In vitro repair of gaps in bacteriophage T7 DNA

An in vitro system based upon extracts of Escherichia coli infected with bacteriophage T7 was used to study the mechanism of double-strand break repair. Double-strand breaks were placed in T7 genomes by cutting with a restriction endonuclease which recognizes a unique site in the T7 genome. These molecules were allowed to repair under conditions where the double-strand break could be healed by (i) direct joining of the two partial genomes resulting from the break, (ii) annealing of complementary versions of 17-bp sequences repeated on either side of the break, or (iii) recombination with intact T7 DNA molecules. The data show that while direct joining and single-strand annealing contributed to repair of double-strand breaks, these mechanisms made only minor contributions. The efficiency of repair was greatly enhanced when DNA molecules that bridge the region of the double-strand break (referred to as donor DNA) were provided in the reaction mixtures. Moreover, in the presence of the donor DNA most of the repaired molecules acquired genetic markers from the donor DNA, implying that recombination between the DNA molecules was instrumental in repairing the break. Double-strand break repair in this system is highly efficient, with more than 50% of the broken molecules being repaired within 30 min under some experimental conditions. Gaps of 1,600 nucleotides were repaired nearly as well as simple double-strand breaks. Perfect homology between the DNA sequence near the break site and the donor DNA resulted in minor (twofold) improvement in the efficiency of repair. However, double-strand break repair was still highly efficient when there were inhomogeneities between the ends created by the double-strand break and the T7 genome or between the ends of the donor DNA molecules and the genome. The distance between the double-strand break and the ends of the donor DNA molecule was critical to the repair efficiency. The data argue that ends of DNA molecules formed by double-strand breaks are typically digested by between 150 and 500 nucleotides to form a gap that is subsequently repaired by recombination with other DNA molecules present in the same reaction mixture or infected cell.     

Removal by human apurinic/apyrimidinic endonuclease 1 (Ape 1) and Escherichia coli exonuclease III of 3'-phosphoglycolates from DNA treated with neocarzinostatin, calicheamicin, and gamma-radiation

DNA strand breaks with terminal 3'-phosphoglycolate groups are produced by agents that can abstract the hydrogen atom from the 4'-carbon of DNA deoxyribose groups. Included among these agents are gamma-radiation (via the OH radical) and enediyne compounds, such as neocarzinostatin and calicheamicin. However, while the majority of radiation-induced phosphoglycolates are found at single-strand breaks, most of the phosphoglycolates generated by these two enediynes are found at bistranded lesions, including double-strand breaks. Using a 32P-post-labelling assay, we have compared the enzyme-catalyzed removal of phosphoglycolates induced by each of these agents. Both human apurinic/apyrimidinic endonuclease 1 (Ape 1) and its Escherichia coli homolog exonuclease III rapidly removed over 80% of phosphoglycolates from gamma-irradiated DNA, although there appeared to be a small resistant subpopulation. The neocarzinostatin-induced phosphoglycolates were removed more slowly, though not to completion, while the calicheamicin-induced phosphoglycolates were extremely refractory to both enzymes. These data suggest that unless other enzymes are capable of acting upon the phosphoglycolate termini at enediyne-induced double-strand breaks, such termini will be resistant to end rejoining repair pathways.     

In vivo damage and recA-dependent repair of plasmid and chromosomal DNA in the radiation-resistant bacterium Deinococcus radiodurans

Deinococcus radiodurans R1 and other members of this genus share extraordinary resistance to the lethal and mutagenic effects of ionizing radiation. We have recently identified a RecA homolog in strain R1 and have shown that mutation of the corresponding gene causes marked radiosensitivity. We show here that following high-level exposure to gamma irradiation (1.75 megarads, the dose required to yield 37% of CFU for plateau-phase wild-type R1), the wild-type strain repairs > 150 double-strand breaks per chromosome, whereas a recA-defective mutant (rec30) repairs very few or none. A heterologous Escherichia coli-D. radiodurans shuttle plasmid (pMD68) was constructed and found to be retained in surviving D. radiodurans R1 and rec30 following any radiation exposure up to the highest dose tested, 3 megarads. Plasmid repair was monitored in vivo following irradiation with 1.75 megarads in both R1/pMD68 and rec30/pMD68. Immediately after irradiation, plasmids from both strains contained numerous breaks and failed to transform E. coli. While irradiation with 1.75 megarads was lethal to rec30 cultures, a small amount of supercoiled plasmid was regenerated, but it lacked the ability to transform E. coli. In contrast, wild-type cultures showed a cell division arrest of about 10 h, followed by exponential growth. Supercoiled plasmid was regenerated at normal levels, and it readily transformed E. coli. These studies show that D. radiodurans retains a heterologous plasmid following irradiation and repairs it with the same high efficiency as its chromosomal DNA, while the repair defect in rec30 prevents repair of the plasmid. Taken together, the results of this study suggest that plasmid DNA damaged in vivo in D. radiodurans is repaired by recA-dependent mechanisms similar to those employed in the repair of chromosomal DNA.     

Effects of the enediyne C-1027 on intracellular DNA targets

We examined DNA damage induced by the enediyne-containing antitumor antibiotic C-1027 in intracellular nuclear and mitochondrial DNA targets using the episome-containing cell line 935.1. Strand-scission activity of the C-1027 holoantibiotic was measured by the topological forms conversion assay in episomal and mitochondrial DNA, as well as in cell-free plasmid DNA. Genomic DNA damage was quantitated by filter elution analysis. Comparisons were made to the well-characterized enediyne neocarzinostatin. From these studies, mixed single- and double-strand breaks were observed not only in cell-free, plasmid DNA but also in intracellular episomal, mitochondrial, and genomic DNA at low nanomolar concentrations. C-1027 cleaved DNA 285-fold more efficiently in cells than in a cell-free environment, and displayed preference for intracellular DNA species in the following rank order: episome > mitochondrial DNA > genomic. NCS also damaged the non-histone-associated mitochondrial DNA, but not the episome. Cleavage of the 935.1 cell episome by C-1027 occurred at specific sites including the BPV origin of replication and E6/E7 open reading frame regions, as well as the MMTV LTR promoter region.     

Hydroxyl radicals and DNA double-strand breaks in X-irradiated E. coli

We have used glycerol to study the relationship between hydroxyl radicals, one of the primary radiolytic products, and the production of DNA double-strand breaks in selected E. coli strains. Our results suggest that when bacteria are irradiated at doses up to about 120 Gray, hydroxyl radicals produce DNA lesions, but not double-strand breaks.     

Comparison of strand breaks in plasmid DNA after positional changes of Auger electron-emitting iodine-125

To elucidate the kinetics of the induction of DNA strand breaks by low-energy Auger electron emitters, we compared the yields of DNA breaks in supercoiled pUC19 DNA after the decay of 125I (1) in proximity to DNA after minor-groove binding (125I-iodoHoechst 33342, 125IH) and (2) at a distance from DNA (125I-iodoantipyrine, 125IAP). Iodine-125 bound to the minor groove in DNA or free in solution is equally effective per decay in producing single-strand breaks (SSBs), while 125I bound to the minor groove is 6.7-fold more efficient than 125I free in solution in producing double-strand breaks (DSBs) (1.08 +/- 0.13 compared to 0.16 +/- 0.01 DSB/decay). Consequently, SSB to DSB ratios for 125IAP and gamma radiation (20.7 +/- 2.9 and 43.8 +/- 1.5, respectively) are greater than that for 125IH (2.9 +/- 0.4). Finally, the decay of 125IH leads to fragmentation of plasmid DNA beyond SSBs and DSBs.     

bcl-2 protein inhibits etoposide-induced apoptosis through its effects on events subsequent to topoisomerase II-induced DNA strand breaks and their repair

Previous studies have shown that bcl-2 overexpression can inhibit apoptosis induced by DNA-damaging agents widely used in cancer chemotherapy, including X-irradiation, alkylating agents (hydroperoxycyclophosphamide, etc.), and topoisomerase II inhibitors (etoposide, etc.). However, little is known about the mechanism by which bcl-2 overexpression inhibits apoptosis triggered by these agents. In this study, we examined whether bcl-2 overexpression could have effects on etoposide-induced DNA damage and its repair. For these experiments, we developed CH31 clones (mouse B-cells) stably transfected with human bcl-2 sense plasmids and compared these clones with a parental CH31 clone or CH31 clones with antisense plasmids. Overexpression of bcl-2 protein inhibited etoposide-induced apoptosis and cytotoxicity. However, there was no or little difference in the production and repair of DNA-protein cross-links, DNA single-strand breaks, and double-strand beaks among a parental CH31 clone and CH31 clones with human bcl-2 sense or antisense plasmids. These findings indicate that (a) apoptosis or cytotoxicity induced by etoposide can be separated into early events (formation of double-strand breaks, DNA single-strand breaks, and double-strand breaks) and later events (secondary DNA fragmentation or cell death) and (b) bcl-2 inhibits apoptosis and cytotoxicity induced by etoposide at some steps between these events.     

Homologous, homeologous, and illegitimate repair of double-strand breaks during transformation of a wild-type strain and a rad52 mutant strain of Saccharomyces cerevisiae

Different modes of in vivo repair of double-strand breaks (DSBs) have been described for various organisms: the recombinational DSB repair (DSBR) mode, the single-strand annealing (SSA) mode, and end-to-end joining. To investigate these modes of DSB repair in Saccharomyces cerevisiae, we have examined the fate of in vitro linearized replicative plasmids during transformation with respect to several parameters. We found that (i) the efficiencies of both intramolecular and intermolecular linear plasmid DSB repair are homology dependent (according to the amount of DNA used during transformation [100 ng or less], recombination between similar but not identical [homeologous] P450s sequences sharing 73% identity is 2- to 18-fold lower than recombination between identical sequences); (ii) the RAD52 gene product is not essential for intramolecular recombination between homologous and homeologous direct repeats (as in the wild-type strain, recombination occurs with respect to the overall alignment of the parental sequences); (iii) in contrast, the RAD52 gene product is required for intermolecular interactions (the rare transformants which are obtained contain plasmids resulting from deletion-forming intramolecular events involving little or no sequence homology); (iv) similarly, sequencing data revealed examples of intramolecular joining within the few terminal nucleotides of the transforming DNA upon transformation with a linear plasmid with no repeat in the wild-type strain. The recombinant junctions of the rare illegitimate events obtained with S. cerevisiae are very similar to those observed in the repair of DSB in mammalian cells. Together, these and previous results suggest the existence of alternative modes for DSB repair during transformation which differ in their efficiencies and in the structure of their products. We discuss the implications of these results with respect to the existence of alternative pathways and the role of the RAD52 gene product.     

Functional features of an ssi signal of plasmid pGKV21 in Escherichia coli

A single-strand initiation (ssi) signal was detected on the Lactococcus lactis plasmid pGKV21 containing the replicon of pWV01 by its ability to complement the poor growth of an M13 phage derivative (M13 delta lac182) lacking the complementary-strand origin in Escherichia coli. This ssi signal was situated at the 229-nucleotide (nt) DdeI-DraI fragment and located within the 109 nt upstream of the nick site of the putative plus origin. SSI activity is orientation specific with respect to the direction of replication. We constructed an ssi signal-deleted plasmid and then examined the effects of the ssi signal on the conversion of the single-stranded replication intermediate to double-stranded plasmid DNA in E. coli. The plasmid lacking an ssi signal accumulated much more plasmid single-stranded DNA than the wild-type plasmid did. Moreover, deletion of this region caused a great reduction in plasmid copy number or plasmid maintenance. These results suggest that in E. coli, this ssi signal directs its lagging-strand synthesis as a minus origin of plasmid pGKV21. Primer RNA synthesis in vitro suggests that E. coli RNA polymerase directly recognizes the 229-nt ssi signal and synthesizes primer RNA dependent on the presence of E. coli single-stranded DNA binding (SSB) protein. This region contains two stem-loop structures, stem-loop I and stem-loop II. Deletion of stem-loop I portion results in loss of priming activity by E. coli RNA polymerase, suggesting that stem-loop I portion is essential for priming by E. coli RNA polymerase on the SSB-coated single-stranded DNA template.     

DNA damage from oxidants: influence of lesion complexity and chromatin organization

DNA damage by reactive oxygen species results in a spectrum of DNA lesions including single-strand breaks (ssb) and double-strand breaks (dsb). However, most damage is not lethal, and the location and nature of the DNA damage, in addition to total number of breaks, are likely to be critical in determining ultimate survival. Generally associated only with ionizing radiation, multiply damaged sites (i.e., complex lesions and clusters of complex lesions in DNA) are more likely to be lethal because they are less easily repaired. We examined five drugs known to cause DNA adducts, strand breaks, and reactive oxygen species for their ability to produce complex lesions: 4-nitroquinoline-1-oxide (4NQO), H2O2, doxorubicin, Tirapazamine, and etoposide. As indicators of lesion complexity we compared 1) the ratio of ssb to dsb, 2) the rate of rejoining of single-strand breaks, 3) the relative lethality of the breaks (number of breaks per mean lethal dose), and 4) the ability to produce complex lesions. Tirapazamine, etoposide, and doxorubicin gave dsb/ssb ratios similar to that for X-rays, whereas 4NQO and H2O2 showed dsb/ssb ratios of 200 and 3250, respectively. The number of dsb per LD50 varied from 2.5 to 500 for different drugs. There was no apparent relation between ssb rejoining half-time (3.5-85 min) and relative lethality or lesion complexity. A modified (nonionic detergent) filter elution method confirmed that tirapazamine, like ionizing radiation, produced multiple dsb within single chromatin domains. These data indicate that complex lesions can be produced by a number of different chemicals and suggest that the damage that results in killing by these drugs may be related to production of multiply damaged sites in DNA.     

Supercopy plasmid based on the replicon from the temperate N15 bacteriophage

Mini-plasmids, based on the N15 temperate bacteriophage replicon, are described. One of these, N15-203 linear 13.8 kb plasmid, has anomalously high copy number--more than 250 per one bacterial chromosome and the amount of plasmid DNA comprises about half of the total DNA of a cell. This property of N15-203 plasmid is realized only in the strain lysogenic for a N15 phage and is lost for the circular deletion versions of N15-203. The efficiency of transformation of E. coli C (N15) strain is essentially the same for N15-203 and pUC4K plasmids. Insertion of foreign DNA with a size up to 20 kb into BgIII cloning site of N15-203 plasmid does not decrease significantly efficiency of transformation calculated per number of DNA molecules and the total amount of plasmid DNA in a cell. N15-203 plasmid may be used as a vector for molecular cloning of relatively large DNA fragments, and in those biotechnology processes when productivity depends on a vector's copy number.     

Two-minute miniprep method for plasmid DNA isolation

An extremely rapid method, INSTA-PREP, has been developed to prepare plasmid DNA from 1 to 3 mL miniprep Escherichia coli bacterial cultures. Direct extraction of plasmid DNA from E. coli bacterial cells is achieved by a two-phase solution consisting of phenol-chloroform-isoamyl alcohol and water or buffer with efficient separation of the phases by centrifugation in the presence of the INSTA-PREP gel barrier material. Processing time, from E. coli culture to usable plasmid DNA, is two minutes or less per sample. Supercoiled plasmid DNA yields ranged from 3 to 10 micrograms per mL of culture depending on plasmid copy number. Plasmid DNAs prepared by INSTA-PREP were analyzed and are suitable for use in molecular biology procedures including restriction digestion, ligation with T4 DNA ligase, bacterial transformation, PCR, cultured cell transfection and T7 DNA polymerase or thermostable DNA polymerase-mediated dideoxynucleotide sequencing.     

Mismatch repair by efficient nick-directed, and less efficient mismatch-specific, mechanisms in homologous recombination intermediates in Chinese hamster ovary cells

Repair of single-base mismatches formed in recombination intermediates in vivo was investigated in Chinese hamster ovary cells. Extrachromosomal recombination was stimulated by double-strand breaks (DSBs) introduced into regions of shared homology in pairs of plasmid substrates heteroallelic at 11 phenotypically silent mutations. Recombination was expected to occur primarily by single-strand annealing, yielding predicted heteroduplex DNA (hDNA) regions with three to nine mismatches. Product spectra were consistent with hDNA only occurring between DSBs. Nicks were predicted on opposite strands flanking hDNA at positions corresponding to original DSB sites. Most products had continuous marker patterns, and observed conversion gradients closely matched predicted gradients for repair initiated at nicks, consistent with an efficient nick-directed, excision-based mismatch repair system. Discontinuous patterns, seen in approximately 10% of products, and deviations from predicted gradients provided evidence for less efficient mismatch-specific repair, including G-A-->G-C specific repair that may reflect processing by a homologue of Escherichia coli MutY. Mismatch repair was > 80% efficient, which is higher than seen previously with covalently closed, artificial hDNA substrates. Products were found in which all mismatches were repaired in a single tract initiated from one or the other nick. We also observed products resulting from two tracts of intermediate length initiated from two nicks.     

Genetic transformation of Vitreoscilla sp

Of all the methods customarily used to transform E. coli we found only electroporation to be effective for transformation of the Gram-negative bacterium Vitreoscilla, yielding 5.10(5) transformants/microgram of plasmid DNA. The conditions used were close to those described for E. coli E. coli plasmids are stably maintained in Vitreoscilla. This is the first report of exogenous DNA transfer in Vitreoscilla which opens the way for the application of recombinant-DNA techniques to study this unique group of organisms.     

Resistance to beta-lactam antibiotics and aminoglycosides in gram negative bacteria. 1. Molecular and genetic characterization of R-factors (author's transl)

With frequent use of aminoglycoside antimicrobials and beta-lactam antibiotics in hospitals in the last few years, the number of bacterial strains resistant to these chemotherapeutics increased. Lately, strains of E. coli, Klebsiella, Enterobacter, Serratia, Proteus and Pseudomonas resistant to many antimicrobials (ampicillin, carbenicillin, cephalothin, chloramphenicol, gentamycin, tobramycin, sisomycin, neomycin, paromomycin, kanamycin, streptomycin, spectinomycin, tetracycline, sulphonamides) were isolated from patients of the university hospital in Zuerich. The resistant phenotype of two representative strains (Klebsiella pneumoniae 1 and Serratia marcescens 2) could be transferred by mixed cultivation to E. coli K-12. Multiple resistance of strain 1, and addition, could be transferred to Salmonella typhimurium, Serratia marcescens, Providencia, Proteus mirabilis and Klebsiella pneumoniae in varying frequencies. Transfer to Pseudomonas aeruginosa, however, could not be achieved. Spontaneous instability of resistance was observed in 0.15% of the cells of an overnight brothe culture and in 90% of the cells of a three months old culture. Conjugation, instability and the response to the sex phages MS-2 and If-1 suggested that resistance was mediated by a monomolecular R-factor, belonging to the fi+-type. This suggestion was confirmed by molecular characterization of the resistance plasmids. After transfer of the R-factors of K. pneumoniae 1 (R-FK 1) and Serratia marcescens 2 (R-FK2) into E. coli K-12, plasmid DNA was labelled with (methyl-3H) thymidine, and isolated by isopycnic centrifugation in cesiumchlorid-ethidium-bromide. Analysis of plasmid DNA then was carried out by sedimentation in a 5-20% neutral sucrose gradient together with reference plasmids of known molecular weights and sedimentation constants. The analysis revealed that R-FK1 had a molecular weight of 54 X 10(6) and R-FK2 of 50 X 10(6) daltons. The values were confirmed by contour length measurements of open circular forms with an electron microscope. A comparison of the sedimentation profile of labelled plasmid DNA from strain 1 and 14C-labelled DNA of E. coli K-12 (R-FK1) showed that the wild-type strain contained, besides the large resistance plasmid, at least two smaller "cryptic" plasmids. These smaller plasmid molecules were also found in antibiotic susceptible variants of strain 1, which did not contain the 54 X 10(6) dalton plasmid molecule, responsible for the resistant phenotype. The number of copies of R-FK1 in E. coli K-12 was determined to be 2, indicating stringent control of replication. It is discussed that the growing number of isolations of strains of Escherichia, Klebsiella, Serratia, Proteus, Providencia and Pseudomonas, exhibiting the same resistance phenotype, results from the spread of the R-factor described above among the hospital bacterial flora.     

Induction and mechanism of DNA single- and double-strand breaks by tetracycline/Cu(II) in the absence of light

In the absence of light, tetracycline (TC) induced single- and double-strand breaks in PM2 DNA at micromolar concentrations in combination with CuCl2, whereas TC or CuCl2 alone had no effect. Strand break formation was completely suppressed by catalase and the specific Cu(I) scavenger neocuproine. The extent of strand break formation depended on the ratio of Cu(II):TC. At a ratio of > or = 2 most DNA damage was observed. The influence of the kind of Cu(II)/TC complexation on DNA strand break formation is discussed. The DNA damage in PM2 DNA provoked by TC/CuCl2 was indirectly detected also in human fibroblasts by the induction of DNA repair. The results are discussed with regard to human risk from TC/Cu(II).     

Modelling DNA damage induced by different energy photons and tritium beta-particles

PURPOSE: To model the production of single- and double-strand breaks (ssb and dsb) in DNA by ionizing radiations. To compare the predicted effectiveness of different energy photon radiations and tritium beta-particles. MATERIALS AND METHODS: Modelling is carried out by Monte Carlo and includes consideration of direct energy depositions in DNA molecules, the production of species, their diffusion and interactions with each other and DNA. Computer-generated electron tracks in liquid water are used to model energy deposition and to derive the initial positions of chemical species. Atomistic representation of the DNA in B form with a first hydration shell is used. Photon radiations in the energy range 70keV-1MeV and tritium beta-particles are considered. RESULTS: A tentative increase for dsb yield has been predicted for 70 keV photons and tritium compared with 137Cs. This increase is more pronounced for complex dsb. Double-strand breaks are much more prone compared with ssb to combine with additional strand breaks and base damage, which contributes to break complexity. At least half of DNA breaks are hydroxyl radical mediated. CONCLUSIONS: The developed model makes predictions compatible with features of available experimental data. Break complexity has to be addressed in biophysical modelling when the relative effectiveness of radiations in DNA damage is studied. Obtained data strongly argue against the dominance of direct radiation action in DNA damage in the cellular environment predicted by some theoretical studies.