Daniel Billen

The Role of Hydroxyl Radical Scavengers in Preventing DNA Strand Breaks Induced by X Irradiation of Toluene-Treated Escherichia coli

The production of strand breaks by X rays in cellular DNA can result from direct action of the radiation on DNA or from indirect action. Indirect action may depend on OH radicals, hydrogen atoms, or solvated electrons formed by the radiolysis of intracellular water [for a recent review see Ref. (1)]. From studies on the rates of reaction of chemical scavengers for the OH radical and their protective activities, it has been concluded that the OH radical is the primary cause of indirect DNA strand breakage in cells (1-3). Recently it has been proposed that certain protective organic compounds may function in a more complex way than by simple OH radical removal. Ewing (4-6), using lethality as an end point, has found that under certain conditions of irradiation there is a correlation between radiation protection and the formation of a secondary a-hydroxyl radical. For example, he has shown that t-butanol and t-amyl alcohol, both excellent OH radical scavengers that are not converted to a-hydroxyl radicals, also do not protect Escherichia coli B/r cells if they are X-irradiated in air or 100% N2 but will protect if they are irradiated in 1% 02 (5, 6). The purpose of this study was to determine whether chemical protection against single-strand breaks observed in toluene-treated E. coli subjected to X irradiation in air (7) was due to the removal of OH radicals, or resulted from the production of secondary radicals as proposed by Ewing (4). In toluene-treated cells, DNA strandbreak production can be measured without the complication of strand ligation during or immediately following X-ray exposure since such cells are deficient in DNA ligase activity (7). Details of growth and of the methods of toluene treatment of E. coli strain AB3063Fwere described earlier (7, 8). Irradiation was carried out with a TFI Corporation Gemini Industrial X-Ray Unit, equipped with a 3-mm aluminum filter, at a dose rate of about 1 krad/min. Toluene-treated cells were rapidly thawed and added to 50 mM potassium phosphate buffer, pH 7.4 (a final concentration of 5-10 X 108 cells/ml), plus the radioprotective agent. All preparations were held 10 min at room temperature in sealed 2-ml vials prior to X-ray exposure; during irradiation the samples were held in an ice bath. The 02

The effects of radioprotectors on DNA polymerase I-directed repair synthesis and DNA strand breaks in toluene-treated and X-irradiated Escherichia coli.

In Escherichia coli made permeable to nucleotides by toluene treatment, a DNA polymerase I-directed repair synthesis is induced by exposure to X rays. This repair synthesis may be amplified and easily measured through inhibition of DNA ligase action. In an effort to learn more of the relationship between X-ray-induced strand breaks in cellular DNA and the extent of this repair synthesis, experiments designed to compare the influence of radioprotectors on both strand-break production and repair synthesis have been carried out. The results show that cysteamine, sodium formate, and glycerol not only protect against strand breaks but also reduce DNA polymerase I-directed repair synthesis. However, I-, an efficient hydroxyl radical scavenger, is not as effective a protective agent against strand breaks and does not measurably affect repair synthesis in our system.

Free radical scavenging and the expression of potentially lethal damage in X-irradiated repair-deficient Escherichia coli

When cells are exposed to ionizing radiation, they suffer lethal damage (LD), potentially lethal damage (PLD), and sublethal damage (SLD). All three forms of damage may be caused by direct or indirect radiation action or by the interaction of indirect radiation products with direct DNA damage. In this report I examine the expression of LD and PLD caused by the indirect action of X rays in isogenic, repair-deficient Escherichia coli. The radiosensitivity of a recA mutant, deficient both in pre- and post replication recombination repair and SOS induction (inducible error-prone repair), was compared to that of a recB mutant which is recombination deficient but SOS proficient and to a previously studied DNA polymerase 1-deficient mutant (polA) which lacks the excision repair pathway. Indirect damage by water radicals (primarily OH radicals) was circumvented by the presence of 2 M glycerol during irradiation. Indirect X-ray damage by water radicals accounts for at least 85% of the PLD found in exposed repair-deficient cells. The DNA polymerase 1-deficient mutant is most sensitive to indirect damage with the order of sensitivity polA1 greater than recB greater than or equal to recA greater than wild type. For the direct effects of X rays the order of sensitivity is recA greater than recB greater than polA1 greater than wild type. The significance of the various repair pathways in mitigating PLD by direct and indirect damage is discussed.

The role of DNA polymerases in DNA repair in Escherichia coli: DNA polymerase I-dependent repair following chemical alkylation of permeabilized bacteria☆

Abstract Many mutagens and carcinogens damage DNA and elicit repair synthesis in cells. In the present study we report that alkylation of the DNA of Escherichia coli that have been made permeable to nucleotides by toluene treatment results in the expression of a DNA polymerase I-directed repair synthesis. The advantage of the system described here is that it permits measurement of only DNA polymerase I-directed repair synthesis and serves as a simple, rapid method for determining the ability of a given chemical to elicit “excision-repair” in bacteria. DNA ligation is intentionally prevented in our system by addition of the inhibitor nicotinamide mononucleotide. In the absence of DNA ligase activity, nick translation is extensive and an “exaggerated” repair synthesis occurs. This amplification of repair synthesis is unique for DNA polymerase I since it is not observed in mutant cells deficient in this polymerase. DNA ligase apparently controls the extent of nucleotide replacement by this repair enzyme through its ability to rejoin “nicks” thereby terminating the DNA elongation process. The nitrosoamides N -methyl- N -nitrosourea and N -ethyl- N -nitrosourea, as well as the nitrosoamidines N -methyl- N′ -nitro- N -nitrosoguanidine and N -ethyl- N′ -nitro- N -nitrosoguanidine, elicit DNA polymerase I-directed repair synthesis. Methyl methanesulphonate is especially potent in this regard, while its ethyl derivative, ethyl methanesulphonate, is a poor inducer of DNA polymerase I activity in permeabilized cells.

DNA Polymerase I Is Crucial for the Repair of Potentially Lethal Damage Caused by the Indirect Effects of X Irradiation in Escherichia coli

The radiosensitivity of an Escherichia coli mutant deficient in DNA polymerase I was measured in the presence of OH radical scavengers. The extreme X-ray sensitivity of the mutant could be abolished by OH radical scavengers if a sufficiently high level of radioprotector was present. There was a direct correlation between the OH radical scavenging activity of the chemicals tested (NO2-, n-butanol, glycerol, t-amyl alcohol, and t-butanol) and their protective ability. I interpret the data as showing that the indirect actions of X rays (primarily OH radicals) result in major damage to the bacterial DNA which in large part consists of potentially lethal lesions. This potentially lethal damage is repaired through an enzymatic pathway requiring DNA polymerase I. In the mutant lacking DNA polymerase I, these potentially lethal lesions are expressed as cell lethality.

Chapter 20 The Use of T ween-8 0 -Permeabiliyed Mammalian Cells in Studies of Nucleic Acid Metabolism

Publisher Summary The chapter describes the use of Tween-80 as an agent that produces a reversible permeability to nucleotides in Chinese hamster ovary (CHO) cells. Several techniques are described for rendering mammalian cells permeable to exogenous nucleoside triphosphates for the purpose of studying DNA metabolism. These include cold shock and cold shock in a hypotonic medium. The use of Tween-80 to permeabilize mammalian cells seems to have general applicability. CHO cells are reversibly permeabilized by Tween; this has obvious advantages for studies in which subsequent cell growth is important. Whether other cell types can be reversibly permeabilized probably depends on whether or not appropriate conditions for achieving it are attainable. The possibility of using this technique to introduce enzymes and other proteins into cells has not been explored, however clearly, if such could be done, the usefulness of this permeabilization process would be increased for in situ studies of cellular activities.

Differential sensitivity of DNA replication and repair in permeable Escherichia coli exposed to various monofunctional methylating or ethylating agents

Escherichia coli cells made permeable to deoxynucleoside triphosphates by brief treatment with toluene (permeablized) were used to measure the effect of the following chemical alkylating agents on either DNA replication or DNA repair synthesis: methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), N-methyl-N-nitrosourea (MNU), N-ethyl-N-nitrosourea (ENU), N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and N-ethyl-N′-nitro-N-nitrosoguanidine (ENNG). Replication of DNA in this pseudo-in vivo system was completely inhibited 10–15 min after exposure to MMS at concentrations of 5 mM or higher or to MNU or MNNG at concentrations of 1 mM or higher. The ethyl derivatives of the alkylating agents were less inhibitory than their corresponding methyl derivatives, and inhibition of DNA replication occurred in the following order: EMS < ENNG < ENU. Maximum inhibition of DNA replication by all of the alkylating agents tested except EMS occurred at a concentration of 20 mM or lower. The extent of replication in cells exposed to EMS continued to decrease with concentrations of EMS up to 100 mM (the highest concentration tested). The experiments in which the inhibition of DNA replication by MMS, MNU, or MNNG was measured were repeated under similar assay conditions except that a density label was included and the DNA was banded in CsCl gradients. The bulk of the newly synthesized DNA from the untreated cells was found to be of the replicative (semi-conservative) type. The amount of replicative DNA decreased with increasing concentration of methylating agent in a manner similar to that observed in the incorporation experiments. Polymerase I (Pol I)-directed DNA repair synthesis induced by X-irradiation of permeablized cells was assayed under conditions that blocked the activity of DNA polymerases II and III. Exposure of cells to MNNG or ENNG at a concentration of 20 mM resulted in reductions in Pol I activity of 40 and 30%, respectively, compared with untreated controls. ENU was slightly inhibitory to Pol I activity, while MMS, EMS, and MNU all caused some enhancement of Pol I activity. These data show that DNA replication in a pseudo-in vivo bacterial system is particularly sensitive to the actions of known chemical mutagens, whereas DNA repair carried out by the Pol I repair enzyme is much less sensitive and in some cases apparently unaffected by such treatment. Possible mechanisms for this differential effect on DNA metabolism and its correlation with current theories of chemically induced mutagenesis and carcinogenesis are discussed.

Yield of DNA strand breaks and their relationship to DNA polymerase I-dependent repair synthesis and ligation following x-ray exposure of toluene-treated Escherichia coli

In Escherichia coli made permeable to nucleotides by toluene treatment, a DNA polymerase I-directed repair synthesis is observed. This is an exaggerated repair synthesis which can be abruptly terminated by the addition of the DNA ligase cofactor, nicotinamide adenine dinucleotide. This communication describes experiments which bear on the relationship between measurable strand breaks, DNA polymerase I-directed, exaggerated repair synthesis, and strand-break repair.

On the issue of citation of "relevant" published studies in widely different cell systems.

cially diagnosed cases of CRS that meet a more rigid set of diagnostic criteria: (1) Exposure for at least 3 years with individual measurements confirming at least 1 Gy of marrow dose received; (2) medical follow-up confirming the clinical manifestations considered by Guskova and Baysogdolov to be the criteria of CRS; and (3) absence of other illness with similar symptomatology. When one focuses on this subset of 66 cases, the dose-response relationship between CRS severity, hematopoietic depression and chromosomal aberrations is clearer. Of note is that of