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.

Photoaffinity labelling of a DNA-binding site on the globular domain of histone H5.

We have labelled a DNA-binding site on the globular domain of histone H5 (GH5) by ultraviolet-activated cross-linking of a self-complementary 5-bromodeoxyuridine (5BrU)-substituted oligonucleotide with the sequence 5'-AGCGA5BrUATCGCT-3'. Cross-linking was to His62, mainly to the protein backbone. This observation provides further support for the mode of binding of GH5 to DNA proposed on the basis of the similarity between the X-ray crystal structure of GH5 and the DNA-bound structures of catabolite activator protein and hepatic nuclear factor 3 gamma [Ramakrishnan, V. (1994) Curr. Opin. Struct. Biol. 4. 44-50].

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.

Ultraviolet irradiation of DNA complexed with alpha/beta-type small, acid-soluble proteins from spores of Bacillus or Clostridium species makes spore photoproduct but not thymine dimers.

UV irradiation of complexes of DNA and an alpha/beta-type small, acid-soluble protein (SASP) from Bacillus subtilis spores gave decreasing amounts of pyrimidine dimers and increasing amounts of spore photoproduct as the SASP/DNA ratio was increased. The yields of pyrimidine dimers and spore photoproduct were less than 0.2% and 8% of total thymine, respectively, when DNA saturated with SASP was irradiated at 254 nm with 30 kJ/m2; in the absence of SASP the yields were reversed-4.5% and 0.3%, respectively. Complexes of DNA with alpha/beta-type SASP from Bacillus cereus, Bacillus megaterium, or Clostridium bifermentans spores also gave spore photoproduct upon UV irradiation. However, incubation of these SASPs with DNA under conditions preventing complex formation or use of mutant SASPs that do not form complexes did not affect the photoproducts formed in vitro. These results suggest that the UV photochemistry of bacterial spore DNA in vivo is due to the binding of alpha/beta-type SASP, a binding that is known to cause a change in DNA conformation in vitro from the B form to the A form. The yields of spore photoproduct in vitro were significantly lower than in vivo, perhaps because of the presence of substances other than SASP in spores. It is suggested that as these factors diffuse out in the first minutes of spore germination, spore photoproduct yields become similar to those observed for irradiation of SASP/DNA complexes in vitro.

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.

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.

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."

[Localization of lysine residue in histone H3 forming the thymine-lysine cross-link after UV-irradiation of deoxyribonucleoprotein]. / Lokalizatsiia ostatka lizina v gistone H3, obrazuiushchego timin-lizinovuiu sshivku pri obluchenii dezoksiribonukleoproteida UF-svetom.

A thymine-modified derivative of histone H3 formed as a result of thermal treatment of UV-irradiated (lambda = 254 nm) solution of deoxyribonucleoprotein from calf thymus at low ionic strength was isolated. The peptides obtained by tryptic hydrolysis of modified histone H3 were separated by high pressure liquid chromatography. The amino acid sequence of the peptide containing a lysine residue with covalently linked thymine was determined by the Edman method. It was found that Lys localized at the N-terminus of the histone H3 molecule interacts with DNA within the composition of the deoxyribonucleoprotein.

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.

Protein-protein and protein-DNA interactions in calf thymus nuclear matrix using cross-linking by ultraviolet irradiation.

Nuclear matrices from calf thymus contained 30-50 protein species with one prominent band at 70 kilodaltons tentatively identified by its isoelectric point, apparent molecular weight, charge modification, and abundance as bovine lamin. The amount of DNA present in the matrix fraction was strongly dependent on the extent of digestion of the nuclei by micrococcal nuclease. The size of the DNA was higher than two kilobase pairs, although the chromatin DNA had been digested down to short oligonucleosomes. The lamin band was preferentially dissociated from isolated matrices during repeated treatment by 2 M NaCl or 5 M urea. Irradiation of calf thymus nuclear matrices at 313 nm induced protein-protein and protein-DNA cross-linking, as well as double-strand breaking of DNA, presumably at unprotected, protein-free regions. Lamin protein was more dramatically affected than other protein species by ultraviolet (UV) irradiation. In situ DNA hydrolysis, after the separation of the cross-linked matrix components on polyacrylamide-sodium dodecyl sulfate gels, followed by two-dimensional electrophoresis, showed lamin to be the major protein that was cross-linked to the DNA. Lamin molecules were also cross-linked by UV light to each other to form lamin homo-oligomers. A discrete size DNA fragment of approximately 450 base pairs is protected by lamin homo-oligomers from breakdown during UV irradiation. It is proposed that the direct contact between lamin and DNA found in this study is responsible for anchoring chromatin loops (domains) to a stable, immobile matrix structure.

[The role of histones H3 and H1 in the formation of thymine-lysine cross-links during UV-irradiation of deoxyribonucleoprotein]. / Uchastie gistonov H3 i H1 v obrazovanii timin-lizinovoi shivki pri UF-obluchenii deoksiribonukleoproteida.

The effect of UV light (lambda = 254 nm) on calf thymus DNP at low ionic strengths was studied. It was found that at the irradiation doses used the protein in the DNA-protein complex increases as the irradiation dose rises. Thermal treatment and acid hydrolysis resulted in a predominant release of histones H3 and H1 from the complex. Data from liquid high performance chromatography, amino acid analysis, thin-layer chromatography point to the induction by UV-light of a thymine-lysine bond, whose formation involves DNA thymines and histone lysine residues, predominantly H3 and H1 fractions.

[Molecular mechanisms of interphase death of lymphoid cells. Distribution of nuclease attack sites in chromatin and kinetic complexity of DNA in PDN]. / Molekuliarnye mekhanizmy interfaznoi gibeli limfoidnykh kletok. Kharakter raspredeleniia mest nukleaznoi ataki v khromatine i kineticheskaia slozhnost' DNK v sostave PDN.

On the basis of a quantitative correlation between single--and double-helix fragments of different length within PDN, a study was made of the pattern of distribution of the sites, attacked by nuclease, in the chromatin. It was shown that under the effect of ionizing radiation products of enzymic digestion of the chromatin. It was shown that under the effect of ionizing radiation products of enzymic digestion of the chromatin accumulated in thymocytes due to internucleosome degradation were excised from randomly localized genome sites. The analysis of the reassociation curves did not reveal distinctions in the kinetic complexity of the fractions of PDN and total DNA.

[UV-light-tested protein--DNA interactions in the nuclei during the cell cycle in Physarum polycephalum]. / Testiruemye uf-svetom belok--DNK-kontakty v iadrakh na raznykh stadiiakh kletochnogo tsikla Physarum polysephalum.

The release of DNAs from nuclear preparations isolated at different steps of the cell cycle of the synchronous culture of Physarum polycephalum and irradiated with UV-light for 5 min (lambda 253.7 A, i = 1.8 . 10(2) j . m-2 . min-1) into 1% Na-DS - 1.5 M NaCl solution was studied. The value obtained correlated well with the degree of binding of interacting proteins with DNA in chromatin and was identical for all the preparations tested with the only exception of the nuclei isolated at the end of the S-phase, when a slight but significant (15 +/- 10%) increase in DNA release was observed. The results obtained are discussed in terms of structural transitions and function of chromatin during the cell cycle of the myxomycete Physarum polycephalum.

Mechanism of DNA-histone crosslinks in UV-irradiated nuclei.

Mechanism of the DNA-histone crosslinks in UV-irradiated chicken erythrocyte nuclei has been investigated. It has been demonstrated that the major chemical reaction induced by irradiation of DNA-histone complexes is the crosslinking between lysine and thymine residues and subsequent transfer of thyminyl group from DNA to lysine residues of histones.