Involvement of H1 and other chromatin proteins in the formation of DNA-protein cross-links induced by visible light in the presence of methylene blue.
The formation of DNA-protein cross-links (DPCs), induced by irradiation with visible light, was studied in methylene blue-treated (MB-treated) chromatin and H1-depleted chromatin. The effects of the MB concentration and radiation dose were studied using sodium dodecylsulphate-chloroform-isoamyl alcohol assay and sodium dodecylsulphate-polyacrylamide gel electrophoresis. Under identical experimental conditions, DPC formation was less in H1-depleted chromatin (70%) than in chromatin (92%). The non-histone proteins and core proteins of chromatin contributed towards DPC formation. Of the core proteins, H2A was more cross-linked than H4, whereas the bands for H2B and H3 melted into one in chromatin and H1-depleted chromatin. In both cases, the gel pattern showed the appearance of two new protein bands with approximate molecular weights of 27 kDa and 29 kDa as a result of histone-histone cross-linking. Viscometric studies showed that the dissociation of the compact structure of chromatin in 2 M NaCl was more extensive in irradiated, MB-treated, H1-depleted chromatin than in irradiated, MB-treated chromatin, indicating a reduction in the amount of DPC formation in H1-depleted chromatin.
Lalwani
R
R
Department of Biophysics, Chittaranjan National Cancer Institute, Calcutta, India.
Maiti
S
S
Mukherji
S
S
D000818:Animals; D002843:Chromatin / Q000187:drug effects / Q000528:radiation effects*; D003432:Cross-Linking Reagents; D004273:DNA, Neoplasm / Q000187:drug effects / Q000302:isolation & purification / Q000528:radiation effects*; D004307:Dose-Response Relationship, Radiation; D004591:Electrophoresis, Polyacrylamide Gel; D006657:Histones / Q000187:drug effects / Q000302:isolation & purification / Q000528:radiation effects*; D007700:Kinetics; D008027:Light; D008297:Male; D008751:Methylene Blue / Q000494:pharmacology*; D008970:Molecular Weight; D010777:Photochemistry; D011485:Protein Binding; D012510:Sarcoma 180 / Q000378:metabolism
D002843:Chromatin; D003432:Cross-Linking Reagents; D004273:DNA, Neoplasm; D006657:Histones; D008751:Methylene Blue