The sequence specificity of bleomycin-induced DNA damage in intact cells

Bleomycin causes lesions to DNA in intact cells and in purified DNA under appropriate conditions. Using a middle repetitive DNA sequence called alpha-DNA as a target sequence, we have compared the sequence specificity of bleomycin-induced DNA cleavage in intact human cells and in purified human DNA. Bleomycin induces numerous cleavage sites in alpha-DNA which vary widely in intensity and give rise to a complex pattern of bands on a DNA sequencing gel. Unexpectedly, the intensity and position of bleomycin-induced DNA cleavage sites are very similar in intact human cells and in purified human DNA.     

Effect of DNA repair inhibitors on the induction and repair of bleomycin-induced chromosome damage.

The current status of the L5178Y/TK^+/-→TK^-/- mouse-lymphona mutagenicity assay is described. Dose-survival-mutagenic response data are shown for 43 chemicals. Mutagenicity and cytotoxicity in the presence or absence of non-induced and/or Aroclor-induced rat-liver S-9 are compared for most of these chemicals. 25 of these for which usable carcinogenicity data exist have been used to construct an approximately linear relationship between oncogenic potency in vivo and mutagenic potency in this system in vitro; linearity between these two endpoints extends over a greater than 100 000-fold range in potencies. Several carcinogens which are negative or difficult to detect in the standard Ames assay are mutagenic in this mammalian cell system. These include natulan, sodium saccharin (lot S-1022), p,p′-DDE (a metabolite of DDT), dimethylnitrosamine, diethylnitrosamine and diethylstilbestrol.     

Telomere-related functions of yeast KU in the repair of bleomycin-induced DNA damage

Bleomycins are small glycopeptide cancer chemotherapeutics that give rise to 3'-modified DNA double-strand breaks (DSBs). In Saccharomyces cerevisiae, DSBs are predominantly repaired by RAD52-dependent homologous recombination (HR) with some support by Yku70/Yku80 (KU)-dependent pathways. The main DSB repair function of KU is believed to be as part of the non-homologous end-joining (NHEJ) pathway, but KU also functions in a "chromosome healing" pathway that seals DSBs by de novo telomere addition. We report here that rad52Deltayku70Delta double mutants are considerably more bleomycin hypersensitive than rad52Deltalig4Delta cells that lack the NHEJ-specific DNA ligase 4. Moreover, the telomere-specific KU mutation yku80-135i also dramatically increases rad52Delta bleomycin hypersensitivity, almost to the level of rad52Deltayku80Delta. The results indicate that telomere-specific functions of KU play a more prominent role in the repair of bleomycin-induced damage than its NHEJ functions, which could have important clinical implications for bleomycin-based combination chemotherapies.     

The repair of bleomycin-induced DNA damage and its relationship to chromosome aberration repair.

Previous studies using the technique of premature chromosome condensation indicated that nearly one-half of the bleomycin-induced chromatid breaks and gaps in CHO cells could be repaired within 1 h (repair starting at 30 min) after treatment. Cycloheximide and streptovitacin A (but not hydroxyurea or hycanthone) inhibited chromosome repair. The purpose of this study was to measure the kinetics of DNA repair after bleomycin treatment using the alkaline elution technique and to determine whether various inhibitors could block this repair. After bleomycin treatment, the major proportion of the repair of DNA damage occurred within 15 min, with significant repair evident by 2 min. This fast repair component was inhibited by 0.2% EDTA. A slower repair component was observed to occur up to 60 min after bleomycin treatment. None of the inhibitors tested were found to have a significant effect on the repair of bleomycin damage at the DNA level. Since chromosome breaks were observed not to begin repair until after 30 min while over 50% of the DNA was repaired by 15 min, these results suggest that the DNA lesions that are repaired quickly are not important in the formation of chromosome aberrations. Further, since cycloheximide and streptovitacin A blocked chromosome repair but had little measurable effect on DNA repair, these results suggest that the DNA lesions responsible for chromosome damage represent only a small proportion of the total DNA lesions produced by bleomycin.     

Influence of solute, pH, and incubation temperature on recovery of heat-stressed Wallemia sebi conidia

The influences of glucose, sorbitol, and NaCl in a basal enumeration medium at water activities (aw) from 0.82 to 0.97 on colony formation by sublethally heat-stressed Wallemia sebi conidia were determined. Over this aw range, glucose and sorbitol had similar effects on recovery, whereas at an aw of 0.82 to 0.92, NaCl had a detrimental effect. Colony diameters were generally largest on media containing sorbitol and smallest on media containing NaCl. Maximum colony size and viable population of heat-stressed conidia were observed on media at an aw of ca. 0.92. When the recovery incubation temperature was 20 degrees C, the number of uninjured conidia detected at an aw of 0.82 was reduced compared with the number detected at 25 degrees C, while at 30 degrees C, the number recovered at an aw of 0.97 was reduced. The effect on heat-stressed conidia was magnified. This suggests that W. sebi conidia may be more tolerant of aw values higher than the optimum 0.92 when the incubation temperature is decreased from the near optimum of 25 degrees C and less tolerant of aw values greater than 0.92 when the incubation temperature is higher than 25 degrees C. The sensitivity of heat-stressed conidia increased as the pH of the recovery medium was decreased from 6.55 to 3.71. W. sebi conidia dispersed in wheat flour at aw values of 0.43 and 0.71 and stored for up to 65 days at both 1 and 25 degrees C neither lost viability nor underwent sublethal desiccation or temperature injury.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vibrio anguillarum: Influence of temperature, pH, NaCl concentration and incubation time on growth

An analysis of the effect of growth conditions on the growth (O.D. values) of five Vibrio anguillarum strains showed that the optima were as follows: pH 7, temperature 25°C, NaCl concentration 2%, and O.D. estimates increased with the incubation time. The independent parameters, as well as their interactions significantly influenced the growth of Vibrio anguillarum ( P 0<0001). Only the strain-salinity interaction was not always statistically significant. A restriction of the parameters to a level relevant for Danish marine recipients showed that pH and NaCl concentration (salinity) might be of minor importance while the temperature was always of high significance. The possible impact of these observations on local conditions is discussed.     

Measuring the bleomycin-induced paired damage of two DNA chains by a method of psoralen photocross-linking

DNA of bacteriophage PM2 was allowed to react with bleomycin in the presence of Fe(II) and oxygen and the "paired" DNA lesions of two types were measured: (1) double-strand breaks, (2) lesions converted to double-strand breaks after introducing into the DNA a large number of psoralen cross-links (about 10(-2) per base pair) and alkali treatment. The mean numbers of each lesion type per DNA molecule are found to be proportional to the square of bleomycin concentration over the range of 3 X 10(-7) to 3 X 10(-6) M. These findings indicate that paired lesions are formed as a result of action of two bleomycin molecules at the same DNA site.     

Influence of solute, pH, and incubation temperature on recovery of heat-stressed Wallemia sebi conidia.

The influences of glucose, sorbitol, and NaCl in a basal enumeration medium at water activities (aw) from 0.82 to 0.97 on colony formation by sublethally heat-stressed Wallemia sebi conidia were determined. Over this aw range, glucose and sorbitol had similar effects on recovery, whereas at an aw of 0.82 to 0.92, NaCl had a detrimental effect. Colony diameters were generally largest on media containing sorbitol and smallest on media containing NaCl. Maximum colony size and viable population of heat-stressed conidia were observed on media at an aw of ca. 0.92. When the recovery incubation temperature was 20 degrees C, the number of uninjured conidia detected at an aw of 0.82 was reduced compared with the number detected at 25 degrees C, while at 30 degrees C, the number recovered at an aw of 0.97 was reduced. The effect on heat-stressed conidia was magnified. This suggests that W. sebi conidia may be more tolerant of aw values higher than the optimum 0.92 when the incubation temperature is decreased from the near optimum of 25 degrees C and less tolerant of aw values greater than 0.92 when the incubation temperature is higher than 25 degrees C. The sensitivity of heat-stressed conidia increased as the pH of the recovery medium was decreased from 6.55 to 3.71. W. sebi conidia dispersed in wheat flour at aw values of 0.43 and 0.71 and stored for up to 65 days at both 1 and 25 degrees C neither lost viability nor underwent sublethal desiccation or temperature injury.(ABSTRACT TRUNCATED AT 250 WORDS)     

Repair of bleomycin-induced damage to Escherichia coli DNA. II. Participation of inducible processes

The role of inducible cell functions in repair and mutagenesis after bleomycin-induced DNA damages was studied in Escherichia coli. Influence on these processes of some rec genes as well as sbcB, the structural gene for exonuclease I, was investigated. The data obtained suggest that this enzyme plays a negative role in the repair of DNA damaged by bleomycin. The hypersensitivity of recA mutant to bleomycin and recAlexA-dependence of bleomycin-induced mutagenesis do not suggest any principal differences between UV-induced pyrimidine dimers and apyrimidinic sites in the case of post-replication repair.     

Influence of DNA length on spermine-induced condensation. Importance of the bending and stiffening of DNA

Using DNA restriction fragments of 258 to 4362 base-pairs, we have investigated the influence of the DNA length on the condensation process induced by spermine, with the aid of electric dichroism measurements. The 258- and 436 bp fragments condensed into rod-like particles, while the fragments of 748 bp or more condensed into torus-shaped particles. Our results suggest that a DNA molecule longer than the circumference of the toroids observed previously (680 bp) is required to serve as a nucleus for the growth of the condensed particles. The toroids were more stable in the electric field than the rod-shaped particles, suggesting that rapid fluctuations of the bound spermine counterions can provide one of the main attractive forces yielding to the condensation process. Relaxation time data for the 436 bp fragment revealed that the structure of DNA was altered at a spermine concentration as low as one-tenth of that required for condensation: the DNA became bent in the presence of spermine. Moreover, the field strength dependence of the relaxation times, as well as the fitting of the decay curves at 12.5 kV/cm, showed an increase of the stiffness of the DNA double helix upon spermine addition. We estimated that, in the case of DNA condensation by spermine, a decrease in the measured persistence length may occur, irrespective of the DNA flexibility, owing to the bending of the DNA molecule.     

Influence of DNA length on spermine-induced condensation: Importance of the bending and stiffening of DNA

Using DNA restriction fragments of 258 to 4362 base-pairs, we have investigated the influence of the DNA length on the condensation process induced by spermine, with the aid of electric dichroism measurements. The 258- and 436 bp fragments condensed into rod-like particles, while the fragments of 748 bp or more condensed into torus-shaped particles. Our results suggest that a DNA molecule longer than the circumference of the toroids observed previously (680 bp) is required to serve as a nucleus for the growth of the condensed particles. The toroids were more stable in the electric field than the rod-shaped particles, suggesting that rapid fluctuations of the bound spermine counterions can provide one of the main attractive forces yielding to the condensation process. Relaxation time data for the 436 bp fragment revealed that the structure of DNA was altered at a spermine concentration as low as one-tenth of that required for condensation: the DNA became bent in the presence of spermine. Moreover, the field strength dependence of the relaxation times, as well as the fitting of the decay curves at 12.5 kV/cm, showed an increase of the stiffness of the DNA double helix upon spermine addition. We estimated that, in the case of DNA condensation by spermine, a decrease in the measured persistence length may occur, irrespective of the DNA flexibility, owing to the bending of the DNA molecule.     

Effect of recombinant interferon-alpha on bleomycin-induced chromosome damage in hamster cells

The effect of recombinant interferon-alpha-2a (rIFN-alpha-2a) on the induction of chromosomal aberrations (CAs) by the radiomimetic antibiotic bleomycin (BLM, 5 microg/ml, 30 min, 37 degrees C) in Chinese hamster ovary (CHO) cells was investigated. Recombinant IFN-alpha-2a (4500-180,000IU/ml) was added to the cell cultures 0.5 or 24h before BLM (and left in the culture medium until the end of treatments) or immediately after BLM treatment (and left in the culture medium until harvesting). Cells were sampled at 18 or 2.5h after the end of treatments, in order to determine, respectively, the effect of rIFN-alpha-2a on the total chromosome damage induced by BLM and on the chromosome damage induced by this antibiotic in the G(2) phase of the cell cycle. A statistically significant increase in the frequency of CAs was observed following treatment with BLM (P<0.05), whereas treatments with rIFN-alpha-2a alone did not produce any significant increase of CAs over control values (P>0.05). The yield of CAs by BLM was significantly inhibited by rIFN-alpha-2a (P<0.05, 65.3% maximum inhibition). A strong inhibitory effect (around 80%) of rIFN-alpha-2a on the yield of BLM-induced CAs in the G(2) phase of the cell cycle was also observed. It is suggested that the inhibitory effect of rIFN-alpha-2a on the induction of CAs by BLM is mainly due to the stimulation of DNA synthesis and repair by the cytokine.     

Influence of pH on the conformation and stability of mismatch base-pairs in DNA

A series of self-complementary dodecanucleotide duplexes containing two symmetrically disposed mismatches have been studied by pH-dependent, ultraviolet light melting techniques. The results indicate that A.C, and C.C mismatches are strongly stabilized by protonation and that the degree of stabilization of the A.C mismatch depends greatly on the flanking bases. In one case, a duplex containing two A.C mismatches is more stable than the native sequence below pH 5.5. The G.A mismatch displays conformational flexibility, with a protonated G(syn).A(anti) base-pair occurring in certain base stacking environments but not in others. The A.A and T.C mismatches are not stabilized at low pH. These solution studies correlate well with predictions based on X-ray crystallographic data.     

Roles of the DNA binding proteins H-NS and StpA in homologous recombination and repair of bleomycin-induced damage in Escherichia coli

The DNA binding protein H-NS promotes homologous recombination in Escherichia coli, but the role of its paralog StpA in this process remains unclear. Here we show that an hns mutant, but not an stpA mutant, are marginally defective in conjugational recombination and is sensitive to the double-strand-break-inducing agent bleomycin. Interestingly, the hns stpA double mutant is severely defective in homologous recombination and more bleomycin-sensitive than is the hns or stpA single mutant, indicating that the stpA mutation synergistically enhances the defects of homologous recombination and the increased bleomycin-sensitivity in the hns mutant. In addition, the transduction analysis in the hns stpA double mutant indicated that the stpA mutation also enhances the defect of recombination in the hns mutant. These results suggest that H-NS plays an important role in both homologous recombination and repair of bleomycin-induced damage, while StpA can substitute the H-NS function. The recombination analysis of hns single, stpA single, and hns stpA double mutants in the recBC sbcA and recBC sbcBC backgrounds suggested that the reduction of the hns single or hns stpA double mutants may not be due to the defect in a particular recombination pathway, but may be due to the defect in a common process of the pathways. The model for the functions of H-NS and StpA in homologous recombination and double-strand break repair is discussed.     

Influence of thiol structure on neocarzinostatin activation and expression of DNA damage.

Neocarzinostatin (NCS) is an enediyne antitumor antibiotic that cleaves DNA following a thiol-induced electronic rearrangement to a diradical form. Structure-function studies with 11 thiol-containing compounds were undertaken to clarify the role of the thiol in NCS-mediated DNA damage. The rates of activation of NCS in the presence of DNA with the various thiols approximated a Br?nsted relation (beta = 0.43, r2 = 0.86), which suggests that the basicity/nucleophilicity of the thiol is important to NCS activation. However, an additional contribution to NCS activation may arise from the affinity of the thiol for DNA, since there is a correlation between the concentration of thiol producing maximal DNA damage, assessed by quantitating the topologic forms of plasmid pBR322 following treatment with NCS, and the apparent ability of the thiol to bind to DNA by hydrophobic or electrostatic interactions. The overall second-order rate constants for the activation of NCS were found to be inversely correlated with the thiol optima; a plot of the former versus the reciprocal of the optimal thiol concentration revealed a first-order rate constant of activation of 0.013 s-1 in the presence of DNA. This indicates that maximal DNA damage occurs when NCS is activated with a half-life of 52 s, a relatively slow rate of activation that suggests that NCS binds to DNA before undergoing activation by thiol. Finally, an analysis of strand breaks in pBR322 shows that thiols possessing a carboxylate moiety produce larger quantities of bistranded DNA lesions than their esterified or non-carboxylate-containing counterparts.     

Postnatal effects of incubation length in mallard and pheasant chicks

Eggs of mallard ducks ( Anas platyrhynchos ) and ring-necked pheasants ( Phasianus colchicus ) were incubated in clutches arranged to stimulate embryos to hatch earlier or later than normal. This manipulation of hatching time was achieved by combining eggs of different age in the same clutch. To ensure hatching synchrony, embryos communicate with each other during the last stage of incubation, resulting in either a delay or an acceleration of hatching. Embryos of both species that accelerated their hatching time suffered a higher mortality rate after hatching. Combining mortality with the proportion of hatchlings that suffered from leg deformities, impeding their movements, resulted in a cost also to pheasant chicks delaying their hatching. Chicks of both species accelerating hatching time had a lower minimum mass and a shorter tarsus length than control chicks, whereas chicks delaying hatching time either grew as well or slightly better than control chicks. Mallard chicks had better balance and mobility immediately after hatching the longer they stayed in the egg. This indicates that the period immediately before hatching, is an important period for muscular and organ maturity. Reducing this period results in costs affecting post-hatching survival. The strategy to assure synchronous hatching in mallards and pheasants probably reflect a trade-off between the negative effects of shifting the age at hatching away from normal and differences in predation risk during different stages of reproduction.     

Influence of sequence context and length on the structure and stability of triplet repeat DNA oligomers

Genetic expansion diseases have been linked to the properties of triplet repeat DNA sequences during replication. The most common triplet repeats associated with such diseases are CAG, CCG, CGG, and CTG. It has been suggested that gene expansion occurs as a result of hairpin formation of long stretches of these sequences on the leading daughter strand synthesized during DNA replication [Gellibolian, R., Bacolla, A., and Wells, R. D. (1997) J. Biol. Chem. 272, 16793-7]. To test the biophysical basis for this model, oligonucleotides of general sequence (CNG)(n), where N = A, C, G, or T and n = 4, 5, 10, 15, or 25, were synthesized and characterized by circular dichroism (CD) spectropolarimetry, optical melting studies, and differential scanning calorimetry (DSC). The goal of these studies was to evaluate the influence of sequence context and oligomer length on their secondary structures and stabilities. The results indicate that all single oligomers, even those as short as 12 nucleotides, form stable hairpin structures at 25 degrees C. Such hairpins are characterized by the presence of N:N mismatched base pairs sandwiched between G:C base pairs in the stems and loops of three to four unpaired bases. Thermodynamic analysis of these structures reveals that their stabilities are influenced by both the sequence of the particular oligomer and its length. Specifically, the stability order of CGG > CTG > CAG > CCG was observed. In addition, longer oligomers were found to be more stable than shorter oligomers of the same sequence. However, a stability plateau above 45 nucleotides suggests that the length dependence reaches a maximum value where the stability of the G:C base pairs can no longer compensate the instability of the N:N mismatches in the stems of the hairpins. The results are discussed in terms of the above model proposed for gene expansion.