Analysis of multiple forms of nuclear factor I in human and murine cell lines.

Nuclear factor I (NFI) is a group of related site-specific DNA-binding proteins that function in adenovirus DNA replication and cellular RNA metabolism. We have measured both the levels and forms of NFI that interact with a well-characterized 26-base-pair NFI-binding site. Five different NFI-DNA complexes were seen in HeLa nuclear extracts by using a gel mobility shift (GMS) assay. In addition, at least six forms of NFI were shown to cross-link directly to DNA by using a UV cross-linking assay. The distinct GMS complexes detected were composed of different subspecies of NFI polypeptides as assayed by UV cross-linking. Different murine cell lines possessed varying levels and forms of NFI binding activity, as judged by nitrocellulose filter binding and GMS assays. The growth state of NIH 3T3 cells affected both the types of NFI-DNA complexes seen in a GMS assay and the forms of the protein detected by UV cross-linking.

Structure and mechanism of hydroxyl radical-induced formation of a DNA-protein cross-link involving thymine and lysine in nucleohistone.

Hydroxyl radical-induced formation of a DNA-protein cross-link involving thymine and lysine in calf thymus nucleohistone in vitro is reported. Basic amino acids such as lysine constitute a very high proportion of the amino acids of histones, and help histones to bind to DNA in chromatin. For this reason, basic amino acids are likely to participate in DNA-protein cross-linking. For identification of the thymine-lysine cross-link in nucleohistone, hydroxyl radical-induced cross-linking of thymine to lysine was investigated first using a model system, i.e., an aqueous mixture of thymine and lysine. Hydroxyl radicals were generated by exposure of this mixture to ionizing radiation after N2O saturation. The technique of gas chromatography-mass spectrometry was used to analyze the samples for possible cross-links. One thymine-lysine cross-link was found and its structure was elucidated. Using gas chromatography-mass spectrometry with selected-ion monitoring, this thymine-lysine cross-link was identified in acidic hydrolysates of calf thymus nucleohistone gamma-irradiated in N2O-saturated aqueous solution. The yield of this DNA-protein cross-link was also measured and found to be a linear function of radiation dose between 15 and 200 Gy. This yield amounted to 0.0085 mumol/J. Possible mechanisms for the formation of this DNA-protein cross-link in nucleohistone were proposed.

Electron transfer between protein and DNA in gamma-irradiated deoxyribonucleoprotein.

When deoxyribonucleoprotein-proflavine complexes were studied by electron spin-resonance spectroscopy following gamma-irradiation, it was found that stable free radicals were not formed at random on the complex but were preferentially located on proflavine. Since proflavine intercalalated to DNA bases serves as a final acceptor of electrons liberated by ionization, the result of our experiment was regarded as suggesting that the electron transfer from the protein moiety to the DNA moiety occurred in the irradiated deoxyribonucleoprotein.

Effects of irradiation on RNA synthesis primed by calf thymus deoxyribonucleoprotein treated with salt and salt-urea.

1. The effects of ionizing radiation on the activity of calf thymus templates were examined in a Escherichia coli RNA polymerase system. 2. The template activity of native and 2 M NaCl-5M urea-treated deoxyribonucleoproteins was enhanced by relatively low doses of irradiation, while that of 2 M NaCl-treated deoxyribonucleoprotein was not enhanced by irradiation. 3. The template activity of purified DNA was markedly decreased by irradiation, while that of native deoxyribonucleoprotein, 2 M NaCl-treated, and 2 M NaCl-5 M urea-treated ones were slightly decreased at a higher dose range. The doses for 50% inactivation of these templates were 1.3, 210, 140, and approximately 200 krad, respectively.

Alterations in chromosome structure and variations in the inherent radiation sensitivity of human cells.

Variations in the inherent radiosensitivity of both tumor cells and the normal tissues that surround them play an important role in tumor response to radiation therapy. In vitro studies suggest that variations in radiation sensitivity both between different tissues and within a specific histology are a reflection of differences in the rate and fidelity of rejoining of chromosome breaks. Cells of radiosensitive cell lines rejoin breaks more slowly and with less fidelity than those of more resistant cell lines. Differences in radiation sensitivity are also associated with variations in chromosome structure as detected by nucleoid-based assays. A model is presented to suggest that the radiation sensitivity of a cell line is a reflection of its transcriptional architecture, the number and genomic location of its actively transcribing regions. Also, it is proposed that chromosome breaks induced at or near transcriptionally active regions of the genome are rejoined preferentially and with greater fidelity than breaks induced at other regions of the genome.

DNA-protein crosslinking in normal and solar UV-sensitive ICR 2A frog cell lines exposed to solar UV-radiation.

DNA-protein crosslinks (DPC) were measured following exposure to the solar UV wavelengths produced by a fluorescent sunlamp in ICR 2A frog cells and two solar UV-sensitive mutants derived from this cell line. Approx. 5-7 DPC per 10(10) dalton were induced in these cells by either 150 kJ/m2 of sunlamp UV greater than 315 nm plus photoreactivating light (PRL) or 10 kJ/m2 of sunlamp UV greater than 295 nm. The irradiated cells were then incubated for 0-24 h and the level of DPC measured using alkaline elution. It was found for the ICR 2A cells exposed to sunlamp UV greater than 315 nm that the level of DPC increased about 3-fold during a 2-h postirradiation incubation and then decreased. The mutant cell lines also showed an enhancement in the level of DPC following irradiation, although it was much less pronounced and the levels decreased much more rapidly. In a similar fashion, the level of DPC increased in ICR 2A cells exposed to sunlamp UV greater than 295 nm with more than a 5-fold enhancement after a 4-h incubation. Once again, the mutant cell lines showed an increase in the level of DPC that was smaller and more transient than the effect in the ICR 2A cells. These results suggests that this enhancement in DPC may be indicative of a process that plays a role in cellular survival following solar UV-irradiation.

The effects of continous irradiation on histological picture and deoxyribonucleoprotein content in spleen of rats.

The histological picture and deoxyribonucleoprotein (DNP) content in the spleen of the white male Wistar rats continuously irradiated with the daily dose rates 9.57 mGy (1 R), 95.7 mGy (10 R), 478.5 mGy (50 R) and 657.0 mGy (100 R) were studied. The animals were examined within the day until 60 of irradiation. The number of lymphocytes permanently decreased at the daily dose rates 95.7 mGy and higher ones. The increased activity of reticulum with the simultaneous multiplication of the erythroid cells in the red pulp of spleen was observed at day 10 and 60 of irradiation with 9.57 mGy and 95,7 mGy. Simultaneously DNP content in spleen increased in accordance with the increased activity of reticulum and extramedullar erythropoiesis. The significant decrease in DNP content alter the onset of irradiation with the daily dose rates 478.5 mGy and 957.0 mGy was caused mainly by the fall of lymphocytes. The incidence of eosinophilic leukocytes increased from day 40 irradiation with the daily dose rates 9.57 mGy, 95.7 mGy and 478.5 mGy and decreased with the daily dose rate 957.0 mGy. The plasma cells tended to increase in accordance with the increasing accumulated dose of irradiation with all daily dose rates.

Deoxyribonucleoprotein structure and radiation injury: cellular radiosensitivity is determined by LET infinity -dependent DNA damage in hydrated deoxyribonucleoproteins and the extent of its repair.

For decades, theories of cellular radiosensitivity relied upon the initial patterns of energy deposition to explain radiation lethality. Such theories are unsound: cellular (DNA) repair also underlies cellular radiosensitivity. For the charged particles encountered in deep space, both the types of DNA damage caused in cellular deoxyribonucleoproteins and the efficacies of their repair are dependent on linear energy transfer (LET infinity), and repair efficiency is also influenced by cell and tissue type, i.e., the actual recovery processes involved. Therefore, quality factors derived from radiation quality alone are inadequate parameters for assessing the radiation risks of space flight. Until recently, OH radicals formed in bulk nuclear water were believed to be the major causes of DNA damage that results in cell death, especially for sparsely ionizing radiations. That hypothesis has now been challenged, if not refuted. Lethal genomic DNA damage is determined mainly by energy deposition in deoxyribonucleoproteins, and their hydration shells, and charge (energy) transfer processes within those structures.

Structural changes of deoxyribonucleoprotein fibres following gamma-irradiation under aerobic and hypoxic conditions.

The DNP fibres gamma-irradiated under aerobic condition showed a reduction of their diameter, while no remarkable changes were observed in the DNP fibres irradiated under hypoxic condition by scanning electron microscopy.

Cellular radiation biology in consolidation and transition.

Cellular radiation biology currently is undergoing changes common to all science in which the understanding in one area is becoming solidified while in another area conclusion of the classical phase is being engendered by the needs of modern thought. Aspects of these changing circumstances are discussed here from the standpoint of the roles played by direct and indirect action in cell death and the position that promulgation of the correct explanations of the radiosensitivities of mammalian cells can be facilitated if use of such classical operational definitions as sublethal and potentially lethal damage is discontinued. The latter consideration will be supported by a summary of the responses of synchronous populations of the L5178Y S/S murine leukaemic lymphoblast to 20Ne, 28Si, 40Ar, 56Fe and 93Nb ions of energies broadly in the region of 500 MeV/u.

Isolation of modified histone H3 from ultraviolet-irradiated deoxyribonucleoprotein by reversed-phase high-performance liquid chromatography.

A rapid reversed-phase high-performance liquid chromatography procedure for the isolation of histone H3 and/or of thymine modified at the lysine residue histone H3 from uv-irradiated deoxyribonucleoprotein and DNA-protein complex is reported. The system utilizes a C8 Ultrasphere macroporous column and an acetonitrile "inverse or negative gradient."

[Effect of UV-light on the structure of soluble deoxyribonucleoprotein-200 A]. / Vliianie uf-sveta na struktupu rastvorimogo dezoksiribonukleoproteida 200 A.

The effects of UV-light (253,7 nm) on the structure of DNP and its protein and nucleic components were studied. The formation of protein-DNA covalent bonds in DNP-200 A at low ionic strength was confirmed. Under certain irradiation conditions more than 80% of protein may be linked to the DNA; all histone fractions were linked to the same extent and at the same rates. The local denaturation of DNA in the region of photo-induced thymine-thymine dimers and other photoadducts dramatically changed the rate and specificity of the effects of staphylococcal nuclease, which directly affected the composition and size of the fragments formed. A possible application of UV-irradiated DNP for various structural investigations is discussed.

[Changes in DNA-histone contacts under thermal denaturation of DNP and nucleosomes]. / Izmenenie kontakov DNK - gistony pri termicheskoi denaturatsii DNP i nukleosom.

The UV (253.7 nm)-induced binding of the protein to DNA in DNP and nucleosomes at low ionic strength within the temperature range of 5--85 degrees C was studied. A two-step increase in th degree of protein binding was revealed, which was assumed to correspond to two structural conversions in the nucleoproteins during their denaturation. It was shown that the appearance of additional sites of core histone binding in the premelting region is induced by local changes in the DNA structure. None of the four histone fractions was shown to bind preferentially over the whole temperature range studied, which suggests that the structural changes occur all over the length of DNA in the nucleoproteins.

Changes in the circular dichroic spectrum of calf thymus solubilized chromatin caused by ultraviolet irradiation.

UV irradiation of the chromatin caused an increase of the positive circular dichroic band in the vicinity of 275 nm (corresponding to DNA) and a deepening of the negative band of proteins at about 225 nm. These changes in the circular dichroic spectrum are monotonous in the range of doses studied (less than 6 X 10(4) J.m-2). The increase of the positive circular dichroic band probably reflects the occurrence of local conformational changes in DNA, which include changes in base position (tilting, distance from helix axis) in the close neighborhood of photoproducts. The presence of photoproducts in chromatin reduces changes in its circular dichroic spectra with temperature.

Effect of gamma irradiation on dye-DNH binding.

This paper attempts to analyse the effect of (a) gamma radiation and (b) the presence of histones in the form of nucleohistones (DNH) on the modes of ligand (Proflavine, Pf) binding to DNH. Effects were investigated by spectrophotometry. A certain dose of radiation which causes appreciable DNA denaturation has much less effect on DNH. There is a 'threshold' dose beyond which DNH denaturation is directly proportional to dose as in the case of DNA. The histones in DNH act as a shield and at higher doses get dissociated to expose the double helix to radiations. Ligand binding has a further stabilizing effect against radiation damage to DNH.