Alkaline Sucrose Gradient Sedimentation Research Articles

Overexpression of the adenovirus type 12 (Ad12) pTP or E1A gene facilitates Ad12 DNA replication in nonpermissive BHK21 hamster cells.

In the adenovirus type 12 (Ad12) hamster cell system, abortive virus infection is one of the factors associated with the highly efficient oncogenesis in newborn Syrian hamsters. We have shown earlier that the replication and efficient late transcription of the Ad12 genome are blocked in Syrian hamster cells. Some of the early Ad12 functions are transcribed in these cells, although at a minimal rate. In the present study, we demonstrate that low expression levels of the E1A and precursor to terminal protein (pTP) genes of Ad12 seem to be responsible for the lack of Ad12 DNA replication in hamster cells. The Ad12 genes for the E1A functions or for pTP were tethered to the strong early promoter of the human cytomegalovirus and transfected into BHK21 cells. Subsequently, these cells were infected with Ad12 virions. In Ad12-infected BHK21 cells, which overexpressed pTP or E1A, full-length Ad12 DNA was de novo synthesized, as documented by metabolic labeling with [3H]thymidine and by zone velocity sedimentation in alkaline sucrose gradients followed by gel electrophoresis of the 3H-labeled DNA and Southern blot hybridization to 32P-labeled Ad12 DNA. Transfection of the cloned E1A region of Ad2 yielded similar results. The newly synthesized Ad12 DNA was covalently linked to pTP. The Ad12 DNA binding protein (DBP) and DNA polymerase (pol) genes were transcribed at levels similar to those in merely Ad12-infected cells. In pTP or E1A gene-transfected and Ad12-infected BHK21 cells, marginal levels of late Ad12 mRNA were detectable. Late Ad12 proteins were, however, not synthesized. Apparently, Ad12 DNA replication in hamster cells is rendered impossible due to insufficient threshold levels of the viral E1A and/or pTP.

DNA damage-inducible and RAD52-independent repair of DNA double-strand breaks in Saccharomyces cerevisiae.

Chromosomal repair was studied in stationary-phase Saccharomyces cerevisiae, including rad52/rad52 mutant strains deficient in repairing double-strand breaks (DSBs) by homologous recombination. Mutant strains suffered more chromosomal fragmentation than RAD52/RAD52 strains after treatments with cobalt-60 gamma irradiation or radiomimetic bleomycin, except after high bleomycin doses when chromosomes from rad52/rad52 strains contained fewer DSBs than chromosomes from RAD52/RAD52 strains. DNAs from both genotypes exhibited quick rejoining following gamma irradiation and sedimentation in isokinetic alkaline sucrose gradients, but only chromosomes from RAD52/RAD52 strains exhibited slower rejoining (10 min to 4 hr in growth medium). Chromosomal DSBs introduced by gamma irradiation and bleomycin were analyzed after pulsed-field gel electrophoresis. After equitoxic damage by both DNA-damaging agents, chromosomes in rad52/rad52 cells were reconstructed under nongrowth conditions [liquid holding (LH)]. Up to 100% of DSBs were eliminated and survival increased in RAD52/RAD52 and rad52/rad52 strains. After low doses, chromosomes were sometimes degraded and reconstructed during LH. Chromosomal reconstruction in rad52/rad52 strains was dose dependent after gamma irradiation, but greater after high, rather than low, bleomycin doses with or without LH. These results suggest that a threshold of DSBs is the requisite signal for DNA-damage-inducible repair, and that nonhomologous end-joining repair or another repair function is a dominant mechanism in S. cerevisiae when homologous recombination is impaired.

Reduced global genomic repair of ultraviolet light-induced cyclobutane pyrimidine dimers in simian virus 40-transformed human cells

The p53 tumor-suppressor gene has been implicated in the inducible activation of excision repair of ultraviolet (UV)-induced cyclobutane pyrimidine dimers (CPDs) in human cells. Because the large T antigen (LTAg) of the simian virus 40 (SV40) binds p53 protein and can interfere with its function, it was of interest to study DNA repair in normal human fibroblasts that had been transformed by SV40 compared with that in their nontransformed parental counterparts and to determine whether such transformation attenuated global genomic repair (GGR) of CPDs. Three methods were used to measure GGR in UV-irradiated cells: (i) an immunoassay using monoclonal antibodies specific for CPDs or 6-4 photoproducts (6-4PPs), (ii) zone sedimentation in alkaline sucrose gradients to measure the average DNA strand size after specific nicking at CPD sites in duplex DNA with T4 endonuclease V (TEV), and (iii) Southern hybridization of TEV-treated DNA with strand-specific mRNA probes to assess removal of CPDs from either strand of a defined genetic sequence in an expressed gene. Whereas repair of 6-4PPs was very similar in paired SV40-transformed and primary fibroblasts, GGR of CPDs was significantly reduced in the SV40-transformed cells. In contrast, SV40 transformation did not appreciably affect the efficiency of transcription-coupled repair. These data support the hypothesis that SV40 transformation can result in reduced levels of GGR, most likely because of the inhibition of normal p53 function by LTAg.

A requirement for recombinational repair in Saccharomyces cerevisiae is caused by DNA replication defects of mec1 mutants.

To examine the role of the RAD52 recombinational repair pathway in compensating for DNA replication defects in Saccharomyces cerevisiae, we performed a genetic screen to identify mutants that require Rad52p for viability. We isolated 10 mec1 mutations that display synthetic lethality with rad52. These mutations (designated mec1-srf for synthetic lethality with rad-fifty-two) simultaneously cause two types of phenotypes: defects in the checkpoint function of Mec1p and defects in the essential function of Mec1p. Velocity sedimentation in alkaline sucrose gradients revealed that mec1-srf mutants accumulate small single-stranded DNA synthesis intermediates, suggesting that Mec1p is required for the normal progression of DNA synthesis. sml1 suppressor mutations suppress both the accumulation of DNA synthesis intermediates and the requirement for Rad52p in mec1-srf mutants, but they do not suppress the checkpoint defect in mec1-srf mutants. Thus, it appears to be the DNA replication defects in mec1-srf mutants that cause the requirement for Rad52p. By using hydroxyurea to introduce similar DNA replication defects, we found that single-stranded DNA breaks frequently lead to double-stranded DNA breaks that are not rapidly repaired in rad52 mutants. Taken together, these data suggest that the RAD52 recombinational repair pathway is required to prevent or repair double-stranded DNA breaks caused by defective DNA replication in mec1-srf mutants.

Repair of mtDNA in Vertebrates

The author's research is supported by National Institutes of Health grants GM29681 and ES04068.

Metabolic fate of [3H]1-beta-D-arabinofuranosyl-5-[(E)-2-bromovinyl]uracil in herpes simplex virus type 1-infected cells.

The metabolic fate of 1-beta-D-arabinofuranosyl-5-[(E)-2-bromovinyl]uracil (BV-araU) in herpes simplex virus type 1-infected cells was studied using tritium-labeled BV-araU. [3H]BV-araU was selectively taken-up by infected cells. Approximately 10% of the total uptake of [3H]BV-araU was recovered from the acid-insoluble fraction at any time post-infection. Both cellular uptake of [3H]BV-araU and its incorporation into the acid-insoluble fraction increased with increasing incubation time through 8 hr post-infection. Uptake of [3H]BV-araU and its incorporation into the acid-insoluble fraction also increased proportionally to the duration of exposure to [3H]BV-araU. An alkaline sucrose gradient sedimentation analysis revealed that the radioactive DNA obtained from cells pulse-labeled with [3H]BV-araU were small DNA fragments which remained at the top following a chasing period in isotope-free medium, whereas that pulse-labeled with [3H]thymidine was chased to a fraction of high molecular weight DNA. Nuclease P1 digestion reduced 99% of the [3H]BV-araU-labeled DNA extracted from infected cells to a low molecular weight. Following digestion of [3H]BV-araU-labeled DNA with micrococcal nuclease and spleen exonuclease, all of the radioactivity was recovered as [3H]BV-araU 3'-monophosphate. Thus, BV-araU strongly inhibits the elongation of viral DNA strands as demonstrated by the alkaline sucrose gradient sedimentation analysis, whereas at least a portion of the [3H]BV-araU is incorporated inside viral DNA strands in infected cells.

Overexpression of insulin-like growth factor (IGF)-I receptor enhances inhibition of DNA replication in mouse cells exposed to x-rays.

Previous studies from our laboratory provided evidence for the operation of signal transduction pathways involving ras, myc, and staurosporine-sensitive protein kinases in the regulation of DNA replication in irradiated cells. Because ras and myc are also involved in the signal transduction elicited in response to ligand activation of growth factor receptors, we wondered whether growth factor receptors are upstream elements in the regulation of DNA replication in irradiated cells. Here, we report on the role of insulin-like growth factor I receptor (IGF-IR) in the regulation of DNA replication in irradiated cells. We compare radiation-induced inhibition of DNA replication in BALB/c 3T3 cells with that in P6 cells. P6 cells are derived from BALB/c 3T3 cells by transfection with a vector expressing IGF-IR, leading to 30-fold overexpression. We observe a significantly stronger inhibition of DNA replication after irradiation in P6 as compared with BALB/c 3T3 cells at all doses examined. Sedimentation in alkaline sucrose gradients shows that the increased inhibition in P6 cells is due to an increased inhibition of replicon initiation, the main controlling event in DNA replication. Staurosprine at 20 nM reduces radiation-induced inhibition of DNA replication in BALB/c 3T3 cells, but has only a small effect in P6 cells. Caffeine at a concentration of 1 mM, on the other hand, removes over 60% of the inhibition in both cell lines. The results implicate IGF-IR in the regulation of DNA replication in irradiated cells, but also suggest differences between cells of different origins in the proteins involved in the regulating signal transduction pathway.

The mechanism of recA polA lethality: suppression by RecA-independent recombination repair activated by the lexA(Def) mutation in Escherichia coli.

The mechanism of recA polA lethality in Escherichia coli has been studied. Complementation tests have indicated that both the 5'-->3' exonuclease and the polymerization activities of DNA polymerase I are essential for viability in the absence of RecA protein, whereas the viability and DNA replication of DNA polymerase I-defective cells depend on the recombinase activity of RecA. An alkaline sucrose gradient sedimentation analysis has indicated that RecA has only a minor role in Okazaki fragment processing. Double-strand break repair is proposed for the major role of RecA in the absence of DNA polymerase I. The lexA(Def)::Tn5 mutation has previously been shown to suppress the temperature-sensitive growth of recA200(Ts) polA25::spc mutants. The lexA(Def) mutation can alleviate impaired DNA synthesis in the recA200(Ts) polA25::spc mutant cells at the restrictive temperature. recF+ is essential for this suppression pathway. recJ and recQ mutations have minor but significant adverse effects on the suppression. The recA200(Ts) allele in the recA200(Ts) polA25::spc lexA(Def) mutant can be replaced by delta recA, indicating that the lexA(Def)-induced suppression is RecA independent. lexA(Def) reduces the sensitivity of delta recA polA25::spc cells to UV damage by approximately 10(4)-fold. lexA(Def) also restores P1 transduction proficiency to the delta recA polA25::spc mutant to a level that is 7.3% of the recA+ wild type. These results suggest that lexA(Def) activates a RecA-independent, RecF-dependent recombination repair pathway that suppresses the defect in DNA replication in recA polA double mutants.

Enhancing effect of heterocyclic amines and ß-carbolines on UV or chemically induced mutagenesis in E. coli

Most heterocyclic amines and ß-carbolines — harman, norharman, harmine, harmaline — enhanced UVC (254 nm) induced mutagenesis without microsomal activation in E. coli B/r WP2. 3-Amino-1,4-dimethyl-5 H-pyrido[4,3- b]indole (Trp-P-1) was most effective and increased UVAB (295–400 nm) induced mutations as well as UVC induced ones. Trp-P-1 enhanced the frequencies of mutations induced by not only UV but also 4-nitroquinoline-1-oxide (4NQO) or 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF2), while it showed little effect on N-methyl- N′-nitro- N-nitrosoguanidine (MNNG) or γ-ray induced mutagenesis. Trp-P-1 decreased the survival of UVC irradiated cells of CM571 recA. However, these effects of Trp-P-1 on UVC induced mutagenesis and lethality were not observed in WP2s uvrA which is excisioon repair deficient. The alkaline sucrose gradient sedimentation analysis demonstrated that Trp-P-1 blocked the incision step in DNA excision repair. Further, pretreatment with Trp-P-1 before UVC irradiation showed no effect on UVC induced mutagenesis. Similar effects were also seen in the case of harman or norharman. These results suggest that heterocyclic amines and ß-carbolines inhibit DNA excision repair directly or indirectly, thus enhancing UV or chemically induced mutagenesis.

Differences in DNA damage induced by mutagenic and nonmutagenic 4-aminoazobenzene derivatives in Escherichia coli

DNA lesions produced in Escherichia coli AB2500 (uvrA) exposed to the carcinogen N-hydroxy-3-methoxy-4-aminoazobenzene (N-OH-3-MeO-AAB) or the noncarcinogen N-hydroxy-2-methoxy-4-aminoazobenzene (N-OH-2-MeO-AAB) were investigated by alkaline sucrose gradient sedimentation and 32P-postlabeling analysis. Alkali-labile sites appeared to be formed equally in cells treated with both aminoazobenzene derivatives. 32P-Postlabeling analysis revealed that the 3-MeO-AAB-DNA adduct level was 25-fold higher than that for 2-MeO-AAB-DNA adducts. In addition to major adducts, 4 minor spots were detected in N-OH-3-MeO-AAB-treated cells, while only one major adduct was found in N-OH-2-MeO-AAB-treated cells. The mutagenecities and cytotoxicities were also determined with E. coli with different repair capacities; we found that repair of 3-MeO-AAB damages is strongly dependent on the UVR repair system. Moreover, N-OH-3-MeO-AAB, but not N-OH-2-MeO-AAB, could induce recA and umuC gene expression, which was higher in uvrA strains than in the wild type.

Effect of arsenite on the DNA repair of UV-irradiated Chinese hamster ovary cells.

Arsenite, an ubiquitous human carcinogen, has been shown to enhance the cytotoxicity, mutagenicity and clastogenicity of UV light in mammalian cells. Arsenite may exert its co-genotoxic effects by inhibiting DNA repair. Results from alkaline sucrose gradient sedimentation show that arsenite did not accumulate UV-induced DNA strand breaks in Chinese hamster ovary (CHO) K1 cells as aphidicolin plus hydroxyurea (HU) did. These data indicate that arsenite did not inhibit the activity of DNA polymerase alpha in UV repair. Treatment with arsenite before UV irradiation slightly reduced the DNA strand breaks accumulated by cytosine beta-D-arabinofuranoside (AraC) plus HU. This effect implies that arsenite only slightly inhibited the incision of UV-induced DNA adducts. The low molecular weight DNA accumulated by post-UV incubation with AraC plus HU shifted to high molecular weight upon the incubation of cells in drug-free medium, but this shifting was prohibited by the presence of arsenite. This suggests that arsenite inhibited the rejoining of DNA strand breaks. When a pulse-chase labelling procedure was applied on UV-irradiated cells, the chain elongation of nascent DNA was strongly inhibited by post-incubation with arsenite. These data show that arsenite inhibited post-replication repair in UV-irradiated cells. Therefore, the steps inhibited by arsenite in UV-induced DNA repair in CHO K1 cells are different from human fibroblasts in which the inhibition of excision of pyrimidine dimers by arsenite was reported to be the major target.

CORRELATION BETWEEN CELL SURVIVAL AND DNA SINGLE‐STRAND BREAK REPAIR PROFICIENCY IN THE CHINESE HAMSTER OVARY CELL LINES AA8 AND EM9 IRRADIATED WITH 365‐nm ULTRAVIOLET‐A RADIATION

Cell survival parameters and the induction and repair of DNA single-strand breaks were measured in two Chinese hamster ovary cell lines after irradiation with monochromatic UVA radiation of wavelength 365 nm. The radiosensitive mutant cell line EM9 is known to repair ionizing-radiation-induced single-strand breaks (SSB) more slowly than the parent line AA8. EM9 was determined to be 1.7-fold more sensitive to killing by 365-nm radiation than AA8 at the 10% survival level, and EM9 had a smaller shoulder region on the survival curve (alpha = 1.76) than AA8 (alpha = 0.62). No significant differences were found between the cell lines in the initial yields of SSB induced either by gamma-radiation (as determined by alkaline sucrose gradient sedimentation) or by 365-nm UVA (as determined by alkaline elution). For measurement of initial SSB, cells were irradiated at 0.5 degrees C to minimize DNA repair processes. Rejoining of 365-nm induced SSB was measured by irradiating cells at 0.5 degrees C, allowing them to repair at 37 degrees C in full culture medium, and then quantitating the remaining SSB by alkaline elution. The repair of these breaks followed biphasic kinetics in both cell lines. EM9 repaired the breaks more slowly (t1/2 values of 1.3 and 61.3 min) than did AA8 (t1/2 values of 0.9 and 53.3 min), and EM9 also left more breaks unrepaired 90 min after irradiation (24% vs 8% for AA8). Thus, the sensitivity of EM9 to 365-nm radiation correlated with its deficiency in repairing DNA lesions revealed as SSB in alkaline elution.(ABSTRACT TRUNCATED AT 250 WORDS)

Saturation of DNA Repair Measured by Alkaline Elution

To determine whether the half-times (T1/2) of the DNA repair processes measured by alkaline elution increased in a dose-dependent manner, exponentially growing 9L/Ro rat brain tumor cells were irradiated with doses of 15-50 Gy, and their DNA repair kinetics was measured by alkaline elution. At 15 Gy, the DNA repair kinetics was biphasic with the fast phase having a T1/2 approximately 6 min and the slow phase having a T1/2 approximately 42 min. As the dose was increased to 50 Gy, the fast-phase T1/2 remained at approximately 6 min, but the slow-phase T1/2 increased to approximately 87 min. Although a dose-dependent increase in the T1/2 of the slow phase of DNA repair (saturation) was measured by alkaline elution, both the absolute value of the slow-phase T1/2 and the dependency of the slow-phase T1/2 on dose were less than those measured by alkaline sucrose gradient sedimentation in zonal rotors with slow reorienting gradient capability. Thus these two techniques appear either to depend on different hydrodynamic properties of the DNA or to have different coefficients of dependency for the same hydrodynamic properties of the DNA. The lower sensitivity for detection of the dose dependency of DNA repair makes it unlikely that the alkaline elution technique will be useful for quantitatively relating the shape of mammalian cell survival curves to the doses at which saturation of a DNA repair process occurs.

Role of DNA replication in carrier-ligand-specific resistance to platinum compounds in L1210 cells

L1210 cell lines have been described that are sensitive to most platinum compounds (L1210/0), resistant to ethylenediamine (en)-Pt but sensitive to diaminocyclohexane (dach)-Pt (L1210/DDP), and resistant to dach-Pt but sensitive to en-Pt (L1210/DACH). We have examined the effect of the dach and en carrier ligands on the ability of Pt-DNA adducts to inhibit DNA replication. Alkaline sucrose gradient sedimentation was used to determine the influence of both carrier ligands on the inhibition of replicon initiation and DNA chain elongation. Initiation of replicons was inhibited by Pt-DNA adducts to a greater extent than chain elongation in all three cell lines. Inhibition of replicon initiation was affected by the nature of the platinum carrier ligands only in the L1210/DACH cells in which 7.8-fold more dach-Pt adducts than en-Pt adducts were required to reach 63% inhibition. However, a strong carrier ligand effect was observed on the inhibition of DNA chain elongation in both the L1210/DDP and L1210/DACH cell lines. The L1210/DDP cell line required 4-fold more en-Pt adducts than dach-Pt adducts to inhibit DNA chain elongation by 63%. In the L1210/DACH cell line, 2.7-fold more dach-Pt adducts than en-Pt adducts were required for 63% inhibition. The L1210/0 cell line demonstrated no carrier ligand specificity for inhibition of chain elongation. Significant replicative bypass of Pt-DNA adducts was observed even in the L1210/0 cell line in that greater than 50 Pt-DNA adducts per 100 kb were required for 63% inhibition. The same level of inhibition was reached with 1.25 adducts of benzo[a]pyrene diolepoxide I per 100 kb. These data suggest that L1210 cells are capable of substantial replicative bypass of Pt-DNA adducts. Furthermore, the bypass of Pt-DNA adducts is increased in resistant L1210 cells and is markedly dependent on the nature of the platinum carrier ligand.

Preferential DNA repair of alkali-labile sites within the active insulin gene.

DNA damage and repair were studied in a DNA fragment containing the insulin gene after treatment of cells with methylnitrosourea. For these studies, two clonal isolates from the same rat insulinoma cell line which differ in that the insulin gene is transcribed in one (RINr 38) and is silent in the other (RINr B2) were utilized. Both the determination of immunologically reactive insulin released and the expression of insulin mRNA were used to verify that the gene was transcribed in the RINr 38 cells and not in the RINr B2 cells. Repair kinetics for the removal of alkali-labile sites were comparable across the entire genome in the RINr 38 and RINr B2 cells as determined using alkaline sucrose gradient sedimentation and a 32P end-labeling assay for the quantitation of N7-methylguanine. Quantitative DNA blot analysis was utilized to assess the formation and repair of alkali-labile sites within the restriction fragment containing the insulin gene. Alkali-labile sites appeared to be formed equally within the restriction fragment containing the insulin gene in both the RINr 38 and RINr B2 cells. However, at 24 h, 60% of the lesions were removed from the fragment in the RINr 38 cells, where the gene was transcribed, compared to the removal of only 20% in the RINr B2 cells, where the gene was silent. Thus, it appears that alkali-labile sites induced by exposure to methylnitrosourea are repaired more efficiently in the DNA fragment containing the insulin gene when it is actively transcribed.

A comparison of the techniques of alkaline filter elution and alkaline sucrose sedimentation used to assess DNA damage induced by 2-nitroimidazoles

The induction of DNA single-strand breaks (DNA-SSB) in Chinese hamster V79-379A lung fibroblasts by misonidazole or RSU-1069 under both aerobic and hypoxic conditions was examined following incubations for up to 4hr at 310°K using the technique of alkaline filter elution. Incubation with RSU-1069 induces DNA-SSB under both hypoxic and aerobic conditions, whereas incubation with misonidazole induces DNA-SSB only under hypoxia. The yield of breaks is dependent on both agent concentration and contact time. Following identical treatments with these agents, the yield of DNA-SSB (expressed in radiation dose equivalents) determined by alkaline filter elution is about one order of magnitude less than that previously determined by alkaline sucrose gradient sedimentation. In contrast to radiation induced DNA-SSB, alkaline elution is less sensitive than alkaline sucrose gradient sedimentation when determining DNA-SSB induced by RSU-1069 and misonidazole. During the filter elution assay, either increasing cell lysis from 2 to 4 hr, the pH of the lysing buffer from pH 8.7 to 12.5 or the elution buffer from pH 12.2 to 12.5 does not significantly effect the yield of DNA-SSB. Increasing the pH of the lysing or elution buffers to > pH 13 however results in considerable degradation of the DNA, whereby 50–85% of the total DNA passes through the filter with the lysing solution. This effect was similar for DNA from both control and chemically insulted cells. In conclusion, it is apparent that incubation with these agents results in the induction of DNA damage which is expressed as a DNA-SSB only after prolonged treatment under alkaline conditions. Further, the use of alkaline elution to study DNA-SSB damage induced chemically must be treated with caution in the light of these findings.

Heterogeneity of nitrogen mustard-induced DNA damage and repair at the level of the gene in Chinese hamster ovary cells.

We here present a general method to detect alkylation damage in specific genomic regions. Cells are treated with nitrogen mustard or dimethyl sulfate; the DNA is extracted and restricted, and the parental DNA is separated. Strand breaks are created at sites of N-alkylpurines by neutral depurination followed by alkaline hydrolysis. The DNA is then separated on alkaline agarose gels and transferred, and gene fragments are detected after hybridization with specific probes. Using this approach, we have examined damage formation and repair in the active genes dihydrofolate reductase and adenosine phosphoribosyltransferase, in a fragment containing the inactive c-fos gene and in a nontranscribed region downstream from the dihydrofolate reductase gene in Chinese hamster ovary cells. We find variations in the formation of nitrogen mustard adducts in these different regions. Nitrogen mustard adducts are preferentially repaired from the active genes as compared to the inactive gene and the noncoding region. However, we find no preferential damage or repair in these regions of the N7-methylpurines after dimethyl sulfate damage. Thus, there are significant differences in the repair mechanisms for two alkylating agents; this may implicate that there are important differences in the structural alterations in chromatin invoked by these agents. As a comparison to the studies of adduct levels in specific genomic regions, we have examined the overall genome, average adduct formation, and repair by these agents in the hamster cells. We used alkaline sucrose gradient sedimentation, and also a novel approach: quantitation of the DNA smears stained by ethidium bromide in the alkaline gels (used in the gene-selective repair analysis). Both these techniques gave similar data for adduct formation and repair; there was less initial damage formation and repair in the average genome than in specific genomic regions.

Induction and repair of DNA strand breaks in Bacteroides fragilis

Alkaline sucrose gradient sedimentation was used to establish whether strand breakage and repair take place in the DNA of UV-irradiated Bacteroides fragilis during the removal of pyrimidine dimers. A B. fragilis wild-type strain and two of its repair mutants, a mitomycin C sensitive mutant (MTC25) having wild-type levels of UV survival, and a UV-sensitive, mitomycin C sensitive mutant (UVS9), were investigated. Under anaerobic conditions, far-UV irradiation induced metabolically regulated strand breakage and resynthesis in the wild-type strain, but this was markedly reduced in both the MTC25 and UVS9 mutants. Approximately half of the strand breaks generated by the various strains were rejoined during further holding in buffer. Under replicating conditions, complete repair of strand breaks in the wild type was observed. Caffeine treatment under anaerobic conditions caused direct DNA strand breakage in B. fragilis cells but did not inhibit UV-induced breakage or repair.

The role of excision repair in the removal of transient benzo[a]pyrene-induced DNA lesions in Chinese hamster ovary cells

In Chinese hamster ovary (CHO) cells, benzo[a]pyrene induces both persistent and transient lesions that are detected by alkaline sucrose gradient sedimentation analysis (ASG sites). The transient lesions disappear within 15 min while the persistent lesions can be detected for several hours following treatment. Although the persistent ASG sites are believed to be repaired by excision repair, the process responsible for the disappearance of the transient ASG sites is unknown. To determine the contribution of excision repair to the removal of these transient lesions, CHO cells were treated with benzo[a]pyrene (B(a)P) in the presence of the inhibitors of excision repair, araC and novobiocin. The results indicate that: (1) araC inhibits the removal of persistent, but not the transient B(a)P-induced ASG sites; (2) novobiocin, a putative inhibitor of the incision step of DNA excision repair, reduced the number of lesions detected immediately following treatment, indicating that many of these lesions may represent single-strand discontinuities generated during repair; and (3) the lesions detected in the presence of novobiocin disappear rapidly following treatment. Based on these results, we concluded that B(a)P-induced transient ASG sites are repaired by a process other than excision repair.

Photodynamic action of methylene blue: Repair and mutation in Escherichia coli

The effects of the photodynamic action of methylene blue (MB-PDA) on strains of Escherichia coli were investigated to determine whether the dye could be used in photodynamic therapy (PDT). Using the method of alkaline sucrose gradient sedimentation, it was shown that in darkness MB induces a type of prelesion in DNA that transforms into single-strand breaks in alkaline conditions, provided that the dye is present during the processing of the gradient. This prelesion is completely reversible if the cells are washed immediately to remove the dye. However, after illumination with white light, the prelesions become “fixed” stable lesions, irreversible even after successive washings. The lethal damage induced by MB-PDA in E. coli can be repaired by the excision-repair system (about 30%) and by the recA-dependent repair system (about 70%). Polymerase I enzyme participates actively in the repair of the damage. MB-PDA is a weak mutagen and the induction of mutations by this treatment is restricted to high survival rates. Moreover, MB-PDA does not induce the SOS system (an inducible repair system dependent of the recA and loxA genes products), as measured by Weigle reactivation. However, it seems that this treatment can impair the repair systems in E. coli.