Paul Howard-Flanders

Discontinuities in the DNA synthesized in an Excision-defective strain of Escherichia coli following ultraviolet irradiation

Although Escherichia coli K12 uvrA6 is defective in the excision of pyrimidine dimers from its DNA, 37% of cells survive a dose of ultraviolet light which is equivalent to about 50 pyrimidine dimers per 107 nucleotides. The amount of tritiated thymidine incorporated into the DNA of irradiated cells indicates that pyrimidine dimers in the DNA inhibit DNA synthesis but are not permanent blocks. Zone sedimentation of single-strand DNA was performed in alkaline sucrose gradients. To minimize degradation by shearing, the DNA was released from spheroplasts layered on top of the gradients. Newly synthesized, denatured DNA from unirradiated cells sediments with a molecular weight of greater than 100 × 106, whereas the newly synthesized, denatured DNA from cells irradiated with 60 ergs/mm2 has a molecular weight of about 14 × 106. During subsequent incubation of the irradiated cells, the sedimentation rate of the DNA synthesized immediately after irradiation increases and approaches that of normal DNA. However, at any time during this incubation period, the incorporation of tritiated thymidine into fast-sedimenting DNA is minimal, suggesting that the daughter-strand DNA synthesized after ultraviolet-irradiation contains gaps, or alkalilabile bonds. These dicontinuities disappear during further incubation, as a higher rate of sedimentation is found. The number of these daughter-strand defects is similar to the number of pyrimidine dimers in an equivalent length of parental DNA.

Exchanges between DNA strands in ultraviolet-irradiated Escherichia coli☆

Abstract Ultraviolet induced pyrimidine dimers remain in the DNA of excision-defective mutants of Escherichia coli for at least several hours after irradiation. If kept in the dark, the survival of colony-forming ability of these mutant bacteria depends upon recombinational repair, a mechanism which is controlled by the gene recA . In u.v.-irradiated bacteria, newly synthesized DNA strands are of low molecular weight, but these strands are subsequently joined into high molecular weight chains, as is shown by sedimenting the bacterial DNA in alkaline sucrose gradients. These longer chains may contribute to cell survival by acting as templates for the synthesis of high molecular weight strands during further replication. At times greater than 90 minutes after irradiation, the molecular weight of newly synthesized DNA covers a wide range and extends fully to the maximum found with unirradiated cells, thus indicating the presence of high molecular weight templates virtually free from damaged bases. Evidently the u.v. photoproducts tend to remain in the original strands where they were formed, rather than becoming distributed equally among all the strands with each subsequent replication. Recombinational repair may depend upon genetic exchanges between sister duplexes. To see whether such exchanges could be detected after u.v.-irradiation, cells were grown for several generations in medium containing 13 C and 15 N. They were then exposed to various doses of ultraviolet light and grown for less than a generation in light medium containing [ 3 H]thymidine, so that part of their DNA was hybrid in density, with the 3 H label in the light strand. Under these conditions, light 3 H-labeled chains will become linked to heavy strands if exchanges occur between sister duplexes. The DNA was extracted from unirradiated cells, heat denatured and centrifuged in neutral cesium chloride gradients, and was found to separate into heavy and light bands. Denatured DNA from u.v.-irradiated cells did not separate completely, but contained strands of intermediate density which separated into heavy and light components only after shearing to 5 × 10 5 molecular weight, thus indicating that segments of molecular weight greater than 5 × 10 5 had been exchanged. To judge from the fraction of the 3 H-labeled molecules that were of intermediate density, one exchange occurred for every one to two pyrimidine dimers in the DNA replicated in the 3 H-labeled medium. Sister exchanges involving a single strand of each duplex, may insert the correct bases sequence into the gap opposite each pyrimidine dimer, and thus promote the reconstruction of a genome with the complete base sequence needed for the survival of colony-forming ability.

DNA REPAIR AND GENETIC RECOMBINATION: STUDIES ON MUTANTS OF ESCHERICHIA COLI DEFECTIVE IN THESE PROCESSES.

A theory for the mechanism of enzymic repair of DNA containing ultraviolet (UV) photoproducts in UV-irradiated bacteria was proposed in 1964 (1, 2). Subsequent experiments have lent further support to this theory and have provided evidence that it may be applicable to the enzymic repair of products formed in DNA by a number of different mutagens, including alkylating agents and X-rays. It has also been shown that DNA repair of a similar kind may play an essential role in genetic recombination. With these latter findings taken into consideration, the following more comprehensive theory for the mechanism and functions of DNA repair can be formulated. Four main steps are involved in DNA repair: 1. Single strands of two-strand DNA are interrupted in either of several ways; (a) by a radiation-induced chain break; (b) by the enzymic excision of damaged bases; or (c) by a recombination enzyme that acts during the early stages of genetic recombination. 2. Nucleotides are released, presumably through the action of an enzyme on the free single-strand ends left by these cuts. This enzyme appears to proceed only for a limited distance in normal cells. 3. The DNA twin helix is reconstructed by a DNA repair polymerase that inserts complementary nucleotides into the gap, adding them onto one of the singlestrand ends. Thus, the end of a single strand serves as initiator, while the intact strand opposite serves as template. 4. The repair is completed by joining the phosphodiester backbone when the last nucleotide is inserted into the gap. The mode of action of this repair mechanism in removing a thymine dimer from DNA is illustrated in Fig. 1. The first section of this paper will review experiments on the mechanisms of DNA repair in Escherichia coli following exposure to irradiation or to mutagens. In the next section the role of DNA repair in genetic recombination will be dis-

Abnormal metabolic response to ultraviolet light of a recombination deficient mutant of Escherichia coli K12.

A given dose of ultraviolet irradiation produces as many pyrimidine-dimer-containing photoproducts in the DNA of the Escherichia coli Rec − strain JC1569 as it does in the related Rec + strain JC1557. Both strains excise dimer-containing photoproducts from their DNA to the same extent. In comparison with JC1557, approximately 30 times as much DNA of the Rec − strain is degraded per unit dose of ultraviolet light. Perhaps as a result of this excess ultraviolet-induced degradation, irradiated Rec − cells do not incorporate appreciable amounts of exogenous thymidine or thymine into DNA and have few survivors following exposure to an ultraviolet dose which almost all Rec + cells survive. A numerical argument is offered to establish the ability of Rec − cells successfully to replace excised pyrimidine-dimer-containing regions of the DNA with normal nucleotide residues. No effect of the rec − mutation in JC1569 was found on the level of activity of the following enzymes: endonuclease I, exonuclease I, exonuclease III and DNA phosphatase, and DNA polymerase and exonuclease II.

Macromolecular Repair and Free Radical Scavenging in the Protection of Bacteria against X-Rays

Among the substances that modify the effects of X-rays on cells are oxygen (1-3), nitric oxide (4-7), N-ethylmaleimide (8), sulfhydryls (9-15), and alcohols (1619). To explain the actions of these substances, several authors have suggested hypothetical reaction schemes for the events leading to the formation of injuries in vital macromolecules within the cell (20-24). Similarly, experiments on the survival of the biological activity of X-irradiated bacteriophage under a wide range of sulfhydryl concentrations have led to the formulation of a reaction scheme for the X-ray injury to phage DNA (25), which, in a simplified form, is as follows:

Genetic control of DNA breakdown and repair inE. coli K-12 treated with mitomycin C or ultraviolet light

SummaryUltraviolet light sensitive mutants ofE. coli defective at theuvrA,uvrB oruvrC locus showed increased sensitivity to the lethal effects of mitomycin C when compared with theuvr+ parental strain. In addition, DNA breakdown after treatment of cells with either mitomycin C or with ultraviolet light was greater in the parental strain carrying the activeuvr+ genes than inuvr mutants. Thus, injuries produced by either mitomycin C or by ultraviolet light may be repaired by the same molecular mechanism which has been proposed and which involves defect excision, single strand breakdown and reconstruction of the DNA.

Postreplication repair in E. coli: Strand exchange reactions of gapped DNA by RecA protein

SummaryWe have used a sensitive gel electrophoresis assay to detect the products of Escherichia coli RecA protein catalysed strand exchange reactions between gapped and duplex DNA molecules. We identify structures that correspond to joint molecules formed by homologous pairing, and show that joint molecules are converted by RecA protein into heteroduplex monomers by reciprocal strand exchanges. However, strand exchanges only occur when there is a 3′-terminus complementary to the single stranded DNA in the gap. In the absence of a complementary free end, the two DNA molecules pair and short heteroduplex regions are formed by localised interwinding.

Reactions of Deoxyribonucleic Acid Radicals with Sulfhydryl Compounds in X-Irradiated Bacteriophage Systems

The effects of oxygen and nitric oxide on the inactivation of T2 phage by x rays were investigated both when the free phage was suspended in nutrient broth and when it had adsorbed to the host E. coli B. It was found that, when protected from indirect radiation effects by nutrient broth, the free phage is protected 1.8-fold by hydrogen sulfide and mercaptoethanol, but that this protection is reversed by oxygen or nitric oxide. At higher concentrations of sulfhydryl (20 to 200 mM), the protection is no longer fully reversed by the gases. Analysis of the results indicates that, as regards effect on x-rayinactivation, oxygen and the sulfhydryl are in competition. It is suggested that the substances compete for radioinduced phage DNA radicals, and that anoxic protection is the result of reaction between the sulfhydryl and the phage DNA in which the sulfhydryl donates a hydrogen atom. It appears that the sulfhydryls react about one-thirtieth as rapidly as oxygen with the DNA radicals. The results are discussed in terms of reaction mechanisms. (P.C.H.)

Preferential sensitization of anoxic bacteria to x-rays by organic nitroxide-free radicals.

Living organisms are generally sensitized to the lethal effects of ionizing radiation if either oxygen (1, 2) or nitric oxide (3, 4) is present during irradiation. They are also sensitized by pretreatment with N-ethylmaleimide (5), a sulfhydryl-binding agent, presumably as the result of the reduction in the level of free sulfhydryls in the cell. The sensitizing action of oxygen and nitric oxide, however, seems to be due to the high reactivity of these compounds with radiation-induced free radicals produced in vital molecules within the cell. It has been pointed out that anoxic protection may be a cause of the failure of the treatment of some forms of cancer by ionizing radiation. Oxygen, because of its high metabolic turnover, cannot diffuse far from circulating blood, and, since neoplastic growth tends to cause congestion and interference with the local blood supply, islands of malignant cells can become anoxic and therefore more resistant to damage by ionizing radiation. Some of these cells may therefore survive and serve as foci for regrowth (6, 7). In a search for other suitable substances that might sensitize anoxic cells, we were guided by the following requirements. For clinical application, a sensitizing agent would have to be chemically unreactive, stable in oxygen, nontoxic at concentrations at which it sensitized significantly, not rapidly metabolized, not pharmacologically active, and highly water-soluble rather than fat-soluble. Moreover, since nitric oxide and oxygen are both free radicals (in so far as they contain unpaired electrons), it seemed that an additional requirement would be that the compound should be a free radical. Since the organic nitroxide-free radicals (7-10) appeared to fulfill many of these requirements, we have studied the radiosensitizing action of three of these compounds. A preliminary account of the sensitizing action of the nitroxides on anoxic bacteria has been published (11). We report here a further investigation of their effects on both aerobic and anoxic bacteria and review the possible mechanisms involved.

Duplex-duplex interactions catalyzed by recA protein allow strand exchanges to pass double-strand breaks in DNA

Using gapped circular DNA and homologous duplex DNA cut with restriction nucleases, we show that E. coli RecA protein promotes strand exchanges past double-strand breaks. The products of strand exchange are heteroduplex DNA molecules that contain nicks, which can be sealed by DNA ligase, thereby effecting the repair of double-strand breaks in vitro. These results show that RecA protein can promote pairing interactions between homologous DNA molecules at regions where both are duplex. Moreover, pairing leads to strand exchanges and the formation of heteroduplex DNA. In contrast, strand exchanges are unable to pass a double-strand break in the gapped substrate. This apparent paradox is discussed in terms of a model for RecA-DNA interactions in which we propose that each RecA monomer contains two nonequivalent DNA-binding sites.