DNA Repair-deficient Mutants Research Articles

Characterization of an Escherichia coli Mutant (radB101) Sensitive to γ 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.

MODE OF ACTION OF α‐TERTHIENYL ON ESCHERICHIA COLI: EVIDENCE FOR A PHOTODYNAMIC EFFECT ON MEMBRANES

AbstractThe photodynamic effects of α‐terthienyl (αT) in near‐UV light (UV‐A) on Escherichia coli showed close agreement with the light absorption of αT at different wavelengths suggesting that αT is the primary absorbing molecule responsible for the photosensitized reaction. Studies with DNA repair deficient mutants of E. coli indicated that the bactericidal action of αT/UV‐A was not mediated by DNA damage, in direct contrast to the well‐known photosensitizer, 8‐methoxypsoralen. By using a closed borosilicate glass reaction vessel and various gas mixtures, it was demonstrated that photosensitization of both E. coli and a more resistant bacterium, Pseudomonas aeruginosa, was absolutely dependent on the presence of oxygen. The rate of killing by αT/UV‐A showed a rather small dependence on preincubation temperatures, with quite rapid killing at 5°C, suggesting that the movement of αT across the cytoplasmic membrane of E. coli is not the rate limiting step in killing and perhaps is not even necessary for killing. Sodium dodecyl sulphate‐polyacrylamide gels of cell membrane proteins after 15 and 30min of treatment with αT/UV‐A showed substantial random crosslinking of these proteins. The results taken overall suggest that αT is a photodynamic photosensitizer which exerts its primary effect at the level of the cytoplasmic membrane.

Direct screening of living mammalian cell colonies for the identification of DNA repair deficient mutants by a Multiwire Proportional Chamber

Mammalian cell mutants with defective repair of UV-induced DNA damage can be identified by the lack of unscheduled incorporation of radioactive thymidine into DNA after UV irradiation. With a specially designed windowlessMultiWireProportionalChamber (MWPC), colonies with absent or low incorporation of3H-thymidine can be precisely located within a population of normally labelled colonies. The main advantages of MWPC analysis over other more conventional methods for radioactive measurements are: (i), cells are not harmed by the analysis, so that previous replica plating of colonies becomes unnecessary; (ii), measurements are quantitative, hence discrimination between colonies is not subjective; (iii), location of unlabelled, non-repairing colonies is rapid and can be automatized. The method therefore appears suitable for the isolation of mammalian cell mutants with a block in DNA repair.

Azaserine: survival and mutation in Escherichia coli

Azaserine is an antineoplastic agent, mutagen and carcinogen that is known to inhibit purine metabolism. Comparison of mutation in stationary-phase cultures of E. coli WP2 and a series of its DNA repair-deficient mutants exposed to azaserine showed that the effects of the compound closely mimicked those caused by UV light indicating, therefore, that azaserine-induced mutagenesis occurred via pathways dependent upon the recA and lexA genes. Comparison of survival of these strains showed that potentially lethal DNA lesions induced by azaserine were corrected by the excision, recombination, and rec-lex repair systems. These results show that azaserine causes DNA damage as well as inhibition of purine metabolism.

A MWPC with a cathode coupled delay line read-out as radioactivity detector for DNA repair studies

A non selective method for the isolation of DNA repair-deficient mutants in mammalian cells is discussed. The method requires radioactive labelling of the short DNA sequences synthesized during repair of damaged regions. Mutants should be recognized by the absence of radioactive incorporation into thier DNA. A multiwire proportional chamber (MWPC) is proposed as a suitable radioactivity detector. The performance of a MWPC prototype with a cathode coupled delay line read-out is described and is shown to be adequate for this application. The main avaantages of a MWPC are reviewed with respect to other methods used for β− radioactivity counting of biological samples, such as liquid scintillators or autoradiography: the proposed detection method is non destructive for the cells, which are being kept alive for further biological studies; furthermore many cell clones can be screened within a reasonable time.

Sensitivity of DNA repair deficient mutants ofEscherichia colito freezing and thawing

Sensitivity of DNA repair deficient mutants of<i>Escherichia coli</i>to freezing and thawing

Sensitivity of DNA repair deficient mutants of Escherichia coli to freezing and thawing

Sensitivity of DNA repair deficient mutants of Escherichia coli to freezing and thawing

Evidence for the inducibility of the uvrB operon.

A new tool for probing the regulation of non-essential gene products has recently been developed1—this is a derivative of bacteriophage Mu into which the Escherichia coli genes for lactose metabolism have been fused (referred to here as Mu(lac, amp)). The DNA of this defective virus can insert randomly into the chromosome of a cell and mutants canbe retrieved in which the β-galactosidase gene carried on the phage is under the control of the regulatory elements of the operon in which it now resides.We used this phage to generate DNA repair-deficient mutants, one of which washighly UV-sensitive due to insertion of the Mu(lac, amp) into the uvrB regionof the chromosome. When exposed to UV light, this strain produces β-galactosidase at a much greater rate than unexposed cells. This suggests that the uvrB operon is inducible. These results were initially rather surprising. Although various components of the enzyme systems involved in Weigle-reactivation2,3, post-replication repair4–6 and repair of alkylation damage7,8 had been established as inducible, the products of the uvrA, uvrB and uvrC genes which collectively effect nucleotide excision repair9–11, seemed to be firmly established as a constitutively synthesized repair system. This conclusion was drawn because the activity of these enzymes can be easily detected in uninduced cells9–11; and mutations in the genes recA and lexA, which affect the regulation of most of the inducible repair systems12, have relatively little effect on uvrA, uvrB -mediated excision repair13. Thus the uvrA and uvrB gene products seemed to be produced constitutively at maximal or near maximal rates. However, recent reports suggest a role for the uvrA and uvrB gene products in various inducible repair systems6,14–17. Because these studies all relied on indirect measurements of induction, it could never be determined if the results reflected induction of other components of these systems or if the uvrA or uvrB gene products themselves were regulated. The insertion mutant described here allowed us to monitor expression of the uvrB operon directly and provided evidence for regulated synthesis of the uvrB gene product.

A screening method for isolating DNA repair-deficient mutants of CHO cells.

A simple procedure for isolating mutagen-sensitive clones of CHO cells was developed and applied in mutant hunts in which colonies were screened for hypersensitivity to killing by ultraviolet radiation (UV, ethyl methanesulfonate (EMS), or mitomycin C (MMC). Each of two UV-sensitive clones studied in detail had a D37 dose of 1.0 J/m2 compared to 7.0 J/m2 for the wild-type cells, and each was shown to have no detectable repair replication following exposure to UV doses of up to 26 J/m2. Although these mutants resemble xeroderma pigmentosum human mutants with respect to their repair defect and cross-sensitivity to the carcinogen 4-nitroquinoline-1-oxide, one of two clones (UV-20) is characterized by extreme hypersensitivity to MMC (80-fold as compared to the wild type). Clones having hypersensitivity to alkylating agents, but not UV, were obtained using MMC and EMS. In the latter case the two clones had significantly increased sensitivity to the killing action of 60Co gamma-rays.

20 Fragmentation of cis-platinum(II) diamminodichloride-induced filaments of wild type and DNA repair-deficient mutants of Escherichia coli

It has been previously reported that the ultraviolet sensitivity of recA strains of Escherichia coli in the dark is suppressed by a plasmid pKY1 which carries the phr gene, suggesting that this is due to a novel effect of photoreactivating enzyme (PRE) of E. coli in the dark (Yamamoto et al., 1983a). In this work, we observed that an increase of UV-resistance by pKY1 in the dark is not apparent in strains with a mutation in either uvrA, uvrB, uvrC, lexA,recBC or recF. The sensitivity of recA lexA and recA recBC multiple mutants to UV is suppressed by the plasmid but that of recA uvrA, recA uvrB an recA uvrC is not. Host-cell reactivation of UV-irradiated λ phage is slightly more efficient in the recA/pKY1 strain compared with the parental recA strain. On the other hand, the recA and recA/pKY1 strains do not differ significantly in the following properties: Hfr recombination, induction of λ by UV, and mutagenesis. We suggest that dark repair of PRE is correlated with its capacity of excision repair.

Synergistic killing of Escherichia coli by near-UV radiation and hydrogen peroxide: distinction between recA-repairable and recA-nonrepairable damage

Wild-type cells and six DNA repair-deficient mutants (lexA, recA, recB, recA, recB, polA1, and uvrA) of Escherichia coli K-12 were treated with near-ultraviolet radiation plus hydrogen peroxide (H2O2). At low H2O2 concentrations (6 X 10(-6) to 6 X 10(-4) M), synergistic killing occurred in all strains except those containing a mutation in recA. This RecA-repairable damage was absent from stationary-phase cells but increased in logarithmic cells as a function of growth rate. At higher H2O2 concentrations (above 6 X 10(-4) M) plus near-ultraviolet radiation, all strains, including those with a mutation in recA, were synergistically killed; thus, at high H2O2 concentrations, the damage was not RecA repairable.

Suggestive evidence for relationship between sensitivity and repair capability in rat ascites hepatoma cells treated with the antitumor agent, 1-(gamma-chloropropyl)-2-chloromethylpiperidine hydrobromide.

Antitumor activity of 1-(gamma-chloropropyl)-2-chloromethylpiperidine hydrobromide (CAP-2) was studied in vivo and in vitro, using various rat ascites hepatoma cell lines. Among eight ascites hepatoma cell lines, AH-13 was extremely sensitive both to in vivo antitumor and to in vitro lethal action of the agent, whereas AH-44 was resistant in both cases. The sensitivity of ascites hepatoma cell lines to CAP-2, nitrogen mustard N-oxide, 4-nitroquinoline 1-oxide, and ultraviolet ray in vitro was widely different but their relative sensitivities were very similar against these agents. For all the agents, AH-13 was inactivated very rapidly and AH-109A moderately, whereas AH-44 was relatively resistant. These results indicate that the sensitivity of the cells to CAP-2 may be closely related to their repair-capability of damaged DNA. Similar experiments using various DNA repair-deficient mutants of Escherichia coli B strain demonstrated that the repair-deficient mutants were several times more sensitive to CAP-2 than the wild type strain. From these results, it may be concluded that CAP-2 induces DNA lesions repairable by the same repair mechanisms that work on pyrimidine dimers.