Distribution of Newly Synthesized Histones during DNA Replication

Abstract

A procedure has been developed which allows the separation of chromatin into two fractions highly enriched in preexisting and in newly synthesized DNA, respectively. The procedure includes the following steps. (a) Partial substitution of thymine with 5–bromouracil in the newly synthesized DNA strand. (b) Labelling of proteins with 14 C‐labelled amino acids and DNA with [ 3 H]thymidine during the period of DNA replication. (c) Introduction of breaks in the new DNA strand at the points containing 5‐bromouracil by irradiation at 313 nm. (d) Denaturation of chromatin at pH 12.2 and separation of the intact from the fragmented material by centrifugation in alkaline sucrose density gradient. (e) Purification of the two fractions by a recentrifugation under the same conditions. The results from such experiments have shown that during DNA replication the preexisting histones remain bound to the parental DNA strand while the newly synthesized histones join the new DNA strand.