Predominance of core histones in formation of DNA--protein crosslinks in gamma-irradiated chromatin.

Abstract

Chromatin and a subunit ofchromatin containing a complex of DNA and the core histones-H2A, H2B, H3, and H4-have been prepared from cultured Chinese hamster cells.Comparison of the formation of radiation-induced DNA-protein crosslinks in whole chromatin with that in the DNA-core histone complex has demonstrated that the core histones are the specific proteins involved in crosslinking. y irradiation of the chromatin subunit in the presence of radical scavengers has shown the hydroxyl radical to be the most effective aqueous radical intermediate for the promotion of crosslinking and the solvated electron and superoxide radical to be essentially ineffective.DNA-protein crosslinks are formed when whole cells or mixtures of DNA and cell protein in vitro are subjected to y or UV irradiation (1,2 It is generally accepted that, in eukaryotic cells,,nuclear DNA is organized as nucleosomes, consisting of a core region, containing the histon-es H2A, .H2B, H3, and H4, wrapped around with DNA; these core regions are joined by-linker DNA, probably. associated with H1 histone. Isolated together with the 'DNA and histones in purified chromatin are nonhistone chromosomal proteins (NHCP), probably consisting of as many as 100 structural, enzymic, and regulatory proteins, of which 15-20 are major constituents.Chromatin and a subunit of chromatin containing a complex of DNA and the core histones have been isolated from cultured Chinese hamster cells. On the basis ofcomparing radiation-induced crosslinking in the whole chromatin and in the chromatin subunit, we have now identified the core histones as the specific chromosomal proteins predominantly involved in crosslinking to DNA. Furthermore, y irradiation of the chromatin subunit in the presence of radical scavengers has confirmed the efficacy of the hydroxyl radical for the promotion of formation of crosslinks, and the inefficacy ofthe solvated electron and superoxide radical (3). MATERLALS AND METHODSCell Culture and Labeling. Chinese hamster lung fibroblasts (V79-753 cell line from J. A. Belli, Harvard Medical School) were grown as described (4). Eagle's minimal essential medium was supplemented with 15%.fetal bovine serum, 4% NCTC-109 medium (M. A. Bioproducts, Walkersville, MD), 2 mM L-glutamine, 0.1 mM of each nonessential amino acid, penicillin at 50 units/ml, and streptomycin at 50 Ag/ml. Cells were grown as monolayers in 150-cm2 plastic flasks at 370C in an atmosphere of 5% CO2. They were grown for two generations (18 hr) prior to harvesting in a medium containing [methyl-3H]thymidine (New England Nuclear) at.a concentration of0.25 ,uCi/ml (1 Ci = 3.7 x -101 becquerels) to label the DNA, and in some experiments, in a medium containing L-[U-'4C]lysine (New En-gland Nuclear) at a concentration of 0.5 ,Ci/ml to label the proteins.Preparation of Chromatin and a Chromatin Subunit. The isolation of chromatin from the cell pellet has been described in detail (4). Nuclei were prepared.by the method ofHymer and Kuff (5), in which cells were suspended in hypotonic sucrose b...

Reaction mechanisms in the radiolysis of peptides, polypeptides and proteins

“…are exposed to y and UV radiation (Smith, 1976;Mee and Adelstein, 1979;Mee and Adelstein, 1981;Cress and Bowen, 1983).…”

1,N 2-Deoxyguanosine Adducts of Acrolein, Crotonaldehyde, and trans-4-Hydroxynonenal Cross-link to Peptides via Schiff Base Linkage

“…The histones and nuclear matrix proteins are the predominant substrates involved in DPC formation (7)(8)(9), and chromatin structure significantly affects cross-linking efficiency (10 -12). Not surprisingly, aldehydes with established DPC-forming ability disrupt DNA replication for the SV40 minichromosome following exogenous exposure (13), suggesting that DPC damage presents a major obstacle to the mammalian DNA replication (and transcription) machinery.…”

Loss of Cellular Transformation Efficiency Induced by DNA Irradiation with Low-Energy (10 eV) Electrons

“…It has been shown that the nucleosomal core histones (H2A, H2B, H3 and H4) are the main proteins involved in the formation of DNA-protein cross-links in irradiated chromatin. 27 Although DSBs and other cluster lesions, as well as DNA-protein cross-links have the potential to be lethal, the relative contribution of each lesion to the loss of DNA functionality induced by LEEs in living cells is not known. Since LEEs have a very short-range in condensed matter (5-10 nm), 3 it is not possible to irradiate the DNA of living cells with them.…”

“…[22][23][24][25][26] IR can also induce cross-links between DNA and nearby biomolecules, including proteins, which can cause cell death, mutagenesis or carcinogenesis. 27 If not correctly repaired, the formation of DNA-protein cross-links within the cell may disrupt DNA transcription, replication, and, ultimately, cell division. It has been shown that the nucleosomal core histones (H2A, H2B, H3 and H4) are the main proteins involved in the formation of DNA-protein cross-links in irradiated chromatin.…”