X-ray-induced base sequence damage in primate alphoid DNA

Radiation-induced single-strand breaks were found throughout the 172 bp repeat units of African green monkey component alpha DNA. Two kinds of 3'-ends of 5'-32P-labeled restriction fragments were found, as previously described by others. After irradiation in vitro, the yield of single-strand breaks was 4 X 10(-5) breaks/nucleotide/Gy, as determined by analyses in DNA sequencing type gels. Protection from X-ray damage was found when the DNA received 150 Gy in the presence of 2-mercaptoethanol. The results demonstrate a very sensitive quantitative means to study the role of indirect effects of ionizing radiation on strand-break induction and protection at the base sequence level. Component alpha DNA was isolated from irradiated CV-1 cells and was analyzed for single-strand breaks. Under these conditions the frequency of breaks was less than the frequency obtained when purified DNA was irradiated. The methodology is presented because of its relevance to the study of DNA strand breakage in living cells.     

Local dynamics of DNA probed with optical absorption spectroscopy of bound ethidium bromide.

We have studied the local dynamics of calf thymus double-helical DNA by means of an "optical labeling" technique. The study has been performed by measuring the visible absorption band of the cationic dye ethidium bromide, both free in solution and bound to DNA, in the temperature interval 360-30 K and in two different solvent conditions. The temperature dependence of the absorption line shape has been analyzed within the framework of the vibronic coupling theory, to extract information on the dynamic properties of the system; comparison of the thermal behavior of the absorption band of free and DNA-bound ethidium bromide gave information on the local dynamics of the double helix in the proximity of the chromophore. For the dye free in solution, large spectral heterogeneity and coupling to a "bath" of low-frequency (soft) modes is observed; moreover, anharmonic motions become evident at suitably high temperatures. The average frequency of the soft modes and the amplitude of anharmonic motions depend upon solvent composition. For the DNA-bound dye, at low temperatures, heterogeneity is decreased, the average frequency of the soft modes is increased, and anharmonic motions are hindered. However, a new dynamic regime characterized by a large increase in anharmonic motions is observed at temperatures higher than approximately 280 K. The DNA double helix therefore appears to provide, at low temperatures, a rather rigid environment for the bound chromophore, in which conformational heterogeneity is reduced and low-frequency motions (both harmonic vibrations and anharmonic contributions) are hindered. The system becomes anharmonic at approximately 180 K; however, above approximately 280 K, anharmonicity starts to increase much more rapidly than for the dye free in solution; this can be attributed to the onset of wobbling of the dye in its intercalation site, which is likely connected with the onset of (functionally relevant) DNA motions, involving local opening/unwinding of the double helix. As shown by parallel measurements of the melting curves, these motions precede the melting of the double helix and depend upon solvent composition much more than does the melting itself.     

Study of different types of chlorophyll a aggregates in solutions and films by absorption and luminescence derivative spectroscopy]

The second derivative of absorption, fluorescence and fluorescence excitation spectra of chlorophyll a in concentrated solutions and films was investigated. More than 14 forms of pigment aggregates, which can be divided into two types--with narrow 8-10nm) and wide (25-40nm) low temperature (-196 degrees C) spectra bands, were found. For the most part of the aggregated forms, the position and half width of the bands, as well as the Stokes shift and relative quantum yield were determined. The comparison of the spectral characteristics points to the indentity of the aggregates and corresponding native forms of Chl. a. It is shown that the universal relationship between absorption and fluorescence bands in applicable to the aggregates of the two types and the energy of resonance interaction between monomers in the aggregates is evaluated.     

Reduced repair of X-ray-induced DNA lesions in cells without functioning DNA polymerase alpha

X irradiation of cells induces damage in the DNA, which can be detected as fragmentation of the DNA in alkali. To examine whether DNA polymerase alpha plays a role in the X-ray-induced fragmentation of the DNA, cells with and without functioning DNA polymerase alpha have been compared. We have used the drug aphidicolin, which is a specific inhibitor of polymerase alpha. The results show that DNA of aphidicolin-treated cells is more easily fragmented in alkali than DNA of untreated cells. This is paralleled by a lower repair replication in cells without functioning DNA polymerase alpha. Hence polymerase alpha is involved in the repair process of lesions induced by X irradiation.     

Repair of X-ray-induced DNA damage in aging human diploid cells

Human diploid cells cultured in vitro provide an excellent model system for the study of aging. In this study, we examined the formation and rejoining of DNA single-strand breaks (SSBs) induced by X-rays in human lung diploid fibroblasts during senescence, by using a modified alkaline elution method. For detecting the formation and rejoining of DNA SSBs, conventional [¹⁴C]thymidine (TdR)-labeling and fluorometric methods were applied to dividing cells and to the whole cell population including non-dividing and slowly-dividing cells, respectively. We did not find any significant differences in the rejoining ability of X-ray-induced SSBs in human diploid cells at almost all population doubling levels, although only in terminally senescent cells the rejoining of SSBs seems to proceed more slowly. However, it was observed that the alkaline elution of DNA from unirradiated and X-irradiated cells seems to become faster with increasing in population doubling number, although there were no remarkable differences in the elution rates of DNA as measured by the [¹⁴C]TdR-labeling method and those measured by the fluorometric method. These results seem to suggest that the molecular size of DNA in human diploid cells in culture decreases with aging.     

Probing DNA superstructure in human quiescent lynphocytes by X-ray-induced double-strand breakage

Summary Human quiescent lymphocytes were lysed onto neutral sucrose gradients in order to sediment subsequently the nuclear DNA released within nucleoids. The position of nucleoids in the centrifuge tubes was detected fluorometrically by using the dye, ethidium bromide, and the height of the fluorescent peak was taken as a measure of DNA content. X-irradiation of lymphocytes, before their lysis, altered the DNA content of nucleoids and their sedimantation rate in accord with the view that(1) nuclear DNA is attached along its length at distance corresponding to 1.7 × 10¹⁰ g/mol, amd that(2) X-ray-induces double-strand breakage releases DNA fragments at random. Incubation at 37° C of irradiated lymphocytes restored the amount of attached DNA as it would be expected from an intracellular repair process for DNA double-strand breaks.     

Scanning force microscopy studies of X-ray-induced double-strand breaks in plasmid DNA

We present an analysis of X-ray-induced damage in ΦX174 plasmid DNA, applying doses between D = 250 and 1,500 Gy. To analyse this damage in detail, the distribution of plasmid fragments after irradiation have been determined by scanning force microscopy. The results show that even for the lowest dose of D = 250 Gy, a significant amount of double-strand breaks are observed. For increasing dose, the percentage of small fragments increases and is accompanied by a shortening of the average fragment length from < L> = 1,400 nm for a dose of D = 250 Gy to < L> = 1,080 nm after irradiation with D = 1,500 Gy. The most crucial parameter, the average number of double-strand breaks per broken plasmid (<DSB_b> ) has been determined for the first time for the applied doses. The results show that the average number of DSBs per broken plasmid <DSB_b> increases almost linearly from a value of <DSB_b> = 1.3 after irradiation with D = 250 Gy to <DSB_b> = 1.7 after exposure to D = 1,500 Gy. The presented results show that the amount of DSBs induced by X-ray radiation in plasmid DNA can be calculated with high accuracy by means of scanning force microscopy, providing relevant information regarding the interaction of X-rays with DNA molecules.

Rapid rejoining of X-ray-induced DNA single-strand breaks in tuberous sclerosis fibroblasts

The formation and rejoining rates of X-ray-induced DNA single-strand breaks (SSBs) were examined in radiosensitive and non-radiosensitive fibroblast lines from patients with tuberous sclerosis (TS), and fibroblasts from normal individuals, using the sensitive and quantitative alkaline elution method. No difference was found between these cell lines in the frequency of DNA SSBs directly produced by X-irradiation at any dose up to 750 rad. Kinetic analysis of the rate of rejoining of DNA SSBs after X-irradiation at 500 rad indicated that the rate of rejoining involved at least two components, an initial fast component and a slower component. TS fibroblast lines, either radiosensitive or nonsensitive, were proficient as to DNA SSB repair, but they showed an increased rate of rejoining in the initial fast repair process, when compared to normal fibroblast lines. Although the molecular basis for the accelerated rejoining of DNA SSBs remains unknown, it is possible that the abnormality may be related to a basic defect in TS.     

Bufalin influences the repair of X-ray-induced DNA breaks in Chinese hamster cells

The bufadienolide bufalin, a component of the Chinese medicine chan'su, has been reported to selectively inhibit the growth of various lines of human cancer cells, due at least in part to its specific effect on topoisomerase (topo) II. We have treated Chinese hamster ovary (CHO) cells with doses of bufalin that result in a dramatic reduction in both the level and catalytic activity of topo II without any concomitant induction of DNA damage, as assessed by the comet assay. When cells were pre-treated with bufalin and then irradiated with X-rays, a follow-up study revealed that the kinetics of DNA repair was clearly affected, with a general delay in the restoration of DNA to the situation observed in non-irradiated controls. The possible involvement of topo II in radiation damage repair is discussed.     

G2 phase repair of X-ray-induced chromosomal DNA damage in trichothiodystrophy cells

The repair of X-ray-induced DNA damage during G₂ cell-cycle phase has been examined in lines of skin fibroblasts from three patients with trichothiodystrophy (TTD), one with apparently normal and two with defective nucleotide excision repair (NER). These responses are compared with those of five lines from clinically normal controls, lines from xeroderma pigmentosum (XP), Cockayne syndrome (CS), Down syndrome (DS), and ataxia telangiectasia (AT) patients. Chromosomal DNA repair was measured as the chromatid aberration frequency (CAF) or total number of chromatid breaks and long gaps per 100 metaphase cells, determined 0.5–1.5 h after X-irradiation (53 rad). Chromatid breaks and gaps (as defined herein) represent unrepaired DNA strand breaks. Only one of the TTD lines, TTD 1BR, showed an abnormally high CAF. This line was shown subsequently to be of a different complementation group, representing a new nucleotide excision repair gene. An abnormally high CAF was also observed, as reported previously, in XP-C, AT and DS but not in CS skin fibroblasts. In addition, cell lines were examined for DNA incision activity by an indirect method in which chromatid aberrations were enumerated with or without ara-C, an inhibitor of repair synthesis, added after X-irradiation. All TTD lines had abnormally low incision activity.     

Age-dependent decline in rejoining of X-ray-induced DNA double-strand breaks in normal human lymphocytes

Unstimulated human peripheral blood lymphocytes (HPBL), separated by density centrifugation from anticoagulated whole blood, were X-irradiated (30 Gy) on ice and incubated in medium at 37 degrees C for repair times of 15, 30, and 120 min. Blood donors were 18 normotensive, non-smoking Caucasians aged 23-78, free from overt pathology and not taking any medications. Neutral filter elution was used to assay DNA double-strand break (DSB) induction and completeness of DSB rejoining (plus rejoining of any X-ray-induced alkali-labile sites converted to DSBs in vitro at pH 9.6). After 30 or 120 min repair incubation, the percentage of DSBs rejoined by cells from older donors (aged 66-78 years) was less than half the percentage of DSBs rejoined by cells from younger donors (aged 23-39 and 42-57). When data from the 3 age groups were pooled, the age-related decline in percent DSBs rejoined was significant for repair times 30 min (r = -0.63, p less than 0.005) and 120 min (r = -0.64, p less than 0.005) but not for 15 min (r = -0.04). These age-related declines were observed even though DNA from older donors sustained fewer strand breaks as demonstrated by the negative correlation between donor age and DSB induction (r = -0.65, p less than 0.005). These results suggest that the efficacy of X-ray-induced DSB repair diminishes with in vivo age in unstimulated HPBL.     

An optical method for investigating the microwave absorption characteristics of DNA and other biomolecules in solution

Direct determination of the microwave absorption characteristics of biological molecules in solution by an optical heterodyne technique is described. A visibly transparent sample is irradiated in a spatially nonuniform manner with pulsed microwaves, and the spatial variation in temperature increase measured by detecting the phase chirp impressed on a single-frequency He-Ne laser beam passing through the heated region. Results for several liquids and solutions such as water, methanol, various saline solutions, and solutions of DNA and DNA sodium salt in water are described. Where direct comparison is possible the results agree very well with published values. A significant increase in the absorption of DNA solutions compared with pure water has been observed that is consistent with microwave absorption by the longitudinal mode of the double helix.