Introduction Of Single-strand Breaks Research Articles

Processing of UV damage in vitro by FEN-1 proteins as part of an alternative DNA excision repair pathway.

Ultraviolet (UV) irradiation induces predominantly cyclobutane and (6-4) pyrimidine dimer photoproducts in DNA. Several mechanisms for repairing these mutagenic UV-induced DNA lesions have been identified. Nucleotide excision repair is a major pathway, but mechanisms involving photolyases and DNA glycosylases have also been characterized. Recently, a novel UV damage endonuclease (UVDE) was identified that initiates an excision repair pathway different from previously established repair mechanisms. Homologues of UVDE have been found in eukaryotes as well as in bacteria. In this report, we have used oligonucleotide substrates containing site-specific cyclobutane pyrimidine dimers and (6-4) photoproducts for the characterization of this UV damage repair pathway. After introduction of single-strand breaks at the 5' sides of the photolesions by UVDE, these intermediates became substrates for cleavage by flap endonucleases (FEN-1 proteins). FEN-1 homologues from humans, Saccharomyces cerevisiae, and Schizosaccharomyces pombe all cleaved the UVDE-nicked substrates at similar positions 3' to the photolesions. T4 endonuclease V-incised DNA was processed in the same way. Both nicked and flapped DNA substrates with photolesions (the latter may be intermediates in DNA polymerase-catalyzed strand displacement synthesis) were cleaved by FEN-1. The data suggest that the two enzymatic activities, UVDE and FEN-1, are part of an alternative excision repair pathway for repair of UV photoproducts.

FORMATION OF SINGLE AND DOUBLE STRAND BREAKS IN DNA ULTRAVIOLET IRRADIATED AT HIGH INTENSITY

The induction of single-strand breaks (SSB) by two quantum processes in DNA is well established. We now report that biphotonic processes result in double-strand breaks (DSB) as well. pUC19 and bacteriophage M13 RF DNA were irradiated using an excimer laser (248 nm) at intensities of 10(7), 10(9), 10(10) and 10(11) W/m2 and doses up to 30 kJ/m2. The proportion of DNA as supercoil, open circular, linear and short fragments was determined by gel electrophoresis. Linear molecules were noted at fluences where supercoiled DNA was still present. The random occurrence of independent SSB in proximity to each other on opposite strands (producing linear DNA) implies introduction of numerous SSB per molecule in the sample. If so, supercoiled DNA that has sustained no SSB should not be observed. A model accounting for the amounts of supercoiled, open circular, linear and shorter fragments of DNA due to SSB, DSB and Scissions (opposition of two independently occurring SSB producing an apparent DSB) was developed, our experimental data and those of others were fit to the model, and quantum yields determined for SSB and DSB formation at each intensity. Results showed that high intensity laser radiation caused an increase in the quantum yields for both SSB and DSB formation. The mechanism of DSB formation is unknown, and may be due to simultaneous cleavage of both strands in one biphotonic event or the biased introduction of an SSB opposite a preexisting SSB, requiring two biphotonic events.

Response to Commentary by D. Billen

In a recent Commentary (1), Billen, considering the deleterious effects of ionizing radiation on cells, equates damage produced by this agent on cellular DNA with that which occurs naturally by hydrolysis (2) and by oxidation resulting from metabolism (3). From these considerations Billen concludes that there is a negligible dose of ionizing radiation. This conclusion is not warranted when the quality of the damage induced by radiation is taken into account. There are fundamental differences between the nature of the damage produced by ionizing radiation and that caused by the endogenous processes. In previous publications we have pointed out these differences (4, 5). That work can be summarized: At equal levels of total amounts of DNA damage, hydrolysis (6) or oxidation products (7) are several orders of magnitude less biologically effective than ionizing radiation damage. We assign this difference in effectiveness to the double-strand breaks (DSBs) and locally multiply damaged sites (LMDS) which are induced in DNA by ionizing radiation (8). The damage introduced by hydrolysis (loss of purines, pyrimidines, and amino groups from bases) and oxidation (base alterations and introduction of single-strand breaks (SSBs)) is part of the cell's evolutionary experience. It is not surprising, therefore, that cells have developed several efficient and accurate repair mechanisms for handling these types of damage. DNA DSBs, on the other hand, as Billen points out (1), have not been detected as a result of endogenous processes so that repair of these lesions may not have evolved (an ancillary question here is: Is there a repair gene specific for DSB?). The DSBs produced by background radiation are formed infrequently: At a typical background dose rate of 0.1 cGy per year the average mammalian cell experiences a DNA DSB once every 25 years. It is questionable whether this frequency would be sufficient to educe a repair mechanism. In our comparisons of oxidative damage produced by hydrogen peroxide and ionizing radiation, the special nature of the DNA DSBs induced by the latter agent has been described. We pointed out (9) the ease with which singly damaged sites can be enzymatically repaired and contrasted this facility with the problems facing a cell in its attempts to repair DNA DSBs accurately and efficiently. From these arguments, we would challenge Billen's equating, on purely quantitative grounds, DNA damage produced by ionizing radiation with that caused by natural processes. The quality of the DNA damage is a major aspect which must be taken into account.

DNA of human Raji target cells is damaged upon lymphocyte-mediated lysis.

Human Raji target cells DNA is degraded by the introduction of single-strand breaks (alkali-sensitive sites) upon lymphocyte-mediated lysis. This type of DNA degradation appears earlier and is more extensive in lymphocyte-than in antibody + complement-mediated lysis of Raji cells, regardless of the species of effector lymphocytes (human or mouse). Mouse P815 target cell DNA is extensively fragmented (yielding 200 base pair fragments) when human or mouse lymphocytes are used to lyse P815. Thus, these observations indicate that both human and mouse target cell DNA are affected during lymphocyte-mediated lysis. Moreover, the pattern of DNA degradation in target cells lysed by effector lymphocytes is characteristic of the target cell species, suggesting that DNA degradation proceeds through the activation of target cell endonuclease(s).

The effect of chlorpromazine on transformation in Bacillus subtilis.

In the presence of the widely used tranquilizer, chlorpromazine, transforming DNA of Bacillus subtilis is photoinactivated by long-wave ultraviolet light. The loss of biological activity is predominantly caused by lack of binding of the DNA to recipient cells and the introduction of single-strand breaks in the treated DNA.

Purification and properties of a manganese-stimulated deoxyribonuclease produced during sporulation of Bacillus subtilis.

A DNAase (deoxyribonuclease) was isolated from culture supernatants of sporulating Bacillus subtilis 168. The purified enzyme migrated as a single band during polyacrylamide-gel electrophoresis. The enzyme differs from other DNAases of B. subtilis in molecular weight, metal-ion requirement and mode of action. The enzyme was inactive in the absence of metal ions, and exhibited optimum activity with 10 mM-Mn2+, although Mg2+, Cd2+ and Co2+ could also permit some activity. The pH optimum for the enzyme was pH 7.5, and it degraded linear-duplex DNA or closed-circular-duplex DNA to acid-soluble material. There was little or no activity on single-stranded DNA or rRNA. Sucrose-gradient analysis of the products of DNAase action on bacteriophage T7 DNA showed that endonucleolytic cleavage had occurred by the introduction of single-strand breaks in both strands of the duplex. The molecular weight of the enzyme was determined, by gel filtration on Sephadex G-75, to be 12000.

Detection of strand breaks in phiX 174 RFI and PM2 DNA reacted with ultimate and proximate carcinogens.

Supercoiled DNA duplexes of phages phiX 174 and PM2 were treated in aqueous solution at neutral pH with ultimate and proximate carcinogens. Subsequently, the carcinogen-treated phage DNAs were subjected to velocity sedimentation in neutral and alkaline sucrose to quantitative introduction of single strand breaks. Reaction of phage DNA with the ultimate carcinogens N-methyl-N-nitrosourea (MeNOUr), N-ethyl-N-nitrosourea (EtNOUr), 7-bromomethyl-benza[a]-anthracene, N-acetoxy-2-acetylaminofluorene [(Ac)2ONFln] and K-region oxides for short periods followed by sedimentation in neutral sucrose gradients led to very few breaks. Incubation with the proximate carcinogens N-hydroxy-2-acetylaminofluorene, 2-acetylaminofluorene, 7-methyl-, and 7,12-dimethyl-benza[a]anthracene did not result in breaks. However, when the phage DNAs were reacted with the ultimate carcinogens under the same conditions but subsequently alkali-denatured and sedimented in alkaline sucrose gradients, single strand breaks were readily introduced. Incubation with the proximate carcinogens followed by alkali denaturation and sedimentation in alkaline sucrose showed that only 7,12-dimethyl-benz[a]anthracene and, to a minor extent, 7-methyl-benz[]anthracene caused alkali-inducible breaks. The ability of N-methyl-N'-nitro-N-nitrosoguanidine to effect breakdown of superhelical phage DNA in alkali was found enhanced in the presence of N-acetyl-cysteine.

Properties of uvrE mutants of Escherichia coli K12 I. Effects of UV irradiation on DNA metabolism

Escherichia coli K12 uvrE is a mutator strain which is highly sensitive to ultraviolet (UV) radiation. In an attempt to determine the underlying molecular basis for the UV sensitivity, we have compared a mutant and an isogenic wild type strain with regard to several metabolic responses to 254-nm radiation. The introduction of single-strand breaks into intracellular DNA after irradiation is normal. However, the rate of excision of pyrimidine dimers as well as of DNA degradation and final rejoining of the strand breaks is lower in the mutant as compared to the repair proficient strain. These data suggest that the uvrE gene product may be involved in a reaction between the incision and excision steps in the excision repair process.

Properties of uvrE mutants of Escherichia coli K12 I. Effects of UV irradiation on DNA metabolism

Escherichia coli K12 uvrE is a mutator strain which is highly sensitive to ultraviolet (UV) radiation. In an attempt to determine the underlying molecular basis for the UV sensitivity, we have compared a mutant and an isogenic wild type strain with regard to several metabolic responses to 254-nm radiation. The introduction of single-strand breaks into intracellular DNA after irradiation is normal. However, the rate of excision of pyrimidine dimers as well as of DNA degradation and final rejoining of the strand breaks is lower in the mutant as compared to the repair proficient strain. These data suggest that the uvrE gene product may be involved in a reaction between the incision and excision steps in the excision repair process.

The repair of methyl methanesulphonate-induced lesions in the DNA of a murine lymphoma cell

The introduction of single-strand breaks into the DNA of a murine lymphoma (L5178Y) cell treated in vivo with methyl methanesulphonate (MMS) and the behaviour of these breaks on post-treatment incubation were studied. A large proportion of single-strand breaks present after MMS treatment could be repaired as shown by sedimentation in alkaline sucrose. Two inhibitors of DNA synthesis, hydroxyurea and cytosine arabinoside affected the repair process differently-hydroxyurea had only a small effect while cytosine arabinoside blocked repair and at some doses allowed further degradation of the DNA. It was also found that the level of ‘repair replication’ in the presence of cytosine arabinoside was lower than that found in the presence of hydroxyurea.

The Deoxyribonucleic Acid Nucleotidyltransferase Activities of the Shope Fibroma: Partial Purification and Kinetics

Abstract DNA nucleotidyltransferase was partially purified from Shope fibroma tissue. The final preparation was free of terminal transferase but contained traces of deoxyribonuclease and nonspecific phosphodiesterase. The preparation was active with either native or denatured DNA as template. When assayed with native DNA as template, the activity was purified 61-fold, whereas when denatured DNA was used as template, purification was 179-fold. The Km for deoxyribonucleoside triphosphates for the native DNA- and denatured DNA-primed activities were 0.046 mm and 0.16 mm, respectively. The Km for native DNA varied with the source of the DNA. The denatured DNA reaction alone was inhibited by high concentrations of substrate. The Km values (20 to 25 µg per ml) for denatured DNA were almost the same for denatured DNA from four different sources, but the K's values varied widely (90 to 650 µg per ml). Brief treatment with DNase I greatly increased the Vmax of the native but not the heat-denatured DNA-primed reaction. The Km of the native DNA-primed reaction was not affected. Despite the affinities for template demonstrable kinetically, the DNA nucleotidyltransferases and DNA failed to form complexes that were stable in neutral sucrose gradients. The data for the reaction primed by denatured DNA suggest competition by 2 molecules of denatured DNA for a secondary binding site for template DNA. The increase in Vmax but not in Km of the reaction primed by native DNA after partial digestion of the template by DNase I may have been due to the increased ease of unwinding of the molecule after the introduction of single strand breaks, whereas the failure of the same preparations to exhibit enhanced priming activity after denaturation indicates the necessity for DNA of some minimum length for the denatured DNA-primed enzyme activity. The data suggest that the preparation contains two different DNA nucleotidyl-transferases, but the possibility that a single molecule is responsible for both activities is not ruled out.