The absence of strand-specific repair for the DNA polymerase pol gene in Deinococcus radiodurans

Deinococcus radiodurans is characterized by its extreme resistance to ionizing radiation. Although Mn+2 was taken up by the cells at a high speed, the absorption of Mn+2 by the cells did not affect their survival rate when irradiated by UV light. However, the Mn-induced-cell-division (Mn-CD) cells do lose their resistance to UV radiation. This can be explained by the fact that the Mn-CD cells were less efficient to remove pyrimidine dimers on damaged DNA. We also found that there is no strand-specific repair of the pol gene in this organism.     

Peptidoglycan fine structure of the radiotolerant bacterium Deinococcus radiodurans Sark

Peptidoglycan from Deinococcus radiodurans was analyzed by high-performance liquid chromatography and mass spectrometry. The monomeric subunit was: N-acetylglucosamine-N-acetylmuramic acid-L-Ala-D-Glu-(gamma)-L-Orn-[(delta)Gly-Gly]-D-Ala-D-Ala. Cross-linkage was mediated by (Gly)2 bridges, and glycan strands were terminated in (1-->6)anhydro-muramic acid residues. Structural relations with the phylogenetically close Thermus thermophilus are discussed.     

Mechanism of thermotolerance induction by split-dose hyperthermia in Deinococcus radiodurans DNA repair deficient mutants

We examined the phenomenon of thermotolerance induction in the radioresistant prokaryote, Deinococcus radiodurans, which was initially exposed to 30 min at 52 degrees C followed by various intervals up to 6 h at 30 degrees C in TGY medium and then re-exposed to 52 degrees C for various periods, i.e., split-dose hyperthermia. This thermotolerance induction was analyzed in DNA repair deficient mutants (strain 302, 251, UVS25, rec30 and KH840) and the wild-type strain MR1. The strain UVS25 is a double mutant for the mtcA and uvsD genes, and strain rec30 is a mutant for the deinococcal recA gene. The induction was suppressed to 1/10 and 1/25 in strains UVS25 and rec30 respectively, as compared with the maximum level in the wild-type strain MR1. However, the induction in strain 302 (mutant for the uvrA gene) was not suppressed. Therefore, we conclude that proteins synthesized during the interexposure interval, i.e., the products of the uvsD (UV endonuclease beta) and recA (RecA protein) genes contribute to the induction of thermotolerance in D. radiodurans.     

Reversible induction of ATP synthesis by DNA damage and repair in Escherichia coli. In vivo NMR studies

Early metabolic events in Escherichia coli exposed to nalidixic acid, a topoisomerase II inhibitor and an inducer of the SOS system, were investigated by in vivo NMR spectroscopy, a technique that permits monitoring of bacteria under controlled physiological conditions. The energetics of AB1157 (wild type) and of its isogenic, SOS-defective mutants, recBC, lexA, and DeltarecA, were studied by 31P and 19F NMR before, during, and after exposure to nalidixic acid. The content of the NTP in E. coli embedded in agarose beads and perfused at 36 degreesC was found to be 4.3 +/- 1.1 x 10(-18) mol/cell, yielding a concentration of approximately 2.7 +/- 0.7 mM. Nalidixic acid induced in the wild type and mutants a rapid 2-fold increase in the content of the NTP, predominantly ATP. This induction did not involve synthesis of uracil derivatives or breakdown of RNA and caused cell proliferation to stop. Removal of nalidixic acid after 40 min of treatment rescued the cells and resulted in a decrease of ATP to control levels and resumption of proliferation. However, in DeltarecA cells, which were more sensitive to the activity of the drug, ATP elevation could not be reversed, and ATP content continued to increase faster than in control cells. The results ruled out association between the elevation of ATP and the induction of the SOS system and suggested involvement of a process reminiscent of apoptosis in the stimulation of ATP synthesis. Thus, the presence of the RecA protein was found to be essential for reversing the ATP increase and cell rescue, possibly by its function in repair of DNA damage.     

Novel repair action of vitamin C upon in vivo oxidative DNA damage

The Nef protein of HIV-1 binds to and induces apoptotic cytolysis of uninfected but activated human peripheral blood mononuclear cells (PBMC) and various cell line cells derived from CD4+ T, CD8+ T and B lymphocytes, macrophages, and neutrophils. The Nef-induced apoptosis also occurs with blood cells not expressing CD95 (Fas). The Nef-induced apoptosis as well as Fas-mediated apoptosis was inhibited by acetyl-Try-Val-Ala-Asp-CHO, an IL-1beta converting enzyme (ICE) inhibitor. On the other hand, serine/threonine protein kinase (PK) inhibitors, H-7, fasudil hydrochloride and M3, inhibited the Nef-induced apoptosis, and not the Fas-mediated one, without affecting the cell-binding activity of Nef and Nef-binding capacity of the activated cells. Preincubation of the cells with the drugs before being bound by Nef was required for the inhibition of apoptosis. These results suggest that the PK inhibitors specifically act on a cellular protein involved in the upper stream of signal transduction pathway of the Nef-induced apoptosis, which is different from the Fas-mediated pathway but meets it upstream of ICE. In addition, the drugs suppressed the cellular activation-associated cell surface expression of a putative Nef-binding protein in PBMC, although they had no influence on its expression in cell line cells. These findings suggest the feasibility of clinical use of the PK inhibitors to prevent the development of AIDS by inhibiting the Nef-induced apoptosis of uninfected blood cells.     

In vivo gene transfer and expression in rat stomach by submucosal injection of plasmid DNA

We used electromyographic analysis to determine the muscle activity of the shoulder muscles during the lift-off test and during resisted internal rotation. The activity in the upper and lower subscapularis muscle during a lift-off test from the region of the midlumbar spine was approximately 70% of maximal voluntary contraction. This level was significantly higher than for all the other muscles tested (P < 0.05). The lift-off test with the hand placed in the region of the midlumbar spine resulted in one-third more electromyographic activity in the subscapularis muscle than when the test was modified and performed with the hand at the buttocks region. A resisted lift-off test resulted in higher activities in all the muscles, but only a small increase in the pectoralis major muscle. The pectoralis major muscle was significantly more active during resisted internal rotation with the arm in front of the body. Comparison of activity in the upper subscapularis with that in the lower subscapularis muscle showed no significant differences during any of the tests. This study documents the importance of the subscapularis muscle during the lift-off test and suggests that other potential internal rotators of the humerus have a limited role in maintaining internal rotation when the arm is placed behind the back.     

Ultraviolet-induced DNA damage stimulates topoisomerase I-DNA complex formation in vivo: possible relationship with DNA repair

An antibody-based method was used to examine genomic DNA cleavage by endogenous topoisomerases in living cells. The method quantifies cleavable (covalent) complex formation in vivo after exposure to topoisomerase poisons, as reported previously (D. Subramanian et al., Cancer Res., 55: 2097-2103, 1995). Unexpectedly, exposing cells to UVB irradiation stimulated endogenous topoisomerase I-DNA covalent complex formation by as much as 8-fold, even in the absence of drugs that stabilize the cleavable complex. Covalent complexes are not a result of nonspecific UV protein-DNA cross-linking; rather, they result from the enzymatic activity of topoisomerase I on genomic DNA. Because the action of topoisomerase II on genomic DNA was not affected by UVB exposure, the observation appears to be specific for type I. Topoisomerase I is rapidly mobilized onto the genome (within 12 min after UVB exposure); however, topoisomerase I polypeptide levels did not show a corresponding increase, suggesting that preexisting enzyme is being recruited to sites of DNA damage. Complexes persist up to 5 h post-UV exposure (concurrent with the period of active DNA repair), and their formation is independent of S phase. These findings can be partially explained by the fact that in vitro topoisomerase I activity on UV-damaged DNA tends to favor formation of cleavage complexes; thus, a higher yield of covalent complexes are detected at or near cyclopyrimidine dimer lesions. Because repair-deficient cells are additionally compromised in their ability to recruit topoisomerase I, a direct role for the enzyme in DNA excision repair process in vivo is proposed that may be related to the activity of the xeroderma pigmentosum complementation group D helicase. Finally, these results collectively demonstrate that topoisomerase I is a repair-proficient topoisomerase in vivo.     

Targeted mutagenesis by duplication insertion in the radioresistant bacterium Deinococcus radiodurans: radiation sensitivities of catalase (katA) and superoxide dismutase (sodA) mutants

Deinococcus radiodurans R1 is extremely resistant to both oxidative stress and ionizing radiation. A simple and general targeted mutagenesis method was developed to generate catalase (katA) and superoxide dismutase (sodA) mutants. Both mutants were shown to be more sensitive to ionizing radiation than the wild type.     

The rate of recombination repair and its relationship to the radiation-induced delay in DNA synthesis in Micrococcus radiodurans

Flow microfluorometry has been used to characterize the effects of serum concentration and cell density on the initiation of cell cycle transit of stationary phase (G0) human diploid fibroblasts (strain WI-38). The concentration of serum used to stimulate these cultures had no effect on the time cells began appearing in S (the DNA synthetic period), nor on the synchrony with which they moved around the cell cycle. However, as the serum concentration increased, the fraction of the stationary phase population released from G0 increased. Cell density modulated the ability of serum to stimulate cell cycle traverse. For example, at a cell density of 1.81 X 10(4) cells/cm2, 78% of the population was sensitive to serum stimulation; whereas, when the density was increased to 7.25 X 10(4) cells/cm2, only 27% of the population could be stimulated. This effect of cell density on the serum response is not simply the result of changing the ratio of serum concentration to cell density, but appears to reflect a true modulation of the population's sensitivity to serum stimulation. These results are consistent with the interpretation that the primary action of serum is to determine the transition of cells from a non-cycling G0 state to a cycling state and that cell density determines the proportion of the population capable of undergoing this transition.     

Curing Saccharomyces cerevisiae of the 2 micron plasmid by targeted DNA damage

Powerful mutagenic screens of yeast Saccharomyces cerevisiae have recently been developed which require strains that lack the endogenous 2 micron plasmid (Burns et al., 1994). Here, we describe a simple and reliable method for curing yeast of the highly stable genetic element. The approach employs heterologous expression of a 'step-arrest' mutant of the Flp recombinase. The mutant, Flp H305L (Parsons et al., 1988), forms long-lived covalent protein-DNA complexes exclusively at 2 micron-borne recombinase target sites. In vivo, the complexes serve as sites of targeted DNA damage. Using Southern hybridization and a colony color assay for plasmid loss, we show that expression of the mutant enzyme results in the effective elimination of the 2 micron from cells.     

In vitro increases in plasmid DNA supercoiling by hydrostatic pressure

Cells of the yeast strain Debaryomyces vanrijiae SBUG 770, grown with glucose, converted biphenyl to 4-hydroxybiphenyl as the major metabolite. In addition, 2-hydroxybiphenyl was formed in minor amounts. No further degradation of these substances was detected. However, these monohydroxylated derivatives were oxidised by alkane-grown cells in the presence of the co-metabolic substrate, tetradecane. Under these conditions 2-hydroxybiphenyl was oxidised to 2,5-dihydroxybiphenyl, and 4-hydroxybiphenyl was rapidly metabolised by formation of two major metabolites. One was identified as 3,4-dihydroxybiphenyl. Characterisation of the second product as 4-phenylmuconolactone points to a further metabolism of the initially formed dihydroxylated biphenyl via ortho-ring fission.     

Mitochondrial targeting of human DNA glycosylases for repair of oxidative DNA damage

Oxidative damage to mitochondrial DNA has been implicated in human degenerative diseases and aging. Although removal of oxidative lesions from mitochondrial DNA occurs, the responsible DNA repair enzymes are poorly understood. By expressing the epitope-tagged proteins in COS-7 cells, we examined subcellular localizations of gene products of human DNA glycosylases: hOGG1, hMYH and hNTH1. A gene encoding for hOGG1 which excises 7,8-dihydro-8-oxoguanine (8-oxoG) from DNA generates four isoforms by alternative splicing (types 1a, 1b, 1c and 2). Three tagged isoforms (types 1b, 1c and 2) were localized in the mitochondria. Type 1a protein, which exclusively contains a putative nuclear localization signal, was sorted to the nucleus and lesser amount to the mitochondria. hMYH, a human homolog gene product of Escherichia coli mutY was mainly transported into the mitochondria. hNTH1 protein excising several pyrimidine lesions was transported into both the nucleus and mitochondria. In contrast to the three DNA glycosylases, translocation of the human major AP endonuclease (hAPE) into the mitochondria was hardly observed in COS-7 cells. These results suggest that the previously observed removal of oxidative base lesions in mitochondrial DNA is initiated by the above DNA glycosylases.     

Tests for DNA and chromosomal damage induced by nuclear magnetic resonance imaging

Chinese hamster ovary (CHO) cells in culture were exposed in a nuclear magnetic resonance (NMR) imaging apparatus to a strong magnetic field, pulsed field gradients, and radio frequency emissions. No chromosomal aberrations were induced even after an exposure of approximately 14 hours. No sister chromatid exchanges were induced by four-hour exposures to either low (average 7.2 mW) or high (average 61.2 mW) radio frequency power. When HeLa cells were exposed for 16 hours to an average radio frequency power of 61.2 mW, no inhibition of DNA synthesis was detectable. These data indicate that the conditions used for NMR imaging do not cause genetic damage which is detectable by any of these methods.     

Defective repair of oxidative damage in mitochondrial DNA in Down''s syndrome

BACKGROUND: Synthetic homopyrimidine peptide nucleic acids (PNAs) can bind complementary targets in double-stranded DNA, generating strand-displacement complexes, and so offering an opportunity to modulate specific gene expression. Several issues remain to be addressed before these attributes can be exploited in vivo, however. RESULTS: The kinetics of the interaction between a homopyrimidine PNA and a complementary homopurine target on double-stranded DNA were analyzed in the presence or absence of a preformed strand-displacement complex proximal to the target. The complex was established under low salt conditions by the binding of a different homopyrimidine PNA to a target situated adjacent to the first PNA target. These two targets were placed next to each other on opposite strands at distances of 0, 2, 4 and 8 base pairs apart. The presence of a preformed strand-displacement complex near the target accelerates the binding of PNA to double-stranded DNA in a salt-dependent manner. The influence of salt on the binding rates was also examined. The binding rate is increased by a factor of 1 x exp(70[NaCl]), that is, 16-fold at 40 mM NaCl and more than 10(4)-fold if extrapolated to 140 mM NaCl. This effect is significantly reduced if the two targets are 2 base pairs apart and completely absent if the distance is 4 base pairs or more. CONCLUSIONS: The perturbation of the DNA helix imposed by a PNA strand-displacement complex only propagates a few base pairs. It is therefore possible to target sites in the immediate vicinity of strand invasion complexes specifically. The results presented have implications for the mechanism of strand displacement and for the application of PNA in a genomic context.     

In vivo chromosomal aberrations in bone marrow cells of rats treated with Marshal

In this study, the cytogenetic effects of Marshal (insecticide/nematocide) were investigated in bone marrow cells of rats. The results obtained from animals treated with Marshal were compared with the results of animals treated with ethyl carbamate (EC) and with controls. Concentrations of 12.5, 25 and 50 mg/kg b.wt. of Marshal and 100, 200 and 400 mg/kg b.wt. of EC were used and animals were sampled at three different times (6, 12 and 24 h). Marshal increased the number of chromosomal aberrations (CA) per cell, and the number of cells with abnormalities, at all concentrations and treatment times. Generally, Marshal could increase the number of the abnormal cells and the formation of CA as easily as EC. However, Marshal, at 50 mg/kg b.wt. did not increase the frequency of abnormal cells or CA as strongly as EC, at 400 mg/kg b.wt. for 6 h. Marshal also decreased the mitotic index (MI) compared with the control group. The MI was higher in the group treated with Marshal for 6 h than that treated with EC. However, the effects of Marshal and EC on the MI in the groups treated for 12 and 24 h were similar. We found that the effect of Marshal on the formation of abnormal cells and CA was dependent on concentration and treatment time.     

Deficient repair of UV-induced DNA damage in immature human T cells

As compared with quiescent lymphocytes, human thymocytes were found in the nucleoid sedimentation assay to be deficient in UV-induced DNA repair. Therefore, the age-related repair deficiency of human lymphocytes can result from the increased proportion of immature T cells occurring with age.     

pR plasmid replication provides evidence that single-stranded DNA induces the SOS system in vivo

The aim of this study was to quantitate factors affecting the initial "peak" of the pulmonary artery (PA) drug concentrations after i.v. bolus drug administration, which is a determinant of the subsequent drug uptake into both the lungs and other well-perfused organs. Indocyanine green (ICG) was used as a marker drug in anaesthetized (1.5% halothane) sheep prepared with an inferior vena cava injection catheter and a large-gauge pulmonary artery blood sampling catheter. For three ranges of cardiac output, 2.5-mg doses of ICG were injected in the following combinations: 10 ml injected over 1, 5 or 10 s; 5 or 25 ml injected over 1 s. On-line PA ICG concentrations were recorded for approximately 60 s using a densitometer. The mean maximum PA ICG concentrations (2-8 mg litre-1), the mean times at which they occurred (7-18 s after injection) and the time lags before ICG was detected in the PA (4-9 s), were inversely related to cardiac output, but linearly related to the time over which the injection was made. The area under the curve of the peak was related inversely to cardiac output only, while the aspect ratio of the peak was related inversely to the time over which the injection was made only. The injectate volume had no effect on any of the measured values. We conclude that, in some circumstances, the rate of injection of drugs with narrow margins of safety should be tailored to the cardiac output of an individual.     

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.