Deposition of histone onto the replicating chromosome: newly synthesized histone is not found near the replication fork.

Abstract

Communicated by David M. Prescott, April 19,1976 ABSRACr We have studied the site of deposition of newly synthesized histone. It appears to be randomly distributed over the chromosomal material and does not become associated specifically with immediately post-replicational DNA, nor is it deposited in discrete continuous regions distal to the sites of DNA synthesis. The newly synthesized DNA, however, rapidly acquires a complement of chromosomal proteins; presumably, preexisting histones must migrate to become associated with post-replicational DNA. Histones are synthesized in the cytoplasm (1, 2) of dividing cells in S phase (3,4). They are subsequently transferred into the nucleus with great rapidity (5-8). Both preexisting and new histones become associated with the doubled complement of chromosome DNA in a random manner (9), so that previous interactions with specific strands or DNA molecules are not necessarily maintained. A priori, it seemed likely that incoming histone would become associated with newly synthesized DNA either just ahead of or just after the replication fork. In view of this likelihood, a number of theories have been proposed for histone F1 phosphorylation which occurs within a few minutes after admission to the nucleus. These theories deal with changes in histone and chromatin conformation required for replication which are likely to occur in the neighborhood of the replication fork. This seemed quite feasible if DNA replication occurred on the periphery of the nucleus as had been suggested by earlier work (10, 11). However, the clear-cut demonstration that DNA synthesis is fully active within the body of the nucleus posed fascinating problems of how the positively charged, incoming histones could circumnavigate the bulk of the chromosomal material on its passage to an inner replication fork (12, 13).We have asked specific questions concerning the site of deposition of incoming histones and we will argue that newly synthesized histones do not in fact become associated specifically with DNA in the region of the replication forks, but rather become randomly associated with the bulk chromatin. We further conclude that the newly synthesized DNA assumes its full complement of histone from preexisting molecules which presumably migrate from other chromosomal sites. This migration is possibly triggered by the incoming flux of histone into the nucleus. For the 30-sec pulse experiments, the cells were concentrated to a cell density of 4 X 107 cells per ml (20 ml total volume) prior to incubation with 2.0 mCi of either [3H]lysine or [3H]thymidine. The incubation was stopped by addition to 180 ml of cold medium; the cells were washed once and then resuspended in warm medium at a cell density of 4 X 105 cells per ml for the chase. After the cells had been harvested, they were washed once in an equal volume of 0.1 M sucrose, 0.2 mM Na2HPO4, pH 7.4, at 40 and quickly frozen in dry ice-ethanol. Identical results were observed if the cells were not frozen prior to isolation of chromatin, which indica...

Global Regulation of Post-translational Modifications on Core Histones

“…Likewise, previous mapping identified serine 1 as the main phosphorylated residue on H4, and this site is most likely occupied in the 11,083-Da form. H4 acetylation at lysines 5 and 12 and phosphorylation of serine 1 have been suggested to be cytoplasmic events occurring on free histones (32)(33)(34)(35). However, our isolation procedure involves precipitation of DNA-associated proteins; therefore the phosphorylation and deacetylation events we observe occur on chromatin.…”

Monomethylation of Lysine 20 on Histone H4 Facilitates Chromatin Maturation

“…The progression of the DNA replication machinery disrupts the nucleosome in front of the replication fork, which is then reassembled onto the newly synthesized DNA strands in a random manner (15,20). The remaining gaps are subsequently filled up with newly translated histone molecules, leading to a mosaic pattern of new and old histone octamers (2,21). This process is assisted by the action of nuclear chaperones, which bind to the histones before deposition (14,34).…”

“…The newly deposited histones are more loosely associated with the DNA than the bulk histones and mature slowly into a more stable chromatin structure. Assembly is achieved via an ordered deposition of H3 and H4, followed by the binding of H2A/H2B dimers and finally the interaction of the linker histone H1 with the chromatin fiber (19)(20)(21)(22)49).Posttranslational modifications of histone molecules are generally considered to play an important role during the establishment and maintenance of chromatin structures. The combination of histone modifications has been proposed to constitute a "histone code" (23, 55), which is involved in the maintenance of epigenetic information.…”

“…The newly deposited histones are more loosely associated with the DNA than the bulk histones and mature slowly into a more stable chromatin structure. Assembly is achieved via an ordered deposition of H3 and H4, followed by the binding of H2A/H2B dimers and finally the interaction of the linker histone H1 with the chromatin fiber (19)(20)(21)(22)49).…”

Stimulation of DNA Replication from the Polyomavirus Origin by PCAF and GCN5 Acetyltransferases: Acetylation of Large T Antigen

“…MNase digestion of extracts of TSPy5 virusinfected Cos7 cells indicated that the TSPy5 genome was assembled into chromatin, and restriction enzyme analysis of TSPy5 minichromosomes revealed that the Py ori core region was occupied by nucleosomes (data not shown). To inhibit extensive DNA replication and SV40 virion assembly that might disrupt histone modifications caused by Gal4 fusion proteins expressed in the Cos7 cells (49), aphidicolin was added to the cells at 16 h after infection. To validate ChIP assays as a means to study TSPy5 chromatin acetylation, we analyzed whether TSA treatment of Cos7 cells infected with TSPy5 viruses changed the acetylation status of TSPy5 chromatin.…”