Abstract
The fate of parental nucleosomes during the replication of chromatin templates was studied using a modification of the cell-free SV40 DNA replication system. Plasmid DNA molecules containing the SV40 origin were assembled into chromatin with purified core histones and fractionated assembly factors derived from HeLa cells. When these templates were replicated in vitro, the resulting progeny retained a nucleosomal organization. To determine whether the nucleosomes associated with the progeny molecules resulted from displacement of parental histones during replication followed by reassembly, the replication reactions were performed in the presence of control templates. It was observed that the progeny genomes resulting from the replication of chromatin templates retained a nucleosomal structure, whereas the progeny of the control DNA molecules were not assembled into chromatin. Additional experiments, involving direct addition of histones to the replication reaction mixtures, confirmed that the control templates were not sequestered in some form which made them unavailable for nucleosome assembly. Thus, our data demonstrate that parental nucleosomes remain associated with the replicating molecules and are transferred to the progeny molecules without displacement into solution. We propose a simple model in which nucleosomes ahead of the fork are transferred intact to the newly synthesized daughter duplexes.
Highlights
The fate of parental nucleosomes during the repli- to this question will require a detailed understanding of how cation of chromatintemplates was studiedusing a mod- chromatin is assembled on progeny DNA duplexes following ification of the cell-free SV40DNA replication system
Parental nucleosomes histones to the replication reaction mixtures, con- could be completely displaced during passage of the fork and firmed that the control templates were not sequestered reassemble at random on the the daughter duplexes
Tal nucleosomesremain associated with thereplicating molecules and are transferred to the progeny molecules without displacement into solution
Summary
Containsan SV40 replication origin sequence identical to that of pKP55.HNO. Relaxation of DNATemplates-50 pg of negatively supercoiled plates for replication, we developed a cell-free assembly sysplasmid (pLC106,pKP55.HN0, or pKP55) was relaxed by incubation tem from HeLa cells dependent on the addition of exogenous with 2 units/pgcalf thymus DNA topoisomerase I The concentration of histones used to assemble 250 ng of DNA isindicated by the Omission of creatine phosphate, the major contributor to the ionic strength of the reaction mixture, decreased the efficiency of assembly markedly (eighth lane).Previous studies of chromatin assembly in whole cell extracts of HeLa cells had noted that M P was not required but that maximal assembly OC-. Plasmid DNA incubated in anassembly reaction mixture lacking added histones was digested to completion by incubationwith 1 unit of micrococcal nuclease for 20 min (lanes 1-4). These data indicate that tcheell-free The products of replication of the chromatin templates system that we have developed is capable of assembling plas- were visualized by autoradiography (Fig. 3A). We carried out containing theSV40 origin (pLC106)were preassembled into numerous restriction digests of the product DNA to test for
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