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.     

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.     

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.     

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.     

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.     

Role of the umuC gene in postreplication repair in UV-irradiated Escherichia coli K-12 uvrB

The role of the umuC gene product in postreplication repair was studied in UV-irradiated Escherichia coli K-12 uvrB cells. A mutation at umuC increased the UV radiation sensitivities of uvrB, uvrB recF, uvrB recB, and uvrB recF recB cells; it also increased the deficiencies in the repair of DNA daughter-strand gaps in these strains, but it did not affect the repair of DNA double-strand breaks that arose from unrepaired DNA daughter-strand gaps. We suggest that the umuC gene product is involved in a minor system for the repair of DNA daughter-strand gaps, possibly the repair of overlapping DNA daughter-strand gaps.     

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.     

Role of DNA polymerase I in postreplication repair: a reexamination with Escherichia coli delta polA.

Using strains of Escherichia coli K-12 that are deleted for the polA gene, we have reexamined the role of DNA polymerase I (encoded by polA) in postreplication repair after UV irradiation. The polA deletion (in contrast to the polA1 mutation) made uvrA cells very sensitive to UV radiation; the UV radiation sensitivity of a uvrA delta polA strain was about the same as that of a uvrA recF strain, a strain known to be grossly deficient in postreplication repair. The delta polA mutation interacted synergistically with a recF mutation in UV radiation sensitization, suggesting that the polA gene functions in pathways of postreplication repair that are largely independent of the recF gene. When compared to a uvrA strain, a uvrA delta polA strain was deficient in the repair of DNA daughter strand gaps, but not as deficient as a uvrA recF strain. Introduction of the delta polA mutation into uvrA recF cells made them deficient in the repair of DNA double-strand breaks after UV irradiation. The UV radiation sensitivity of a uvrA polA546(Ts) strain (defective in the 5'----3' exonuclease of DNA polymerase I) determined at the restrictive temperature was very close to that of a uvrA delta polA strain. These results suggest a major role for the 5'----3' exonuclease activity of DNA polymerase I in postreplication repair, in the repair of both DNA daughter strand gaps and double-strand breaks.     

Suppression of the UV-sensitive phenotype of Escherichia coli recF mutants by recA(Srf) and recA(Tif) mutations requires recJ+.

Mutations in recA, such as recA801(Srf) (suppressor of RecF) or recA441(Tif) (temperature-induced filamentation) partially suppress the deficiency in postreplication repair of UV damage conferred by recF mutations. We observed that spontaneous recA(Srf) mutants accumulated in cultures of recB recC sbcB sulA::Mu dX(Ap lac) lexA51 recF cells because they grew faster than the parental strain. We show that in a uvrA recB+ recC+ genetic background there are two prerequisites for the suppression by recA(Srf) of the UV-sensitive phenotype of recF mutants. (i) The recA(Srf) protein must be provided in increased amounts either by SOS derepression or by a recA operator-constitutive mutation in a lexA(Ind) (no induction of SOS functions) genetic background. (ii) The gene recJ, which has been shown previously to be involved in the recF pathway of recombination and repair, must be functional. The level of expression of recJ in a lexA(Ind) strain suffices for full suppression. Suppression by recA441 at 30 degrees C also depends on recJ+. The hampered induction by UV of the SOS gene uvrA seen in a recF mutant was improved by a recA(Srf) mutation. This improvement did not require recJ+. We suggest that recA(Srf) and recA(Tif) mutant proteins can operate in postreplication repair independent of recF by using the recJ+ function.     

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.     

Amplified Rnase H Activity in Escherichia coli B/R Increases Sensitivity to Ultraviolet Radiation

Strains of E. coli B/r transformed with the plasmid pSK760 were found to be sensitized to inactivation by ultraviolet radiation (UV) and to have elevated levels of RNase H activity. Strains transformed with the carrier vector pBR322 or the plasmid pSK762C derived from pSK760 but with an inactivated rnh gene were not sensitized. UV-inactivation data for strains having known defects in DNA repair and transformed with pSK760 suggested an interference by RNase H of postreplication repair: uvrA cells were strongly sensitized, wild-type and uvrA recF cells were moderately sensitized and recA cells were not sensitized; and minimal medium recovery was no longer apparent in sensitized uvrA cells. Biochemical studies showed that post-UV DNA synthesis was sensitized and that the smaller amounts of DNA synthesized after irradiation, while of normal reduced size as indicated by sedimentation position in alkaline sucrose gradients, did not shift to a larger size (more rapidly sedimenting) upon additional incubation. We suggest an excess level of RNase H interferes with reinitiation of DNA synthesis on damaged templates to disturb the normal pattern of daughter strand gaps and thereby to inhibit postreplication repair.     

Characterization of an Escherichia coli mutant (radB101) sensitive to gamma and uv radiation, and methyl methanesulfonate

After N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis of Escherichia coli K-12 (xthA14), and X-ray-sensitive mutant was isolated. This sensitivity is due to a mutation, radB101, which is located at 56.5 min on the E. coli K-12 linkage map. The radB101 mutation sensitized wildtype cells to gamma and uv radiation, and to methyl methanesulfonate. When known DNA repair-deficient mutants were ranked for their gamma-radiation sensitivity relative to their uv-radiation sensitivity, their order was (starting with the most selectively gamma-radiation-sensitive strain): recB21, radB101, wild type, polA1, recF143, lexA101, recA56, uvrD3, and uvrA6. The radB mutant was normal for gamma- and uv-radiation mutagenesis, it showed only a slight enhancement of gamma- and uv-radiation-induced DNA degradation, and it was approximately 60% deficient in recombination ability. The radB gene is suggested to play a role in the recA gene-dependent (Type III) repair of DNA single-strand breaks after gamma irradiation and in postreplication repair after uv irradiation for the following reasons; the radB strain was normal for the host-cell reactivation of gamma- and uv-irradiated bacteriophage lambda; the radB mutation did not sensitize a recA strain, but did sensitize a polA strain to gamma and uv radiation; the radB mutation sensitized a uvrB strain to uv radiation.     

Non-mutagenic and mutagenic post-replicative DNA repair in prokaryotic and eukaryotic cells

The review is devoted to mechanisms of repair gaps in DNA daughter strand, formed during the stall of moving replication forks and restart of replication in cells after the action of DNA damaging agents (predominantly--UV light). The repair of daughter DNA, or postreplication DNA repair (PRR), is realized by error-free (non-mutagenic) and error-prone (mutagenic) pathways. The former is a recombination repair, or recombination between two sister duplexes. By this way the major part of postreplication gaps is eliminated. The second way is related with the induction of SOS-response. In Escherichia coli cells mutagenic SOS-response is realized by proteins RecA, UmuD, UmuC, DNA-polymerase III holoenzyme and others. In E. coli some mutagenic enzymes--DNA-polymerase IV (the product of dinB gene) and DNA-polymerase V (the product of umuDC genes) have been recently discovered. In Saccharomyces cerevisiae cells postreplicative translesion synthesis is realized by newly discovered enzymes deoxycytidilmonophosphatetransferase (encoded by REV1 gene), DNA-polymerase zeta (encoded by REV3 gene), DNA-polymerase eta (encoded by RAD30 gene). All the three enzymes share a great homology with UmuC enzyme of E. coli. DNA polymerase eta correctly inserts adenine residues in the daughter strand opposite noncoded thymine residues in cyclobutane pyrimidine dimer. Based on RAD6 gene of S. cerevisiae, human cells hREV1, hREV3 and hRAD30A have been obtained to encode, respectively, deoxycytidiltransferase, DNA-polymerase zeta and DNA-polymerase eta. It has been shown that the defect of PRR DNA in xeroderma pigmentosum variant is associated with DNA-polymerase eta deficiency. This defect is corrected by the extract of intact HeLa cells. The importance of newly discovered enzymes in the system of mechanisms of DNA repair and replication is discussed.     

Suppression of Escherichia coli recF mutations by recA-linked srfA mutations.

Suppressors of recF (srfA) were found by selection for resistance to mitomycin C and UV irradiation in a recB21 recC22 sbcB15 recF143 strain. srfA mutations map in recA and are dominant to srfA+. They suppress both the DNA repair and the recombination deficiencies due to recF mutations. Therefore, RecA protein which is altered by the srfA mutation can allow genetic recombination to proceed in the absence of recB, recC, and recF functions. recF is also required for induction of the SOS response after UV damage. We propose that recF+ normally functions to allow the expression of two recA activities, one that is required for the RecF pathway of recombination and another that is required for SOS induction. The two RecA activities are different and are separable by mutation since srfA mutations permit recombination to proceed but have not caused a dramatic increase in SOS induction in recF mutants. According to this hypothesis, one role for recF in DNA repair and recombination is to modulate RecA activities to allow RecA to participate in these recF-dependent processes.     

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.     

Survival of recombination-deficient mutants of Escherichia coli during incubation with nalidixic acid.

The ability of several Escherichia coli strains deficient in recombination (rec) to survive in the presence of nalidixic acid was determined. Genetic blocks of the RecBC or the RecF pathways resulted in increased sensitivity to nalidixic acid when compared with the wild-type strain. Mutants lacking functional recA, recL, or recB recC recF genes showed the most rapid decrease in colony-forming ability when incubated with nalidixic acid. However, the uvrB gene also plays a role in maintaining cell viability.     

Repair of cross-linked DNA and survival of Escherichia coli treated with psoralen and light: effects of mutations influencing genetic recombination and DNA metabolism.

Repair of cross-linked DNA was studied in Escherichia coli strains carrying mutations affecting DNA metabolism. In wild-type cells, DNA strands cut during cross-link removal were rejoined during a subsequent incubation into high-molecular-weight molecules. This rejoining was dependent on gene products involved in genetic recombination. A close correlation was found relating recombination proficiency, the rate of strand rejoining, and formation of viable progeny after DNA cross-linking by treatment with psoralen and light. Wild-type cells and other mutants which were Rec+ (sbcB, recL, recL sbcB, recB recC sbcA, recB recC sbcB, xthA1, and xthA11) rejoined cut DNA strands at a rate of 0.8 +/- 0.1 min -1 at 37 degrees C and survived 53 to 71 cross-links per chromosome. recB, recC, recB recC, recF, or polA strains showed reduced rates of strand rejoining and survived 4 to 13 cross-links per chromosome. Recombination-deficient strains (recA, recB recC sbcB recF, recB recL) and lexA failed to rejoin DNA strands after crosslink removal and were unable to form colonies after treatments producing as few as one to two cross-links per chromosome. Strand rejoining occurred normally in cells with mutations affecting DNA replication (dnaA, danB, dnaG, and dnaE) under both permissive and nonpermissive conditions for chromosome replication. In a polA polB dnaE strain strand rejoining occurred at 32 degree C but not at 42 degree C, indicating that some DNA synthesis was required for formation of intact recombinant molecules.     

Recombinational DNA repair: the ignored repair systems

The recent finding of a role for the recA gene in DNA replication restart does not negate previous data showing the existence of recA-dependent recombinational DNA repair, which occurs when there are two DNA duplexes present, as in the case for recA-dependent excision repair, for postreplication repair (i.e., the repair of DNA daughter-strand gaps), and for the repair of DNA double-strand breaks. Recombinational DNA repair is critical for the survival of damaged cells.     

Effects of the Escherichia coli recF suppressor mutation, recA801, on recF-dependent DNA-repair associated phenomena

In recb recC sbcB mutants genetic recombination is dependent upon the recF gene. recA801, recA802 and recA803 (formerly called srfA mutations) were originally isolated as mutations that suppress recombination deficiency caused by a recF mutation in a recB recC sbcB genetic background. Since the recA801 mutation also suppressed some of the UV sensitivity due to recF143, we sought to determine what DNA-repair pathways were actually being restored by the recA801 mutation in this genetic background. In this paper we show that the suppression of recF143 by recA801 does not extend to the recF143-mediated defects in induced repair of UV-damaged phages. In addition, we show that recA801 suppresses only slightly the recF143-associated defect in induced expression of the SOS-regulated muc genes of pKM101. These results suggest that recA801 suppresses primarily the RecF pathway of recombinational repair.