Fate of newly synthesized histones shortly after interruption of DNA replication.
The synthesis and association of histones with chromatin were studied using MH-134SC cells in suspension culture. Cultures containing approximately equal numbers of cells were pulse-labeled with [3H]lysine at various times after the interruption of DNA synthesis with hydroxyurea. Each culture was mixed with a fixed volume of a culture generally labeled with [14C]lysine at the time of harvesting. Acid-soluble proteins extracted from different subcellular fractions of cells labeled under various conditions were compared by electrophoresis on polyacrylamide gels containing acetic acid and urea. All types of chromatin histones were labeled nearly equally as [14C]marker histones by a 15 min pulse under normal conditions, except that a considerable portion of pulse-labeled H4 was in highly acetylated forms. Addition of hydroxyurea at the start of the pulse markedly reduced the labeling of H3 and H4, but affected the labeling of the other histones only slightly. When DNA synthesis was inhibited before the start of the pulse, labeling of all histones decreased significantly. The addition of hydroxyurea was found to cause transient accumulation of newly synthesized proteins in the cytoplasmic soluble fraction; these were characterized as H3 and H4 from their metabolic properties and their electrophoretic mobilities on sodium dodecyl sulfate-polyacrylamide gels. The results suggest that association of newly synthesized H3 and H4 histones is closely coupled with ongoing DNA replication. The implications of the results for the mechanism of formation of new nucleosomes are discussed.
D002460:Cell Line; D002843:Chromatin / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis; D004261:DNA Replication / Q000187:drug effects*; D006657:Histones / Q000096:biosynthesis / Q000378:metabolism*; D006918:Hydroxyurea / Q000494:pharmacology*; D007700:Kinetics; D008970:Molecular Weight
D002843:Chromatin; D006657:Histones; D004247:DNA; D006918:Hydroxyurea