Abstract
This work analyzes the involvement of arginines in copper/H2O2-induced DNA breakage. Copper is a highly redox active metal which has been demonstrated to form compounds with arginines. For this aim we used mixtures of pGEM3 DNA plasmid and two types of H1 histones which differ only in their arginine content. The sperm H1 histone from the annelid worm Chaetopterus variopedatus (arginine content 12.6 mol% K/R ratio 2) and the somatic H1 histone from calf thymus (arginine content 1.8 mol% and K/R ratio 15). Copper/H2O2-induced DNA breakage was observed only in presence of sperm H1 histones, but it was more relevant for the native molecule than for the deguanidinated derivative (K/R ratio 14), in which 80% of arginine residues were converted to ornithine. Further, copper induced proteinase K resistance and increase of DNA binding affinity on native sperm H1 histones. These observations are consistent with a copper induced reorganization of the side-chains of arginine residues. Copper, instead, did not affect DNA binding affinity of somatic and deguanidinated H1 histones, which show similar K/R ratio and DNA binding mode. These results indicate that arginine residues could affect these H1 histones properties and provide new insights into copper toxicity mechanisms.
Highlights
Copper ions play important roles in many chemical and biochemical processes and are required for cellular respiration, peptide amidation, neurotransmitter biosynthesis, pigment formation and connective tissue strength[1]
Sperm H1 histone was extracted from the sperm chromatin of the annelid worm Chaetopterus variopedatus (Ch.v.); somatic one was from calf thymus (C.T.)
The same analysis performed on deguanidinated derivatives of sperm H1 histones, in which 80% of arginine were converted in ornithine residues (K/R = 14), showed a DNA binding mode more similar to that of somatic H1 histones and not influenced by CuCl2 (Fig. 1c)
Summary
Copper ions play important roles in many chemical and biochemical processes and are required for cellular respiration, peptide amidation, neurotransmitter biosynthesis, pigment formation and connective tissue strength[1]. ·OH produces (additional) piperidine- sensitive base damage and/or strand breaks at short range from the original Cu(II)-binding site It has been reported[20] that a class of binuclear and trinuclear copper complexes show high selectivity in oxidizing DNA at ss/ds DNA junctions, but an efficient cleavage was not observed for ss or ds DNA alone, and that the flexibility of the DNA strand is an important factor in the ss/ds junction selectivity. DNA bases may participate in the formation of DNA-protein cross-links in chromatin[21] and electron transfer occurs from the histone to DNA, leading to DNA damage[23] It seems that some interactions of DNA with peptides can increase metal/H2O2 induced DNA breakage[24] and/or these reactions can lead to oxygen activation that in turn can proceed in vivo around and inside the cell nucleus[25]. Since proteins are the most abundant target within cells for radicals such as ·OH31 and heavily oxidized proteins generally show decreased susceptibility to proteolytic attack by most proteinases[32,33], we studied the relevance of arginine residues in Cu(II)-induced proteinase K resistance of these two types of H1 histones because in literature several studies have suggested that Cu(II) ions convert some proteins to a proteinase K-resistant conformation[34,35]
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