The use of ultraviolet Light in the fractionation of chromatin containing unsubstituted and bromodeoxyuridine-substituted DNA

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Abstract
Two procedures are described for the fractionation of chronatin containing unsubstituted (LL) DNA and DNA unifilarly substituted with bromodeoxyuridine (a). The two procedures rely upon the sensitivity of bromodeoxyuridine-containing DNA to UV light to induce either strand hreakage or protein crosslinking. When a mixture of LL and a chromatin is irradiated with UV light, the a DNA fragments into molecules of smaller molecular weight than the I DNA and crosslinka more chromosomal protein than the LL DNA. LL and a chromatin can be fractionated on the basis of size by centrifuging through a neutral sucrose gradient. The a DNA-protein adducts that are generated by the UV light have a unique buoyant density and may be isolated by isopycnic centrifugation in cs2SO4 The ability to fractionate LL. and HL chromatin permits certain studies on the structure of replicating chromatin.