Protein arrangement in the DNA grooves in chromatin and nucleoprotamine in vitro and in vivo revealed by methylation.

Abstract

The influence of histones, protamine, and some polypeptides bound to DNA on the methylation with [3H]dimethyl sulfate of the N-7 atom of guanine in the major groove, and the N-3 atom of adenine in the minor groove of DNA double helix has been studied. The effect on the methylation pattern of the primary and secondary structure of DNA has also been investigated. The comparative kinetic measurements of methylation of DNA and chromatin in the presence of dGMP as an internal standard has been studied. It has been shown that the presence of histones in chromatin causes a rather small effect on methylation of the DNA grooves: histones leave the minor groove well exposed and shield the major groove of DNA against methylation only by 14%. The increase in the velocity of DNA methylation in ice at – 10°C has been demonstrated. The methylation of ascites tumor cells and fish sperm in the frozen state, when the excision of the methylated bases by repair enzyme is repressed, allowed testing of the chromatin and nucleoprotamine structure directly in the cells. At – 10°C in these cells, as well as in isolated chromatin, histones and protamine preferentially shield the major groove of DNA to the same extent (14–21%). Histones and protamine appear therefore to interact with DNA in a similar way in vitro and in vivo. The results of these studies suggest that histones and protamine seem to lie mainly outside the DNA grooves, do not occupy the minor groove and are only partly buried in the major groove. This may make the DNA grooves, especially the minor one, well exposed and accessible to specific interaction of DNA in chromatin with other proteins.