The metabolism of histone fractions: Phosphorylation and synthesis of histones in late G 1-arrest

Abstract

The biosynthesis and phosphorylation of histone fractions were measured in synchronized CHO Chinese hamster cells arrested in late G 1 by hydroxyurea treatment. Hydroxyurea was found to inhibit the initiation of both DNA and histone synthesis, thus confirming the conclusion that it arrests cells in G 1 slightly before the G 1 S boundary. However, hydroxyurea did not inhibit the phosphorylation of histone f1 or histone f2a2. The phosphorylation of histone f1, which normally is absent in early G 1, begins 2 hr prior to DNA synthesis. In the presence of hydroxyurea, f1 phosphorylation occurs on schedule at this same time in G 1, resulting in significant G 1-phase f1 phosphorylation. This offers strong evidence that (a) f1 phosphorylation is not restricted to S phase; (b) “old” f1 which was synthesized in previous cell cycles is phosphorylated in G 1 before “new” f1 which is synthesized in S phase; and (c) G 1-phase f1 phosphorylation does not require new histone or new DNA synthesis. Histone f1 phosphorylation was observed to occur at accelerated rates in S phase over phosphorylation rates observed in late G 1-arrest. Data support the proposal that three different levels of f1 phosphorylation occur during the cell cycle: (1) a G 1-related phosphorylation of “old” f1; (2) an S-related phosphorylation of both “old” and “new” f1; and (3) a superphosphorylation of f1 associated with chromosome condensation during the G 2 to M transition. It is also possible that a limited proportion of f1 may be phosphorylated in G 1, perhaps at the initial DNA synthesis sites, and that an increased proportion of f1 is phosphorylated in S as DNA is synthesized. Similarities between the kinetics of histone f1 phosphorylation and the association of DNA with lipoprotein in synchronized control and hydroxyurea-treated cells suggest an involvement of f1 phosphorylation in cell-cycle-dependent chromatin structural changes.