Isolation of newly replicated chromatin by using shallow metrizamide gradients

Abstract

The properties of chromatin containing newly synthesized DNA and protein were investigated. A fraction of chromatin enriched in newly replicated DNA was isolated by means of its increased density in metrizamide relative to bulk chromatin. The DNA of this fraction appeared to be packaged into nucleosomes but at a reduced nucleosomal spacing. Although pulse-labeled DNA was present in this dense fraction, nucleosomes labeled with short pulses of arginine or acetate were of normal density. The data presented are consistent with the conclusion that newly replicated DNA is associated with preexisting histones in a short-lived, compact structure whereas newly synthesized histones are deposited at normal spacing some distance from the replication fork.