Chromatin structural changes in synchronized cells blocked in early S phase by sequential use of isoleucine deprivation and hydroxyurea blockade

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Abstract
The loss of histone H1 from chromatin and the structure of chromatin from Chinese hamster (line CHO) cells blocked in early S phase were investigated by sequential use of isoleucine deprivation G1 block and 1 mM hydroxyurea (HU) blockade. Measurements of H1 content in the cell and histone turnover indicate that H1 is lost from the cell and that there is negligible replacement synthesis of H1 during the period of the S-phase block. As H1 is lost, chromatin appears to undergo structural change. After 10 h of HU block, the new DNA and a portion of the old DNA have measured nucleosome repeat lengths (37.degree. C digestion) which are less than those of controls and similar to those observed by Annunziato and Seale for new immature chromatin in the absence of HU. By 24 h of HU block, nearly all of the chromatin has assumed a pseudoimmature conformation in which the nucleosome cores appear to be more closely packed along the DNA chain, but the new DNA is slightly more resistant than old DNA to attack by micrococcal nuclease. Electrophoretic analysis of nucleoprotein particles produced by micrococcal nuclease digestion of nuclei indicates that the distribution of mononucleosome species changes during HU block and some mononucleosome species appear to be enriched in normally minor proteins which may determine the electrophoretic mobility of the nucleoprotein particles in agarose-acrylamide gels. The results raise the possibility that during the early stages of replication (or prior to the passage of the replication fork), H1 is dissociated from initiated replicons and H1 does not reassociate in a concerted fashion with the H1-depleted chromatin until this replication fork has passed and, perhaps, a substantial portion of the replicon was replicated.