussed in relation to the molecular exchanges needed to produce viable recombinants.     

The Salmonella typhimurium LT2 uvrD gene is regulated by the lexA gene product.

The uvrD gene product apparently plays a role in the repair of UV damage, in mismatch repair, and in genetic recombination. A lower level of expression of the Salmonella typhimurium LT2 uvrD gene was observed in maxicells prepared from an Escherichia coli strain that contained a lexA+ plasmid than in maxicells prepared from an E. coli strain that lacked functional LexA protein. These results suggest that the uvrD+ gene is repressed by the LexA protein and is thus a member of the set of genes whose expression is increased by "SOS"-inducing treatments.     

A mutant allele gam18, participating in the RecF repair path in Escherichia coli K-12]

Plasmid pGam18 carrying one of the cloned mutant loci, responsible for enhanced radiation resistance in the strain Escherichia coli Gamr444, was shown to increase resistance to the lethal effect of gamma-rays with a dose modification factor DMF = 2. Enhanced resistance was observed in wild-type cells and in the mutant recBC sbcB, but not recFBC sbcA. This indicates the involvement of a product of the gam18 locus in the RecF pathway of recombinational repair. The protective effect of plasmid pGam18 against radiation was completely abolished by mutations in the most RecF pathway genes (recF, recJ, recR, recO, recQ, recN, and ruvB). However, three mutations in the uvrD gene, which encodes DNA helicase II and belongs to the RecF pathway, can be partially complemented by plasmid pGam18. These data suggest that the mutant allele gam18 affects the DNA helicase II activity at the presynaptic stage of the RecF pathway-mediated repair of DNA double-stranded breaks induced by gamma-irradiation.     

The partial recA-lexA dependence of the adaptive response of Escherichia coli to exposure to methylmethane sulfonate

A study was made of the adaptive response to methylmethane sulfonate (MMS) in E. coli. (18 strains of B, WP2, and H/r30 groups, including three strains of bacteria with pKM101 plasmid). The adaptation of wild type cells and uvrA- and uvrB- mutants to non-lethal concentrations of MMS (10-30 mkg/ml during 90-120 min) leads to a significant increase in their resistance to lethal MMS concentrations (10-30 mM for 10-120 min): the dose modifying factor (DMF) being 1.5-1.8. In single recA or lexA mutants (or double recA uvr- and lexA uvr- mutants) the efficiency of adaptive response to MMS was significantly lower: the DMF being 1.1-1.2. In Bs-1 gamma R strain with intragenic suppressor of lexA gene the adaptive response efficiency was the same as in B/r (recA+lexA+) strain. There is no adaptive response to MMS in polA- strains. The adaptive response to MMS in E. coli is different from that to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and N-methylnitrosourea (MNM), because in these two cases it is absolutely lexA-recA dependent. It is supposed that a partial recA-lexA dependence of the adaptive response to MMS in E. coli may be due to a specific MMS-induced lethal damage that induces an adaptive repair non-related to the system of recA-lexA-independent adaptive responses to MNNG and MNM. The presence of a plasmid of drug resistance pKM101 exerts no influence on the value, efficiency and recA-lexA-dependence of the adaptive response of E. coli to MMS.     

Recombination is essential for viability of an Escherichia coli dam (DNA adenine methyltransferase) mutant

Double mutants of Escherichia coli dam (DNA adenine methyltransferase) strains with ruvA, ruvB, or ruvC could not be constructed, whereas dam derivatives with recD, recF, recJ, and recR were viable. The ruv gene products are required for Holliday junction translocation and resolution of recombination intermediates. A dam recG (Holliday junction translocation) mutant strain was isolated but at a very much lower frequency than expected. The inviability of a dam lexA (Ind(-)) host was abrogated by the simultaneous presence of plasmids encoding both recA and ruvAB. This result indicates that of more than 20 SOS genes, only recA and ruvAB need to be derepressed to allow for dam mutant survival. The presence of mutS or mutL mutations allowed the construction of dam lexA (Ind(-)) derivatives. The requirement for recA, recB, recC, ruvA, ruvB, ruvC, and possibly recG gene expression indicates that recombination is essential for viability of dam bacteria probably to repair DNA double-strand breaks. The effect of mutS and mutL mutations indicates that DNA mismatch repair is the ultimate source of most of these DNA breaks. The requirement for recombination also suggests an explanation for the sensitivity of dam cells to certain DNA-damaging agents.     

UV protection and mutagenesis in uvrD, uvrE and recL strains of Escherichia coli carrying the pKM101 plasmid.

The effect of the pKM101 plasmid on UV mutagenesis and survival was examined in DNA-repair-deficient strains of E. coli carrying the uvrD, uvrE and recL mutations. Although enhancement of UV mutagenesis by pKM101 was found in all 3 strains, UV protection was only observed in the uvrD strain. We conclude that the plasmid not only requires lexA+ recA+ functions of the cell, but also those of uvrE+ recL+ for its UV-protective effect.     

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.