Chemical and enzymatic analysis of covalent bonds between peptides and chromosomal DNA

Abstract

DNA from Ehrlich ascites tumor (EAT) cells and from human placenta was examined for covalent bonds between hydroxy amino acid residues in peptides and nucleotide phosphate groups. The residual proteinaceous material in highly purified DNA was radiolabelled with 125Iodine and the linking-groups between peptides and nucleotides released by combined protease and nuclease treatment were investigated with respect to their chemical and enzymatic stabilities. The residual nucleotide(s)-peptide(s) fraction from DNA isolated after prolonged alkaline cell lysis and phenol extraction contains mainly alkali and acid-stable but phosphodiesterase-sensitive peptide-nucleotide complexes which indicates phosphodiesters between tyrosyl residues in peptides and nucleotide phosphates. In contrast, the linking-group fraction from DNA isolated under native conditions contains additional peptide components. (a) Phospho-peptides that co-purify with DNA but that are not covalently bound to nucleotides. (b) A fraction of peptides that is released from nucleotides by alkali in a time and concentration-dependent reaction. Evidence is presented indicating that the latter fraction involves phospho-triesters between hydroxy amino acid residues in peptides and internucleotide phosphates. The phosphodiesters between hydroxy amino acids and nucleotide phosphates representing the predominant class of peptide-nucleotide complexes in alkali-denatured DNA are most likely side products of peptide-nucleotide phospho-triester hydrolysis.

High salt- and SDS-stable DNA binding protein complexes with ATPase and protein kinase activity retained in chromatin-depleted nuclei

“…At present we have no evidence for a potential relation between the newly discovered high salt-and SDS-stable complexes and covalent DNA-polypeptide complexes described previously (4)(5)(6)(7)(8)(9). However, it has been suggested by many authors that different types of anchorage structures with varying functions may exist, e.g.…”

“…2b). Thus, at present, we have no experimental evidence for a potential relationship between the polypeptides co-isolating with DNA during the salting-out procedure and those bound to DNA by covalent nucleotide-peptide bonds previously detected in phenol-extracted DNA (4,5).…”

“…Previously we found that a small but persistent amount of nuclear polypeptides remains associated with eukaryotic DNA submitted to the most rigorous deproteinization methods, including treatment with SDS, proteinase K and phenol. The chemical stability of this DNA-peptide interaction is apparently based on covalent bonds between hydroxy amino acid residues:in polypeptides and internucleotide phosphates (4,5). Complexes of this type seem to reside preferentially on highly repetitive DNA sequences which have been cloned (6)(7)(8) and shown to be highly enriched in residual nuclear matrix DNA (9).…”

Ribonucleoprotein-masked nicks at 50-kbp intervals in the eukaryotic genomic DNA

“…In the latter experiments, additional labile sites of different configuration may have been revealed (23), because the ends of those shorter DNA fragments, observed at more intensively denaturing conditions, were not labeled by Pol I. Alternatively, secondary changes might have been generated in that or in our system, leading to different end structures. Notwithstanding the differences, our observations and those of refs.…”

Short DNA sequences from the cytoplasm of mouse tumor cells induce immortalization of human lymphocytes in vitro.

“…The salt-stable DNA-protein interaction of nuclear DNA-polypeptide complexes was recently shown to be due to phosphotriester bonds between hydroxyamino acid residues in peptides and internucleotide phosphates of the DNA (20). Covalent bonds between DNA and polypeptides would explain the unusual stability of the cytoplasmic complexes, in particular, the resistance to detergents, high salt concentrations, or alkali (unpublished results).…”