CONSERVATIVE SEGREGATION OF TETRAMERIC UNITS OF H3 AND H4 HISTONES DURING NUCLEOSOME REPLICATION

We have specifically investigated the behavior of H3 and H4 histones during the replication cycle of MF-134SC cells. Mononucleosomes obtained from cells density-labeled with IDU or dense amino acids in the presence of appropriate radiolabeled precursors were applied to sucrose gradients containing 0.3 M NaC1 and 4M urea for rate zonal centrifugation. This allowed the resolution of dense and normal subnucleosome particles composed of DNA and two molecules each of H3 and H4 without any measurable interparticle histone exchange. On labeling with dense amino acids and radiolabeled lysine, a distinct peak of radiolabeled dense paericles was obtained. In contrast, pre-radiolabeled H3 and H4 remained in the normal subnucleosome peak region even after one generation time of culturing with dense amino acids. These data indicate the formation of (H3-H4)2 tetramers composed entirely of new H3 and H4 molecules as well as the conservation of prwe-existing tetramemrs. Density lasbeling for 1 h with IdU in the presence of radiolabeled lyside yielded a distinct peak of radiolabeled dense particles, indicatin the deposition of new tetramers on newly replicated DNA. Similar rate zonal analysis of subnucleosome particles obtained from cells prelabeled for 1 h with radiolabeled lysine followed by various IdU-labeling schedules in nonisotopic media yielded data suggesting that tetramers once deposited do not move about randomly during the replication cycle. A possible mode of nucleosome replication is discussed in the light of the present data. © 1990 COPYRIGHT, 1990 BY THE JOURNAL OF BIOCHEMISTRY.