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 irradiatio n with visible light, was studied in methylene blue-treated (MB-treate d) chromatin and H1-depleted chromatin. The effects of the MB concentr ation and radiation dose were studied using sodium dodecylsulphate-chl oroform-isoamyl alcohol assay and sodium dodecylsulphate-polyacrylamid e gel electrophoresis. Under identical experimental conditions, DPC fo rmation was less in Hi-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-linke d than H4, whereas the bands for H2B and H3 melted into one in chromat in and H1-depleted chromatin. In both cases, the gel pattern showed th e appearance of two new protein bands with approximate molecular weigh ts of 27 kDa and 29 kDa as a result of histone-histone cross-linking. Viscometric studies showed that the dissociation of the compact struct ure of chromatin in 2 M NaCl was more extensive in irradiated, MB-trea ted, HI-depleted chromatin than in irradiated, MB-treated chromatin, i ndicating a reduction in the amount of DPC formation in Hi-depleted ch romatin.