Single-Strand Breaks in Deoxyribonucleic Acid and Viability Loss During Deoxyribonucleic Acid Synthesis Inhibition in Escherichia coli

The effects of deoxyribonucleic acid (DNA) synthesis inhibition brought about in four different ways-thymidine starvation, nalidixic acid, hydroxyurea, and dnaB mutation-were examined in isogenic strains of Escherichia coli K-12. Three parameters were examined to determine whether there are strict correlations among them: (i) the extent of DNA synthesis inhibition; (ii) cell survival; and (iii) the rate of breakage of DNA molecules. There was no significant correlation between the extent of DNA synthesis inhibition and the rate of viability loss caused by the four DNA synthesis inhibitors, nor was there a strict correlation between the rate of occurrence of single-strand breaks in DNA and loss of viability. During treatment with hydroxyurea (0.1 M), no viability loss was observed and little, if any, single-strand breakage of DNA occurred. Both thymidine starvation and nalidixic-acid (20 mug/ml) treatment resulted in viability loss and breakage of DNA. For these latter two inhibitors, the two events appeared to be associated because greater rates of both viability loss and DNA breakage were observed for nalidixic acid compared with thymidine starvation. However, viability loss need not be associated with extensive breakage of DNA as demonstrated with a temperature-sensitive DNA synthesis mutant; at 39 C, viability loss occurred at a high rate without significant DNA breakage. With the other agents, the amount of DNA breakage accumulated when a cell population has sustained an average of one lethal hit was estimated to be about 30 single-strand breaks per genome. Differences in chromosomal and episomal breakage rates were observed.     

Direct Enzymatic Repair of Deoxyribonucleic Acid Single-Strand Breaks in Dormant Spores

With the alkaline sucrose gradient centrifugation method, it was found that dormant spores of Clostridium botulinum subjected to 300 krads of gamma radiation showed a distinct decrease in deoxyribonucleic acid (DNA) fragment size, indicating induction of single-strand breaks (SSB). A two- to threefold difference in radiation resistance of spores of two strains of C. botulinum, 33A (37% survival dose [D(37)] = 110 krads) and 51B (D(37) = 47 krads), was accompanied by relatively larger DNA fragments (molecular weight 7.9 x 10(7)) obtained during extraction from the radiation-resistant strain 33A and smaller DNA fragments (molecular weight 1.8 x 10(7)) obtained under identical conditions from radiation-sensitive strain 51B. The apparent number of DNA SSB produced by 300 krads in strains 33A and 51B was 0.37 and 3.50, respectively, per 10(8) daltons of DNA. Addition of 0.02 M ethylenediaminetetraacetic acid (EDTA) to spore suspensions during irradiation doubled the apparent number of SSB in strain 33A but had no effect on strain 51B. In vivo, 0.02 M EDTA present during irradiation to 100 to 300 krads decreased survival of spores of 33A by about 30% but had little or no effect on 51B. Survival of 33A was also reduced by about 45% when the spores were irradiated while frozen in dry ice (-75 C) and, after irradiation, immediately exposed to 0.03 M EDTA for 1 h to inhibit repair in the dormant spores. These results suggest that the highly radiation-resistant strain 33A may be able to accomplish repair of SSB during irradiation or after irradiation under nonphysiological conditions, i.e., in the dormant state. This repair can be inhibited by EDTA. Sedimentation patterns show that DNA from spores of both strains 33A and 51B did not show any postirradiation repair during the first 6 h of germination, as opposed to Bacillus subtilis spores, which exhibit repair immediately after germination. These observations suggest the existence of direct repair in physiological dormant spores of strain 33A in the cryptobiotic resting state in the absence of germination. The repair seems to be similar to that of polynucleotide ligase activity shown to be operative in some vegetative cells. Apparently radiation-sensitive strains such as 51B and B. subtilis are generally poor in DNA repair enzyme activity under conditions of spore dormancy, which may account for the approximately threefold difference in radiation sensitivity or DNA fragility of different strains, or both.     

Repair of Single-Strand Deoxyribonucleic Acid Breaks in Ultraviolet Light-Irradiated Haemophilus influenzae

The wild-type strain and mutants of Haemophilus influenzae, sensitive or resistant to ultraviolet light (UV) as defined by colony-forming ability, were examined for their ability to perform the incision and rejoining steps of the deoxyribonucleic acid (DNA) dark repair process. Although UV-induced pyrimidine dimers are excised by the wild-type Rd and a resistant mutant BC200, the expected single-strand DNA breaks could not be detected on alkaline sucrose gradients. Repair of the gap resulting from excision must be rapid when experimental conditions described by us are employed. Single-strand DNA breaks were not detected in a UV-irradiated sensitive mutant (BC100) incapable of excising pyrimidine dimers, indicating that this mutant may be defective in a dimer-recognizing endonuclease. No single-strand DNA breaks were detected in a lysogen BC100(HP1c1) irradiated with a UV dose large enough to induce phage development in 80% of the cells.     

Thymine Starvation and Single-Strand Breaks in Chromosomal Deoxyribonucleic acid of Escherichia coli

Single-strand breaks, as measured by the McGrath and Williams procedure, occur in chromosomal deoxyribonucleic acid of Escherichia coli cells during thymine starvation.     

Deoxyribonucleic Acid Damage by Monofunctional Mitomycins and Its Repair in Escherichia coli

Exposure of Escherichia coli to the antibiotic mitomycin C (MTC) at a concentration of 0.5 mug/ml caused cross-linkage between complementary strands of deoxyribonucleic acid (DNA). Derivatives of mitomycin, 7-methoxymitosene (7-MMT) and decarbamoyl mitomycin C (DCMTC), at a level as high as 20 mug/ml formed no cross-links between DNA strands. Ultraviolet light-sensitive mutants of E. coli K-12 bearing uvrA, uvrB, uvrC, or recA mutations were more sensitive to the lethal action of 7-MMT and of DCMTC than was the wild-type strain. Treatment of wild-type cells with these antibiotics resulted in the production of single-strand breaks in DNA, which were repaired upon incubation in a growth medium. Such breaks in DNA were not produced in the uvrA and the uvrB mutants. In the uvrC mutant, single-strand breaks were produced by 7-MMT or by DCMTC, but these breaks were not repaired upon incubation. These results are discussed in connection with the mechanism for removal of pyrimidine dimers in ultraviolet-irradiated bacteria.     

Ionizing Radiation Damage to the Folded Chromosome of Escherichia coli K-12: Repair of Double-Strand Breaks in Deoxyribonucleic Acid

The extremely gentle lysis and unfolding procedures that have been developed for the isolation of nucleoid deoxyribonucleic acid (DNA; K. M. Ulmer et al., J. Bacteriol. 138:475-485, 1979) yield undamaged, replicating genomes, thus permitting direct measurement of the formation and repair of DNA double-strand breaks at biologically significant doses of ionizing radiation. Repair of ionizing radiation damage to folded chromosomes of Escherichia coli K-12 strain AB2497 was observed within 2 to 3 h of post-irradiation incubation in growth medium. Such behavior was not observed after post-irradiation incubation in growth medium of a recA13 strain (strain AB2487). A model based on recombinational repair is proposed to explain the formation of 2,200 to 2,300S material during early stages of incubation and to explain subsequent changes in the gradient profiles. Association of unrepaired DNA with the plasma membrane is proposed to explain the formation of a peak of rapidly sedimenting material (greater than 3,100S) during the later stages of repair. Direct evidence of repair of double-strand breaks during post-irradiation incubation in growth medium was obtained from gradient profiles of DNA from ribonuclease-digested chromosomes. The sedimentation coefficient of broken molecules was restored to the value of unirradiated DNA after 2 to 3 h of incubation, and the fraction of the DNA repaired in this fashion was equal to the fraction of cells that survived at the same dose. An average of 2.7 double-strand breaks per genome per lethal event was observed, suggesting that one to two double-strand breaks per genome are repairable in E. coli K-12 strain AB2497.     

Ultraviolet- and X-Ray-Induced Responses of a Deoxyribonucleic Acid Polymerase-Deficient Mutant of Escherichia coli

Escherichia coli K-12, polAl(-) is a mutant strain whose extracts are deficient in Kornberg deoxyribonucleic acid (DNA) polymerase activity. We have compared the mutant and parental strains on the basis of a number of responses to ultraviolet (UV) and X-irradiation. For both types of radiation, the mutant is more sensitive by approximately the same factor as measured by reduction in colony formation, depression of DNA synthesis, and enhancement of DNA degradation. The rate of repair of X-ray-induced single-strand breaks in the mutant is also slower, as is the repair of breaks after excision repair of UV damage. On the other hand, the mutant has a significant capability to reactivate UV-irradiated lambda phage, although it is almost totally deficient in the ability to carry out UV reactivation. The data indicate that the polAl mutation leaves the cells with some ability to perform excision and strand-rejoining repair but that an exonuclease, whose identity remains obscure, is the agent responsible for the extensive breakdown of the DNA in polAl(-) cells after irradiation.     

Effect of Mutations in Deoxyribonucleic Acid Repair Pathways on the Sensitivity of Escherichia coli K-12 Strains to Nitrofurantoin

Escherichia coli K-12 strains that carry mutations in one or more genes coding for proteins involved in repair of deoxyribonucleic acid lesions are more sensitive to the antibiotic nitrofurantoin than are the nonmutant parent strains.     

Effect of Thymine Starvation on Deoxyribonucleic Acid Repair Systems of Escherichia coli K-12

Thymine starvation of Escherichia coli K-12 results in greatly increased sensitivity to ultraviolet light (UV). Our studies, using isogenic strains carrying rec and uvr mutations, have shown the following. (i) Common to all strains tested is a change from multihit to single-hit kinetics of survival to UV after 60 min of thymine starvation. However, the limiting slope of UV survival curves decreases in the rec(+)uvr(+) strain and changes very little in several rec mutant strains and one uvrB mutant strain. Thus, when either the rec or uvr system is functioning alone, the limiting slopes of the UV survival curves are relatively unaffected by thymine starvation. (ii) Thymine starvation does not significantly inhibit repair processes carried out by either repair system alone; i.e., host cell reactivation of irradiated phage (carried out by the uvr system), excision of thymine dimers (uvr), or X-ray repair (rec). (iii) In a rec(+)uvr(+) strain, repair appears to be a synergistic rather than additive function of the two systems. However, after thymine starvation, repair capacity is reduced to about the sum of the repair capacities of the independent systems. (iv) The kinetics of thymineless death are not changed by rec and uvr mutations. This indicates that the lesions responsible for thymineless death are not repaired by rec or uvr systems. (v) Withholding thymine from thy rec(+)uvr(+) bacteria not undergoing thymineless death has no effect on UV sensitivity. Under these conditions one sees higher than normal UV resistance in the presence or absence of thymine. This is due to increased repair carried out by the uvr system. To explain these results we postulate that thymine starvation does not inhibit either the rec or uvr repair pathway directly. Rather it appears that thymine starvation results in increased UV sensitivity in part by inhibiting a function which normally carries out efficient coordination of rec and uvr pathways.     

Physiological Modifications in the Production and Repair of Methyl Methane Sulfonate-Induced Breaks in the Deoxyribonucleic Acid of Escherichia coli K-12

The medium in which Rec(+) strains of Escherichia coli K-12 are grown affected their sensitivity to treatment with methyl methane sulfonate (MMS). Rec(+) cells grown to the stationary phase in glucose-enriched nutrient broth (GNB) were more resistant to MMS than cells grown in nutrient broth (NB). The repair of MMS-induced breaks (or alkali-labile bonds) in the deoxyribonucleic acid (DNA) from E. coli K-12 strains AB1157, AB1886 uvrA6, and SR111 recA13 recB21 grown in GNB and NB media was examined by means of alkaline sucrose gradient centrifugation. It appeared that essentially all of the repair of breaks that occurred, as evidenced by an increase in "molecular weight," took place within 10 min after treatment with MMS under our conditions. Cell survival was highest in cells for which the size of the DNA after the post-treatment incubation was the largest. The largest DNA after post-treatment incubation was found in Rec(+) cells grown in GNB medium. The results suggest that these cells may have an enhanced capacity for repairing breaks in DNA.     

Molecular Recombination in T4 Bacteriophage Deoxyribonucleic Acid II. Single-Strand Breaks and Exposure of Uncomplemented Areas as a Prerequisite for Recombination

Deoxyribonucleic acid (DNA) from several "DNA-deficient" amber mutants was observed to be either nicked (amber 22, 82, 122, and wild type) or cut (amber 453) after injection into a nonpermissive host. This effect was inhibited by chloramphenicol (CM), indicating that it is due to phage-induced enzymes. Although most of the mutants tested for replication in a density-label system were in fact DNA-deficient (amber 22, 82, 122), one (amber 81) was found to replicate almost identically to the wild type, and another (amber 453) was found to assume a hybrid density only. The hybrid moiety was less than, or equal to, one phage equivalent length, and was more efficiently extracted from infected bacteria than was similarly replicated DNA from wild-type phage. Interparental recombination between heavy and light parental DNA was observed for amber 82, 122, and wild type, but was not observed for amber 453; it was inhibited by CM. In contrast to amber 82 and wild type, the amber 453 intracellular DNA does not have single-strand regions, Because amber 453, unlike amber 82 and wild type T4, does not recombine, nicking and exposure of single-strand regions is postulated to be a prerequisite for recombination.     

Survival and induction of recA protein in mitomycin C-treated Escherichia coli rec, lex, or uvr strains

The capability to synthesize recA protein has been tested for Escherichia coli treated with mitomycin C. recA protein was assayed using an immunoradiometric assay (Paoletti, C., Salles, B., and Giacomoni, P. U. (1982) Biochimie 64, 239-246). Mitomycin C-treated wild type E. coli can express recA gene in a similar quantitative fashion, independently of the growth media used in this work; glucose did not inhibit induction of recA protein in cells growing in synthetic media. Wild type E. coli recovering from energy starvation displays a similar qualitative capability to induce the synthesis of recA protein independently of the stage of growth at which the cells are treated with the drug. At midexponential phase, the cells appear to have an enhanced capability to synthesize recA protein. The relationship between survival and capability to synthesize recA protein was explored for E. coli lex, rec, and/or uvr mutants, after treatment with mitomycin C. A good correlation was found, except for a recB mutant and for an ethidium-sensitive strain, both able to produce as much recA protein as the wild type but 100-fold more sensitive to the drug. A similarly satisfactory correlation was found when plotting the survival after UV irradiation versus the capability of synthetizing recA protein with the exception of an uvrA strain and of a lexA strain.     

Effect of Deoxyribonucleic Acid Ligands on Deoxyribonucleases and Deoxyribonucleic Acid Polymerase I of Escherichia coli K-12

Endonuclease I, exonuclease I, and exonuclease II-deoxyribonucleic acid (DNA) polymerase I activities are not vital functions in Escherichia coli, although the latter two enzymes have been indirectly shown to be involved in DNA repair processes. Acridines such as acridine orange and proflavine interfere with repair in vivo, and we find that such compounds inhibit the in vitro activity of exonuclease I and DNA polymerase I but stimulate endonuclease I activity and hydrolysis of p-nitrophenyl thymidine-5′-phosphate by exonuclease II. Another acridine, 10-methylacridinium chloride, binds strongly to DNA but is relatively inert both in vivo and in vitro. These experiments suggest that acridines affect enzyme activity by interacting with the enzyme directly as well as with DNA. Resulting conformational changes in the DNA-dependent enzymes might explain why similar acridines which form similar DNA complexes have such a wide range of physiological effects. Differential sensitivity of exonuclease I and DNA polymerase I to acridine inhibition relative to other DNA-dependent enzymes may contribute to the acridine sensitivity of DNA repair.     

HIV-1Tat蛋白抑制DNA修复和增强细胞辐射敏感性

近年来临床研究发现,艾滋病合并肿瘤患者放疗后产生的正常组织和皮肤毒性反应明显高于普通肿瘤患者.本研究将探讨HIV-1Tat蛋白是否影响细胞对电离辐射敏感性及机理. 两个表达Tat蛋白的细胞系TT2和TE671-Tat均来源于人的横纹肌肉瘤细胞（TE671）并已转染了不同来源的tat基因.使用细胞辐射后克隆形成率检测辐射敏感性,RT-PCR和Western 印迹检测基因表达,彗星电泳和γ-H2AX位点检测DNA双链断裂和修复. TT2和TE671-Tat细胞的辐射敏感性与转染空载体及对照细胞相比明显增加.彗星电泳和γ-H2AX位点检测表明,在表达Tat蛋白的细胞中,辐射诱导DNA双链断裂的修复水平明显降低.通过RT-PCR和Western 印迹检测进一步证实,表达Tat蛋白的细胞中DNA修复蛋白DNA-PKcs的表达被抑制. HIV-1Tat蛋白抑制DNA-PKcs的表达,降低DNA双链断裂的修复,使细胞的电离辐射敏感性增高.本研究为了解AIDS合并肿瘤患者对放射治疗敏感性变化提供了重要信息.     

Production and Repair of Radiochemical Damage in Escherichia coli Deoxyribonucleic Acid; Its Modification by Culture Conditions and Relation to Survival

Late log-phase Escherichia coli B/r cells are 1.6 times more sensitive to killing by X rays than are stationary-phase cells when grown in Brain Heart Infusion (BHI) + glucose. The number of single-chain breaks formed per krad is the same for log- and stationary-phase cells. Stationary-phase cells show a somewhat greater ability to repair single-chain breaks (especially after high doses of X rays) than do log-phase cells. The rapidity and extent of postirradiation deoxyribonucleic acid (DNA) degradation are greater in log-phase cells than in stationary-phase cells. The enhanced viability exhibited by stationary-phase cells thus appears to correlate both with enhanced single-chain break repair and the reduced degradation of DNA. Cells grown to stationary phase in peptone medium (PO cells) are 3.4 times more sensitive to killing by X rays than cells grown to stationary phase in peptone medium supplemented with glucose and phosphate buffer (PG cells). The yield of single-strand breaks is the same for both types of cells (but the absolute yield is about two times higher than in the cells grown in BHI + glucose). The kinetics for the repair of single-chain breaks are the same for both types of cells for about 30 min. After this time period, further repair ceases in the PO cells but continues in the PG cells, provided that glucose is present in the medium. Postirradiation DNA degradation is both more rapid and more extensive in PO cells than in PG cells whether or not glucose is present in the postirradiation incubation medium. The survival of stationary-phase E. coli B/r grown in PO or PG medium is likewise unaffected by the presence of glucose in the plating medium, and thus correlates better with the lower DNA degradation seen in the PG cells than with the increased strand rejoining, since this latter process requires the presence of glucose.     

Mismatch Repair in Escherichia coli Cells Lacking Single-Strand Exonucleases ExoI, ExoVII, and RecJ

In vitro, the methyl-directed mismatch repair system of Escherichia coli requires the single-strand exonuclease activity of either ExoI, ExoVII, or RecJ and possibly a fourth, unknown single-strand exonuclease. We have created the first precise null mutations in genes encoding ExoI and ExoVII and find that cells lacking these nucleases and RecJ perform mismatch repair in vivo normally such that triple-null mutants display normal mutation rates. ExoI, ExoVII, and RecJ are either redundant with another function(s) or are unnecessary for mismatch repair in vivo.     

Spontaneous Lethal Sectoring, a Further Feature of Escherichia coli Strains Deficient in the Function of rec and uvr Genes

Eight recombination-deficient (Rec(-)) mutants of Escherichia coli were studied. Progeny lines were obtained on solid media, by means of micromanipulation, and the colony-forming ability of individual cells was analyzed. Cells of all eight strains gave rise to colony-forming as well as non-colony-forming descendants ("lethal sectoring"). Lethal sectors, i.e., groups of non-colony-forming cells which originate from a common ancestor, appeared with frequencies per generation ranging between 4 and 20% in Rec(-) strains, whereas lethal sectors were rare in Rec(+) strains (less than 1%). A strain carrying a mutation (uvrA6) in one of the genes involved in pyrimidine dimer excision from deoxyribonucleic acid (DNA) showed twice as many lethal sectors per generation as a strain with the genotype uvrA(+). Similarly, a double mutant (AB2480, uvrA6, recA13) showed twice as much spontaneous lethal sectoring as the corresponding Rec(-) strain (uvrA(+), recA13). The kinetics of growth curves obtained in nutrient broth and the frequency of non-colony-forming units in stationary-phase broth cultures indicate clearly that lethal sectors occur in liquid cultures too. The causes for spontaneous lethal sectoring are unknown at present. It seems reasonable to assume that gene uvrA and the rec genes are somehow involved in the repair of spontaneously occurring DNA lesions, since a deficiency in this type of repair may cause lethal sectors. The extent to which spontaneous lethal sectoring (observed in all Rec(-) strains of E. coli studied) may contribute indirectly to the failure to form recombinants is discussed.