Histone H1 deposition and histone-DNA interactions in replicating chromatin.

Abstract

An immunochemical method for analyzing protein interactions with BrdUrd-substituted DNA was used to study binding of histones to nascent DNA in nuclei. The results indicate that in Ehrlich ascites tumor (EAT) cells, histone Hi deposits on newly replicated DNA simultaneously with or immediately after core histone deposition so that in chromatin replicated for 3 min, the stoichiometry of the histones is the same as in bulk chromatin. All histones, and especially histone Hi, interact with nascent DNA more weakly than with bulk chromatin, although the efficiency of interaction via the globular domains seems to be the same for both types of chromatin.Assembly of eukaryotic chromatin during the postreplication period is a complex, multi-step process (1-3). Apparently, each preexisting nucleosome dissociates into one H3/H4 tetramer and two H2A/H2B dimers, which recombine with newly synthesized histones and segregate randomly to either daughter DNA duplex (for an opposing opinion, see refs. 4-7). Additionally, new histones H3/H4 are deposited on the nascent DNA, while the majority of new H2A/H2B dimers are associated with nonreplicated DNA, and new Hi is randomly distributed over chromatin (1,8). Special protein complexes may target the sequential deposition of histones onto DNA, with histones H3 and H4 joining replicating DNA first, followed by addition of histones H2A and H2B (9-12). The structure of new chromatin during the first 10-20 min after DNA replication is distinguishable from that of bulk chromatin. Nascent chromatin is highly sensitive to nonspecific nucleases (13-18). The newly made nucleosomes are unstable, and they easily slide along DNA during nuclease digestion (19), thereby forming closely packed core particles (20,21) and decreasing the size of the nucleosomal repeat (22). The histones themselves, especially the newly synthesized ones, are bound weakly to nascent DNA (e.g., refs. 19 and 23).The higher order structure of newly replicated chromatin is probably unfolded (12,(24)(25)(26). Maturation of newly replicated chromatin is suggested to be mediated by histone Hi, which is known to play an important role in organization of the chromatosome and of the higher order structures of chromatin (27). However, it is not as yet clear when histone Hi is deposited during maturation. Earlier data obtained on fractionated chromatin were interpreted as indicating that nascent histone Hi appeared in chromatin only 10-20 min after DNA replication (12). As discussed by the authors themselves, however, the results could not unambiguously distinguish between random association to bulk chromatin as opposed to late association to nascent chromatin. Moreover, it was only the behavior of the newly synthesized Hi that was monitored, and, as already known, old histones also participate in the formation of newly replicated chromatin. Further studies using micrococcal nuclease digestion have reported that 2-5 min after DNA replication, the mononucleosomes in nascent chromatin are represented mostly by core particles ...

Modeling Studies of Chromatin Fiber Structure as a Function of DNA Linker Length

“…H1 concentrations and NRL values in those cells rapidly evolve to the values present in mature chromatin [13]. The same behavior was observed in Ehrlich ascites tumor cells (EAT) [14], suggesting a relationship between these factors and certain tumors.…”

Role of linker histone in chromatin structure and function: H1 stoichiometry and nucleosome repeat length

“…Whether this reflects a true reduction in NRL or the high mobility of nascent nucleosomes (Smith et al 1984) is uncertain. However, it is clear that newly replicated chromatin is in a state of rapid flux, and rapidly changes to the mature NRL and H1 content (Annunziato et al 1981, Bavykin et al 1993. Interestingly, in a study of replication in synchronized CHO cells in the presence of hydroxyurea, D' Anna et al (1986) reported that the newly synthesized chromatin had a shortened NRL and was depleted in H1.…”

Alterations in Nucleosome Core Structure in Linker Histone-depleted Chromatin

“…Preparation of Nuclei and Core Particles and Protein-DNA Crosslinking Procedure-Nuclei from sea urchin sperm, chicken erythrocytes, mouse ascites, lily bud sepals, and yeast were isolated as described previously (17,18). Histone-DNA cross-linking, isolation of core particles, purification of cross-linked complexes, and two-dimensional gel electrophoresis of [ 32 P]DNA and 125 I-histone-labeled cross-linked complexes were performed as described (17,19).…”