Conditional Lethality of recA and recB Derivatives of a Strain of Escherichia coli K-12 with a Temperature-Sensitive Deoxyribonucleic Acid Polymerase I

We have isolated a strain of Escherichia coli K-12 carrying a mutation, polA12, that results in the synthesis of a temperature-sensitive deoxyribonucleic acid (DNA) polymerase I. The double mutants polA12 recA56 and polA12 recB21, constructed at 30 C, are inviable at 42 C. About 90% of the cells of both double mutants die after 2 hr of incubation at 42 C. Both double mutants filament at 42 C and show a dependence on high cell density for growth at 30 C. In polA12 recB21 cells at 42 C, DNA and protein synthesis gradually stop in parallel. In polA12 recA56 cells, DNA synthesis continues for at least 1 hr at 42 C, and there is extensive DNA degradation. The results suggest that the primary lesion in these double mutants is not in DNA replication per se.     

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.     

Restoration of viability to an Escherichia coli mutant deficient in the 5'----3' exonuclease of DNA polymerase I.

An Escherichia coli polA (Ex) mutant that is usually inviable at restrictive temperatures (43 degrees C) was found to grow normally at 43 degrees C when incubated in the presence of a membrane-containing fraction prepared from E. coli. This membrane fraction causes anaerobic conditions that are necessary but not sufficient for restoration of viability since some component present in the membrane fraction is also required for colony formation at 43 degrees C. The accumulation of small DNA fragments typical of aerobic growth of the polA(Ex) mutant was also seen under anaerobic conditions. The polA(Ex) strain was also much more sensitive than the isogenic wild-type strain to hydrogen peroxide.     

Involvement of the recB-recC Nuclease (Exonuclease V) in the Process of X-Ray-Induced Deoxyribonucleic Acid Degradation in Radiosensitive Strains of Escherichia coli K-12

The ras, polA, exrA, recA, and uvrD3 strains of Escherichia coli K-12 degrade their deoxyribonucleic acid more extensively than wild-type strains after X irradiation. The relationship of the recB-recC nuclease (exonuclease V) to the degradation process in these strains was determined by comparing the degradation response of the original strains with that of strains containing an additional recB21 or recC22 mutation. The initial rate of degradation in ras, polA12, exrA, and recA13 strains after an exposure of 20 to 30 kR was reduced more than 10-fold by the presence of an additional recB21 or recC22 mutation. The extent of degradation in these irradiated strains after 90 to 120 min of incubation was reduced two- to fivefold. In the uvrD3 strain, a recC22 mutation caused a fourfold decrease in initial degradation rate and reduced the extent of degradation after 90 min of incubation by a factor of 1.6. The results are consistent with the statement that the degradation process is normally dependent on exonuclease V activity. However, the observation that 10 to 30% degradation always occurred even in recB or recC strains, which lack this enzyme, suggests that alternative degradation mechanisms exist.     

Genetic Analysis of a Temperature-Resistant Revertant of the Conditional Lethal Escherichia coli Double Mutant polA12 uvrE502

Conditional lethality of the Escherichia coli polA12 uvrE502 double mutant may be overcome by a mutation that has been termed polA350. The polA350 mutation restored the polymerizing activity of deoxyribonucleic acid polymerase I at 42 C in the polA12 mutant and partially suppressed ultraviolet (UV) and methylmethane sulfonate sensitivities of the polA12. Mapping experiments have located polA350 between metE and polA12, very close to the latter. The strain carrying polA12 polA350 and recB21 was viable at 42 C. The effects of the recB21 and polA12 polA350 combination on the UV sensitivity were additive. The triple mutant polA12 polA350 uvrE502 was more UV sensitive than the single uvrE502 mutant.     

Influence of chromosome integrity on Escherichia coli cell division.

The antitumor agent cis-platinum(II)diamminodichloride (PDD) caused wild-type and recA+ deoxyribonucleic acid (DNA) repair-deficient mutant cells of Escherichia coli K-12 to grow as long, multinucleated filaments. At 5 micrograms/ml, the times required for reduction of viability to 37% for wild-type, polA, recB,C, uvrA, and recA organisms were > 200, 200, 120, 25, and 5 min, respectively. Only recA cells exhibited @reckless" degradation of DNA at this concentration of PDD. As shown by sedimentation in alkaline sucrose gradients, generation of single-strand breaks in DNA of the remaining organisms was a major consequence of growth in PDD. Upon incubation in fresh medium after removal of the compound and storage for 4 h at 4 degrees C, a respective lag of 3, 4, 6, and 9 h occurred before filaments of wild-type, polA, recB,C, and uvrA cells commenced cell division. Maintenance at 4 degrees C, which evidently delayed postshift initiation of chromosome replication, was only essential for fragmentation of uvrA filaments. In all cases, these periods of division delay corresponded to those required for restoration of normal chromosomal molecular weight as determined in alkaline sucrose gradients.     

In Vivo Studies of Temperature-Sensitive recB and recC Mutants

Some in vivo properties of Escherichia coli K-12 strains carrying recB270 (formerly recBts1) and recC271 (formerly recCts1) mutations have been determined. Single recB270 and recC271 mutants appear normal at 30 C with regard to ultraviolet and mitomycin C sensitivity, recombination proficiency, and viability. At 43 C these strains become sensitive to ultraviolet and mitomycin C, while showing only a slight decrease in recombination proficiency. The viable titers of the single mutants are somewhat reduced at 43 C. Double mutant strains carrying polA1 and recB270 or recC271 are inviable at 43 C. The double mutant strain (recB270 recC271) is sensitive to both UV and mitomycin C at 30 C, but shows only slightly reduced recombination proficiency. At 43 C the strain resembles absolute recB and recC mutants in all respects. In addition, the double mutant strain exhibits a temperature-induced drop in viable titer. The triple mutant polA1 recB270 recC271 is viable at 30 C. Two hypotheses are advanced to explain these results.     

Inviability of dam recA and dam recB cells of Escherichia coli is correlated with their inability to repair DNA double-strand breaks produced by mismatch repair.

The molecular basis for the inviability of dam-3 recA200(Ts) and dam-3 recB270(Ts) cells was studied. The dam-3 recA200(Ts) cells were inviable in yeast extract-nutrient broth or in minimal medium at 42 degrees C. Although the dam-3 recB270(Ts) cells were inviable in yeast extract-nutrient broth at 42 degrees C, they were viable at 42 degrees C in minimal medium, in which the high salt content suppresses the mutant phenotype caused by the recB270(Ts) mutation at 42 degrees C. Under the growth conditions rendering dam rec cells inviable, the cells accumulated double-strand breaks in their DNA. Introduction of a mutL or mutS mutation restored the viability of dam-3 recB270(Ts) cells grown in yeast extract-nutrient broth at 42 degrees C and eliminated the formation of DNA double-strand breaks in these cells. We conclude that the inability to repair DNA double-strand breaks produced by the mismatch repair process accounts for the inviability of the dam recA and dam recB cells.     

Inhibition of replication gap closure in Escherichia coli by near-ultraviolet light photoproducts of L-tryptophan

Near-ultraviolet photoproducts of l-tryptophan (TP) differentially inhibited deoxyribonucleic acid (DNA) replication in wild-type cells and uvrA, polA1, and recA recB double mutants of Escherichia coli. Wild-type cells labeled in their DNA with [(3)H]thymidine in the presence of TP produced small pieces of DNA (7 x 10(6) daltons), which corresponded in size to late replicative intermediates of discontinuous DNA synthesis. Moreover, when TP was present, it took five times longer to chase the low-molecular-weight DNA pieces into high-molecular-weight DNA. The observation of replicative intermediates in the presence of TP, and their slow chase into high-molecular-weight DNA in the presence of TP, is strong evidence that TP stabilizes replication gaps in E. coli DNA. Although TP slowed DNA replication in wild-type cells, this effect was transient and DNA synthesis eventually resumed at a normal rate. However, in polA1 and recA recB mutants, DNA synthesis was completely inhibited. Determinations of size and total counts of cells incubated in TP suggested that TP uncouples cell division from DNA replication in recA recB mutants, whereas these processes remain coupled in wild-type cells and in uvrA and polA1 mutants. The slow chase of TP-stabilized pieces of DNA in the presence of TP suggested that the selective effect of TP on DNA synthesis and viability in repair-deficient mutants is a result of TP inhibition of replication gap closure.     

Recombinant levels of Escherichia coli K-12 mutants deficient in various replication, recombination, or repair genes.

Escherichia coli strains containing mutations in lexA, rep, uvrA, uvrD, uvrE, lig, polA, dam, or xthA were constructed and tested for conjugation and transduction proficiencies and ability to form Lac+ recombinants in an assay system utilizing a nontandem duplication of two partially deleted lactose operons (lacMS286phi80dIIlacBK1). lexA and rep mutants were as deficient (20% of wild type) as recB and recC strains in their ability to produce Lac+ progeny. All the other strains exhibited increased frequencies of Lac+ recombinant formation, compared with wild type, ranging from 2- to 13-fold. Some strains showed markedly increased conjugation proficiency (dam uvrD) compared to wild type, while others appeared deficient (polA107). Some differences in transduction proficiency were also observed. Analysis of the Lac+ recombinants formed by the various mutants indicated that they were identical to the recombinants formed by a wild-type strain. The results indicate that genetic recombination in E. coli is a highly regulated process involving multiple gene products.     

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).     

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.     

Repair of near-ultraviolet (365 nm)-induced strand breaks in Escherichia coli DNA. The role of the polA and recA gene products.

The action of near-ultraviolet (UV-365 nm) radiation in cellular inactivation (biological measurements) and induction and repair of DNA strand breaks (physical measurements) were studied in a repair-proficient strain and in polA-, recA-, uvrA-, and polA uvrA-deficient strains of Escherichia coli K-12. The induction of breaks in the polA and polA uvrA strains was linear with dose (4.0 and 3.7 X 10(-5) breaks/2.5 X 10(9) daltons/Jm-2, respectively). However, in the recA-, uvrA-, and repair-proficient strains, there was an initial lag in break induction at low doses and then a linear induction of breaks at higher doses with rates of 4.6, 2.8, and 3.2 X 10(-5) breaks/2.5 X 10(9) daltons/Jm-2, respectively. We interpret these strain differences as indicating simultaneous induction and repair of breaks in polymerase 1 (polA)-proficient strains under the 0 degrees C, M9 buffer irradiation conditions that, for maximum efficiency, require both the polA and recA gene products. Strand-break rejoining also occurred at 30 degrees C in complete growth medium. We propose that at least three (and possibly four) distinct types of pathways can act to reduce the levels of 365-nm radiation-induced strand breaks. A quantitative comparison of the number of breaks remaining with the number of lethal events remaining after repair in complete medium at 30 degrees C showed that between one and three breaks remain per lethal event in the wild-type and recA strains, whereas in the polA strain one order of magnitude more breaks were induced.     

Enzymatic production of deoxyribonucleic acid double-strand breaks after ultraviolet irradiation of Escherichia coli K-12.

We have observed the enzymatic production of deoxyribonucleic acid (DNA) doublestrand breaks in Escherichia coli K12 after ultraviolet irradiation. Doublestrand breaks appeared in wild-type, polA1, recB21, recA, and exrA strains after incubation in minimal medium. THE UVRA6 strain showed no evidence of double-strand breakage under the same conditions. Our data suggest that uvr+ cells, which are proficient in the incision step of excision repair, accumulate double-strand breaks in their DNA as a result of the excision repair process, i.e., arising from closely matched incisions, excision gaps, or incisions and gaps on opposite strands of the DNA twin helix. Furthermore, strains deficient in excision repair subsequent to the incision step (i.e., polA, rec, exrA) showed more double-strand breaks than the wild type strain. The results raise the possibility that a significant fraction of the lethal events in ultraviolet-irradiated, repair-proficient (uvr+) cell may be enzymatically-induced DNA double-strand breaks.     

Changes in bleb formation following hyperthermia treatment of Chinese hamster ovary cells

Chinese hamster ovary cells in suspension cultures were heated for various times at 41.5, 43.5, and 45.5 degrees C, and quantitative determinations of microblebbing and macroblebbing of the cell membrane were performed for cells maintained at 4, 25, and 37 degrees C after hyperthermia. The percentage of cells with blebs following heating at 45.5 degrees C was dependent upon the duration of heating with increases from 40% for 5 min to 90% for 30 min. Cells exposed to lower temperatures exhibited less blebbing which was not quantifiable. The changes in bleb formation following 45.5 degrees C were dependent upon the posthyperthermia temperature: a slight decrease of macroblebbing at 25 degrees C, a decrease to 50% by 2 h at 37 degrees C, and a sharp decrease of macroblebbing to less than 10% by 1 h at 4 degrees C. Microblebbing increased slightly at 37 degrees C. When cells were transferred rapidly from the 4 degrees C posthyperthermia incubation to 37 degrees C, the bleb formation percentages returned rapidly to the higher levels which existed before posthyperthermia incubation at the lower temperatures. Gamma irradiation of 20 and 50 Gy produced only a small increase in microblebbing at longer periods (5 to 6 h) but no increase in macroblebbing. The survival of cells heated for 20 min at 45.5 degrees C was decreased 40% for suspension cells maintained at 4 degrees C for 2 to 3 h before incubation at 37 degrees C for colony formation compared to cells immediately incubated at 37 degrees C after heating. The survival of cells maintained at 25 degrees C after heating was not altered in comparison.     

Separate Branches of the uvr Gene-Dependent Excision Repair Process in Ultraviolet-Irradiated Escherichia coli K-12 Cells; Their Dependence upon Growth Medium and the polA, recA, recB, and exrA Genes

The extent of repair of single-strand breaks (incision breaks) induced in the deoxyribonucleic acid (DNA) of Escherichia coli K-12 cells by the uvr gene-dependent excision repair process after ultraviolet (UV) radiation was determined in the wild-type, polA1, recA56, recB21, and exrA strains. The wild-type strain repaired all incision breaks after incident doses of UV radiation (254 nm) of approximately 60 J m(-2) or less when incubated in growth medium, or approximately 15 J m(-2) or less when incubated in buffer. The polA1 strain repaired the incision breaks completely after incident doses of approximately 12 J m(-2) or less when incubated in growth medium, or after approximately 4 J m(-2) when incubated in buffer. The recA13, recB21, and exrA strains showed essentially complete repair after incident doses of 10 to 15 J m(-2) whether the cells were incubated in buffer or growth medium. These results suggest that the uvr gene-dependent excision repair process may be divided into two branches, one which is dependent on the presence of growth medium and also the rec(+)exr(+) genotype, and a second which can occur in buffer (growth medium-independent) and is largely dependent on DNA polymerase I. The presence of chloramphenicol in the growth medium resulted in an inhibition of the growth medium-dependent repair occurring in wild-type and polA1 cells and had little or no effect on the extent of repair observed in recA56, recB21, or exrA cells. The similarities between the growth medium-dependent and -independent branches of excision repair and two known processes for the repair of X-ray-induced single-strand breaks are discussed.     

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.     

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.     

Characterization of Lactococcus lactis UV-sensitive mutants obtained by ISS1 transposition.

Studies of cellular responses to DNA-damaging agents, mostly in Escherichia coli, have revealed numerous genes and pathways involved in DNA repair. However, other species, particularly those which exist under different environmental conditions than does E. coli, may have rather different responses. Here, we identify and characterize genes involved in DNA repair in a gram-positive plant and dairy bacterium, Lactococcus lactis. Lactococcal strain MG1363 was mutagenized with transposition vector pG+host9::ISS1, and 18 mutants sensitive to mitomycin and UV were isolated at 37 degrees C. DNA sequence analyses allowed the identification of 11 loci and showed that insertions are within genes implicated in DNA metabolism (polA, hexB, and deoB), cell envelope formation (gerC and dltD), various metabolic pathways (arcD, bglA, gidA, hgrP, metB, and proA), and, for seven mutants, nonidentified open reading frames. Seven mutants were chosen for further characterization. They were shown to be UV sensitive at 30 degrees C (the optimal growth temperature of L. lactis); three (gidA, polA, and uvs-75) were affected in their capacity to mediate homologous recombination. Our results indicate that UV resistance of the lactococcal strain can be attributed in part to DNA repair but also suggest that other factors, such as cell envelope composition, may be important in mediating resistance to mutagenic stress.