In vivo assembly of newly synthesized histones

Following a labeling period of 2 min, HeLa histones continue to accumulate in chromatin for 10 min, indicating the presence of a histone pool. During the accumulation period, H2A and H2B enter chromatin immediately, while entry of H3 and H4 is more prolonged. Association of newly synthesized core histones with chromatin does not necessarily indicate assembly. When 2-min [3H]lysine-labeled chromatin is exposed to 0.45 M NaCl, nearly half of the newly synthesized histones are dissociated, while mature core histones are stable. H2A and 70% of H2B are salt stable and remain with newly synthesized polynucleosomes. About 30% of H2B, 50% of H4, and all of H3 are salt labile; thus, both the new nucleosomal core histones and salt-labile new core histones are nonstoichiometric. Pulse-labeled core histones are more trypsin-sensitive than mature histones. When the salt-labile, newly synthesized histones are removed, the remaining proteins have the same trypsin sensitivity as bulk core protein. Examination of the tryptic peptides indicated that the increased trypsin sensitivity was due to complete destruction of the loosely associated core histones which undergo a lag prior to assembly. The altered order of appearance of two peptides in stripped, newly assembled nucleosomes indicates that the conformation in these particles is different from that in mature chromatin.