DISRUPTION OF THE NUCLEOSOMES AT THE REPLICATION FORK

The fate of parental nucleosomes during chromatin replication was stud ied in vitro using in vitro assembled chromatin containing the whole S V40 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 as sembly, indicating that the origin has to be free of nucleosomes for r eplication initiation. The chromatin structure of the newly synthesize d daughter strands in replicating molecules was analysed by psoralen c ross-linking of the DNA and by micrococcal nuclease digestion. A 5- an d 10-fold excess of protein-free competitor DNA present during minichr omosome replication traps the segregating histones. In opposition to p ublished data this suggests that the parental histones remain only loo sely or not attached to the DNA in the region of the replication fork. Replication in the putative absence of free histones shows that a sub nucteosomal particle is randomly assembled on the daughter strands. Th e data are compatible with the formation of a H3/H4 tetramer complex u nder 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 co mplex.