Role of the radB gene in postreplication repair in UV-irradiated Escherichia coli uvrB

In UV-irradiated Escherichia coli, the radB101 mutation sensitized uvrB recF cells 4-fold and uvrB recB cells 1.2-fold, but did not sensitize uvrB recB recF cells. The radB mutation had very little effect (1.2-fold or less) on the repair of UV radiation-induced DNA daughter-strand gaps in uvrB cells, but it did cause about a 3-fold deficiency in the repair of the DNA double-strand breaks that arise in association with nonrepaired daughter-strand gaps in UV-irradiated uvrB recF cells. Thus, the radB gene does not appear to be involved in the recF-dependent or recF recB-independent processes for the repair of DNA daughter-strand gaps, but is involved in the recB-dependent postreplication repair of DNA double-strand breaks.     

recF-dependent and recF recB-independent DNA gap-filling repair processes transfer dimer-containing parental strands to daughter strands in Escherichia coli K-12 uvrB.

The processes for repairing DNA daughter-strand gaps were studied in UV-irradiated uvrB, uvrB recB, uvrB recF, and uvrB recB recF cells of Escherichia coli K-12. The dimer-containing parental DNA was found to be joined to daughter strands during postreplication repair in all four strains examined. Therefore, both the major (recF-dependent) and the minor (recF recB-independent) gap-filling processes repair DNA daughter-strand gaps by transferring parental strands into daughter strands.     

recA (Srf) suppression of recF deficiency in the postreplication repair of UV-irradiated Escherichia coli K-12.

The mechanism by which recA (Srf) mutations (recA2020 and recA801) suppress the deficiency in postreplication repair shown by recF mutants of Escherichia coli was studied in UV-irradiated uvrB and uvrA recB recC sbcB cells. The recA (Srf) mutations partially suppressed the UV radiation sensitivity of uvrB recF, uvrB recF recB, and uvrA recB recC sbcB recF cells, and they partially restored the ability of uvrB recF and uvrA recB recC sbcB recF cells to repair DNA daughter-strand gaps. In addition, the recA (Srf) mutations suppressed the recF deficiency in the repair of DNA double-strand breaks in UV-irradiated uvrA recB recC sbcB recF cells. The recA2020 and recA801 mutations do not appear to affect the synthesis of UV radiation-induced proteins, nor do they appear to produce an altered RecA protein, as detected by two-dimensional gel electrophoresis. These results are consistent with the suggestion (M. R. Volkert and M. A. Hartke, J. Bacteriol. 157:498-506, 1984) that the recA (Srf) mutations do not act by affecting the induction of SOS responses; rather, they allow the RecA protein to participate in the recF-dependent postreplication repair processes without the need of the RecF protein.     

A minor pathway of postreplication repair in Escherichia coli is independent of the recB, recC and recF genes

After ultraviolet (UV) irradiation, an Escherichia coli K12 uvrB5 recB21 recF143 strain (SR1203) was able to perform a limited amount of postreplication repair when incubated in minimal growth medium (MM), but not if incubated in a rich growth medium. Similarly, this strain showed a higher survival after UV irradiation if plated on MM versus rich growth medium (i.e., it showed minimal medium recovery (MMR]. In fact, its survival after UV irradiation on rich growth medium was similar to that of a uvrB5 recA56 strain, which does not show MMR or postreplication repair. The results obtained with a uvrB5 recF332::Tn3 delta recBC strain and a uvrB5 recF332::Tn3 recB21 recC22 strain were similar to those obtained for strain SR1203, suggesting that the recB21 and recF143 alleles are not leaky in strain SR1203. The treatment of UV-irradiated uvrB5 recB21 recF143 and uvrB5 recF332::Tn3 delta recBC cells with rifampicin for 2 h had no effect on survival or the repair of DNA daughter-strand gaps. Therefore, a pathway of postreplication repair has been demonstrated that is constitutive in nature, is inhibited by postirradiation incubation in rich growth medium, and does not require the recB, recC and recF gene products, which control the major pathways of postreplication repair.     

Mechanisms for recF-dependent and recB-dependent pathways of postreplication repair in UV-irradiated Escherichia coli uvrB.

The molecular mechanisms for the recF-dependent and recB-dependent pathways of postreplication repair were studied by sedimentation analysis of DNA from UV-irradiated Escherichia coli cells. When the ability to repair DNA daughter strand gaps was compared, uvrB recF cells showed a gross deficiency, whereas uvrB recB cells showed only a small deficiency. Nevertheless, the uvrB recF cells were able to perform some limited repair of daughter strand gaps compared with a "repairless" uvrB recA strain. The introduction of a recB mutation into the uvrB recF strain greatly increased its UV radiation sensitivity, yet decreased only slightly its ability to repair daughter strand gaps. Kinetic studies of DNA repair with alkaline and neutral sucrose gradients indicated that the accumulation of unrepaired daughter strand gaps led to the formation of low-molecular-weight DNA duplexes (i.e., DNA double-strand breaks were formed). The uvrB recF cells were able to regenerate high-molecular-weight DNA from these low-molecular-weight DNA duplexes, whereas the uvrB recF recB and uvrB recA cells were not. A model for the recB-dependent pathway of postreplication repair is presented.     

Postreplicational formation and repair of DNA double-strand breaks in UV-irradiated Escherichia coli uvrB cells

The number of DNA double-strand breaks formed in UV-irradiated uvrB recF recB cells correlates with the number of unrepaired DNA daughter-strand gaps, and is dependent on DNA synthesis after UV-irradiation. These results are consistent with the model that the DNA double-strand breaks that are produced in UV-irradiated excision-deficient cells occur as the result of breaks in the parental DNA opposite unrepaired DNA daughter-strand gaps. By employing a temperature-sensitive recA200 mutation, we have devised an improved assay for studying the formation and repair of these DNA double-strand breaks. Possible mechanisms for the postreplication repair of DNA double-strand breaks are discussed.     

Different effects of recJ and recN mutations on the postreplication repair of UV-damaged DNA in Escherichia coli K-12.

Two mutations known to affect recombination in a recB recC sbsBC strain, recJ284::Tn10 and recN262, were examined for their effects on the postreplication repair of UV-damaged DNA. The recJ mutation did not affect the UV radiation sensitivity of uvrB and uvrB recF cells, but it increased the sensitivity of uvrB recN (approximately 3-fold) and uvrB recB (approximately 8-fold) cells. On the other hand, the recN mutation did not affect the UV sensitivity of uvrB recB cells, but it increased the sensitivity of uvrB (approximately 1.5-fold) and uvrB recF (approximately 4-fold) cells. DNA repair studies indicated that the recN mutation produced a partial deficiency in the postreplication repair of DNA double-strand breaks that arise from unrepaired daughter strand gaps, while the recJ mutation produced a deficiency in the repair of daughter strand gaps in uvrB recB cells (but not in uvrB cells) and a deficiency in the repair of both daughter strand gaps and double-strand breaks in uvrA recB recC shcBC cells. Together, these results indicate that the recJ and recN genes are involved in different aspects of postreplication repair.     

Mechanism of sbcB-suppression of the recBC-deficiency in postreplication repair in UV-irradiated Escherichia coli K-12

Summary The mechanism by which an sbcB mutation suppresses the deficiency in postreplication repair shown by recB recC mutants of Escherichia coli was studied. The presence of an sbcB mutation in uvrA recB recC cells increased their resistance to UV radiation. This enhanced resistance was not due to a suppression of the minor deficiency in the repair of DNA daughter-strand gaps or to an inhibition of the production of DNA double-strand breaks in UV-irradiated uvrA recB recC cells; rather, the presence of an sbcB mutation, enabled uvrA recB recC cells to carry out the repair of DNA double-strand breaks. In the uvrA recB recC sbcB background, a mutation, at recF produced a huge sensitization to UV radiation, and it rendered cells deficient in the repair of both DNA daughter-strand gaps and DNA double-strand breaks. Thus, an additional sbcB mutation in uvrA recB recC cells restored their ability to perform the repair of DNA double-strand breaks, but the further addition of a recF mutation blocked this repair capacity.     

Post-replication repair and recombination in uvrA umuC strains of Escherichia coli are enhanced by vanillin, an antimutagenic compound

Effects of vanillin on UV killing of umuC mutant strains of E. coli were investigated in order to analyze the antimutagenic role of vanillin in mutagenesis. UV-irradiated uvrA umuC cells showed higher survival when plated on medium containing vanillin rather than medium without vanillin. This increased survival associated with exposure to vanillin was observed more clearly in uvrA umuC lexA(Ind-) and uvrA umuC recF strains. However, the effect was inhibited by additional recB recC mutations and completely blocked by an additional recA mutation. As far as tested the increased survival of UV-treated cells by vanillin was dependent on a capacity for genetic recombination. The effect of vanillin on recombination frequency between 2 plasmid DNA, pATH4 (Cmr Tcs) and pBMX7 (Apr Tcs), in a uvrA umuC background was investigated. A significantly higher frequency of plasmid recombination was observed when vanillin was present in the culture medium. These findings suggest that the antimutagenic effect of vanillin is the result of enhancement of a recA-dependent, error-free, pathway of post-replication repair.     

Plasmid pGW16, a derivative of pKM101, increases post-UV DNA synthesis, but sensitises some strains of Escherichia coli to UV

Plasmid pKM101, which carries muc genes that are analogous in function to chromosomal umu genes, protected Escherichia coli strains AB1157 uvrB+ umuC+, JC3890 uvrB umuC+, TK702 uvrB+ umuC and TK501 uvrB umuC against ultraviolet irradiation (UV). Plasmid pGW16, a derivative of pKM101 selected for its increased spontaneous mutator effect, also gave some protection to the UmuC-deficient strains, TK702 and TK501. However, it sensitised the wild-type strain AB1157 to low, but protected against high doses of UV, whilst sensitising strain JC3890 to all UV doses tested. Even though its UV-protecting effects varied, pGW16 was shown to increase both spontaneous and UV-induced mutation in all strains. Another derivative of pKM101, plasmid pGW12, was shown to have lost all spontaneous and UV-induced mutator effects and did not affect post-UV survival. Plasmids pKM101 and pGW16 increased post-UV DNA synthesis in strains AB1157 and TK702, whereas pGW12 had no effect. Similarly, the wild-type UV-protecting plasmids R46, R446b and R124 increased post-UV DNA synthesis in strain TK501, but the non-UV-protecting plasmids R1, RP4 and R6K had no effect. These results accord with the model for error-prone DNA repair that requires umu or muc gene products for chain elongation after base insertion opposite non-coding lesions. They also suggest that the UV-sensitizing effects of pGW16 on umu+ strains can be explained in terms of overactive DNA repair resulting in lethal, rather than repaired UV-induced lesions.     

The nature of DNA damage and its repair after treatment of bacteria with dioxidine

The nature of the lethal effect of antimicrobial drug dioxidin was studied. The treatment of bacterial cells by dioxidin results in an instant repression of DNA synthesis and formation of single strand gaps in DNA molecule. The repair of single strand gaps in polA+ cells involves the DNA polymerase I. The deficit of this enzyme leads to the increased degradation of DNA. The products of the recA, polA1, lexA, recB are relevant for bacterial resistance to dioxidin while the products of uvrA, uvrE and recF genes are not. On the basis of the obtained data dioxidin may be defined as a "gamma-type" agent due to the nature of dioxidin-induced lesions in DNA and their repair.     

Effects of the ssb-1 and ssb-113 mutations on survival and DNA repair in UV-irradiated delta uvrB strains of Escherichia coli K-12.

The molecular defect in DNA repair caused by ssb mutations (single-strand binding protein) was studied by analyzing DNA synthesis and DNA double-strand break production in UV-irradiated Escherichia coli delta uvrB strains. The presence of the ssb-113 mutation produced a large inhibition of DNA synthesis and led to the formation of double-strand breaks, whereas the ssb-1 mutation produced much less inhibition of DNA synthesis and fewer double-strand breaks. We suggest that the single-strand binding protein plays an important role in the replication of damaged DNA, and that it functions by protecting single-stranded parental DNa opposite daughter-strand gaps from nuclease attack.     

Effects of recB21, recF143, and uvrD152 on recombination in lambda bacteriophage-prophage and Hfr by F- crosses.

The effects of the mutation pairs recB21 recF143 and recB21 uvrD152 on the frequency of genetic recombination were investigated in lambda phage-prophage crosses under homoimmune conditions. To prevent recombinants from being formed by the phage red system, these experiments were performed with phages and prophages carrying red and gam mutations. Both spontaneous and damage-induced recombination was measured, the phages being either undamaged or treated with trimethylpsoralen and 360-nm light to cross-link the phage DNA. Control and damaged phages were allowed to infect lysogenic host cells under conditions in which phage gene expression was repressed and phage DNA replication was blocked by lambda immunity. Although the double mutations recB21 recF143 and recB21 uvrD152 reduced recombination in Hfr by F- crosses to 0.3 to 0.02% of the wild-type controls, the presence of these pairs of mutations in the host lysogens had relatively little effect on the results of the phage-prophage crosses. In the latter system, recB21 recF143 reduced spontaneous and damaged-induced recombination by less than threefold whereas recB21 uvrD152 increased it to three times the wild-type level, the increase being attributable to the uvrD mutation. Evidently, the gene products of recB,C uvrD, and recF wee not needed for lambda phage-prophage recombination under repressed conditions.     

recA-dependent DNA repair processes

UV-radiation-induced lesions in DNA result in the formation of: (1) excision gaps (i.e. a lesion is excised, leaving a gap), (2) daughter-strand gaps (i.e. a lesion can be skipped during replication, leaving a gap), and (3) double-strand breaks (i.e. the DNA strand opposite a gap can be cut). In Escherichia coli, the recA gene product is involved in repairs of all three types of lesions--repair of daughter-strand gaps (2) and double-strand breaks (3) constitutes post-replication repair. The evidence suggests, furthermore, that recA-dependent repair of excision gaps (1) produced in DNA replicated prior to UV irradiation (pre-replication repair) appears to occur by similar mechanisms.     

The effect of catalase on recovery of heat-injured DNA-repair mutants of Escherichia coli

The apparent sensitivity of Escherichia coli K12 to mild heat was increased by recA (def), recB and polA, but not by uvrA, uvrB or recF mutations. However, addition of catalase to the rich plating medium used to assess viability restored counts of heat-injured recA, recB and polA strains to wild-type levels. E. coli p3478 polA was sensitized by heat to a concentration of hydrogen peroxide similar to that measured in autoclaved recovery medium. The apparent heat sensitivity of DNA-repair mutants is thus due to heat-induced sensitivity to the low levels of peroxide present in rich recovery media. It is proposed that DNA damage in heated cells could occur indirectly by an oxidative mechanism. The increased peroxide sensitivity of heat-injured cells was not due to a decrease in total catalase activity but may be related specifically to inactivation of the inducible catalase/peroxidase (HPI).     

Involvement of recA and exr Genes in the In Vivo Inhibition of the recBC Nuclease

When Escherichia coli cells are gamma irradiated they degrade their deoxyribonucleic acid (DNA). The DNA of previously gamma-irradiated T4 phage is also degraded in infected cells. The amount of degradation is not only dependent on the dose but also on the genotype of the cell. The amount of degradation is less in cells carrying a recB or a recC mutation, suggesting that most of the DNA degradation is due to the recB(+) and recC(+) gene product (exonuclease V). In some strains a previous dose of ultraviolet (UV) light followed by incubation renders the cells resistant to DNA degradation after gamma irradiation. We have shown this inhibition to take place for infecting T4 phage also. By using six strains of E. coli selected for mutations in the genes recA, exr (or lex), and uvrB, we have been able to show that the preliminary UV treatment produces no change in recA and exr cells for both endogenous DNA degradation and the degradation of infecting irradiated T4 phage DNA, i.e., inhibition was not detected in these strains. On the other hand, wild-type cells and strains carrying mutations of uvrB show inhibition in both types of experiments. Because the recA gene product and the exr(+) (lex(+)) gene product are necessary for the induction of prophage, it is possible that the phenomenon of inducible inhibition requires recA(+) and exr(+) presence. One interpretation of these results is that an inducible inhibitor may be controlled by the exr gene.     

Analysis of the antimutagenic effect of cinnamaldehyde on chemically induced mutagenesis in Escherichia coli

The antimutagenic effect of cinnamaldehyde on mutagenesis was investigated using ten kinds of chemical mutagen in Escherichia coli WP2s (uvr A-). In addition, the frequency of mutation induction by each mutagen in an SOS repair deficient (umuC-) strain was compared with that in a wild-type (umuC+) strain. Cinnamaldehyde greatly suppressed the umuC-dependent mutagenesis induced by 4-nitroquinoline 1-oxide (4-NQO), furylfuramide or captan. However, cinnamaldehyde was less effective against the umuC-independent mutagenesis by alkylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine and ethylmethanesulfonate. On the other hand, no inhibitory effect of cinnamaldehyde was observed on prophage induction or tif-mediated filamentous growth. These results suggest that a cinnamaldehyde does not prevent the induction of the SOS functions. Despite the decrease in the number of revertants, a remarkable increase was observed in the survival of 4-NQO-treated WP2s cells after exposure to cinnamaldehyde. The reactivation of survival suggests the promotion of some DNA repair system by cinnamaldehyde. This enhancement of survival was also observed in uvr B, polA, recF or umuC mutants and less in lexA or recB, C mutants. However, it was not observed in recA mutants. Therefore, we assume that cinnamaldehyde may enhance an error-free recombinational repair system by acting on recA-enzyme activity.     

Effect of rec mutations on viability and processing of DNA damaged by methylmethane sulfonate in xth nth nfo cells of Escherichia coli

The role of recombination genes in the processing of DNA damaged by methlymethane sulfonate (MMS) was examined in an xth nth nfo strain of Escherichia coli K-12. Introduction of a recQ mutation did not increase the cell's sensitivity to MMS treatment. The presence of recF, recJ or recN mutation slightly increased the cell's sensitivity to MMS treatment. The introduction of recA or recB mutation into the cells led to inviability. Taken together, we suggest that replication of DNA containing apurinic/apyrimidinic (AP) sites in vivo will lead to the formation of secondary lesions. The repair of these secondary lesions requires the function of recA and recB genes, but does not appear to require recF, recJ, recQ or recN genes.     

Effect of deficiency in excision repair and umuC function on the mutagenicity with ethylene oxide in the lacI gene of E. coli

The influence of uvrB and umuC genes on the induction of lacI- mutants and nonsense mutants by ethylene oxide (EtO) in the lacI gene of E. coli was studied. The uvrB mutation was characterized by much higher mutation frequencies. In contrast the umuC mutation does not significantly affect the induction kinetics. Thus mutation by EtO is enhanced by the lack of excision repair but not influenced by error-prone repair.