Disruption of the nucleosomes at the replication fork.

Abstract

The fate of parental nucleosomes during chromatin replication was studied in vitro using in vitro assembled chromatin containing the whole SV40 genome as well as salt‐treated and native SV40 minichromosomes. In vitro assembled minichromosomes were able to replicate efficiently in vitro, when the DNA was preincubated with T‐antigen, a cytosolic S100 extract and three deoxynucleoside triphosphates prior to chromatin assembly, indicating that the origin has to be free of nucleosomes for replication initiation. The chromatin structure of the newly synthesized daughter strands in replicating molecules was analysed by psoralen cross‐linking of the DNA and by micrococcal nuclease digestion. A 5‐ and 10‐fold excess of protein‐free competitor DNA present during minichromosome replication traps the segregating histones. In opposition to published data this suggests that the parental histones remain only loosely or not attached to the DNA in the region of the replication fork. Replication in the putative absence of free histones shows that a subnucleosomal particle is randomly assembled on the daughter strands. The data are compatible with the formation of a H3/H4 tetramer complex under these conditions, supporting the notion that under physiological conditions nucleosome core assembly on the newly synthesized daughter strands occurs by the binding of H2A/H2B dimers to a H3/H4 tetramer complex.

DNA Looping Mediates Nucleosome Transfer

“…In contrast, our data of the first transfer distance peaked at 500-700 bp, and do not support histone dissociation and diffusion. Previous studies also provide evidence indicating that during replication, parental histones are not released into solution 7,8,22 , arguing against a diffusion-based mechanism. Fig.…”

“…During DNA replication, dsDNA available for nucleosome transfer is located behind the replication fork on the nascent daughter duplexes, which are poised to accept parental nucleosomes from ahead of the replication fork 22,34,35 . We hypothesize that if nucleosome transfer is dictated by DNA loop formation, transfer should take place on the upstream dsDNA, in a similar manner as demonstrated for the downstream dsDNA.…”

Nucleosome assembly and epigenetic inheritance

“…In contrast, data from some other groups suggest the parental octameric structures are disrupted during the passage of replication fork (Sogo et al, 1986;Fotedar and Roberts, 1989), and some H2A/H2B dimers dissociate from replicating DNA (Jackson, 1987(Jackson, , 1990. Finally, using an improved in vitro replication method, Gruss et al clarified the debate and provided further insights (Gruss et al, 1993). The authors concluded that, a subnucleosomal particle, probably the (H3/H4) 2 tetramer is the transfer unit, which, then assembled with two H2A/H2B dimers to form an intact nucleosome.…”

“…The authors concluded that, a subnucleosomal particle, probably the (H3/H4) 2 tetramer is the transfer unit, which, then assembled with two H2A/H2B dimers to form an intact nucleosome. In addition, 5-10 folds of competitor DNA during replication traps histones transferred to daughter strands, suggests that the histones may only loosely associated with replicating DNA (Gruss et al, 1993). However, a few open questions remain, such as what are the chaperon factors that assist the disassembly/reassembly process of the parent nucleosomes and how they coordinate with chaperons facilitate the deposition of newly synthesized histones.…”

“…The restoration of chromatin structure behind of replication fork can be divided into several steps. First, assembly begins with the incorporation of H3/H4 tetramer, which is followed by the addition of two H2A/H2B dimmers, to form a complete nucleosome structure (Jackson, 1987(Jackson, , 1990Gruss et al, 1993). The incorporation of linker histone was detected 450-650 bp after replication fork passage (Gasser et al, 1996), enabling further folding of higher order structures.…”