A new method for the isolation of replicative chromatin: selective deposition of histone on both new and old DNA.
We have developed a new method for isolating subcellular components after fixation of whole cells with formaldehyde. By a number of criteria we establish that the fixation does not alter or cause rearrangement of nucleosomal structure of either newly replicated or old chromatin. Using this approach we can almost completely resolve newly replicated chromatin from preexisting material on the basis of the difference in density. Newly replicated chromatin (even from cycloheximide-treated cells) appears to contain nucleosomes on both daughter strands. Exploiting the ability to separate newly synthesized chromatin, we have reexamined the question of the deposition of new histone at the replication fork. We find that newly synthesized histones H3 and H4 are deposited onto new DNA and stay in place for a significant time. In contrast new H1 is deposited on old DNA and new H2A-H2B, while they may be transiently bound to new DNA, are largely associated with preexisting chromatin.
D000818:Animals; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D002843:Chromatin / Q000302:isolation & purification* / Q000378:metabolism; D004261:DNA Replication; D003851:Deoxyribonucleases / Q000378:metabolism; D005557:Formaldehyde; D006657:Histones / Q000378:metabolism*; D008114:Liver Neoplasms, Experimental / Q000378:metabolism; D008836:Micrococcal Nuclease / Q000378:metabolism; D009707:Nucleosomes / Q000378:metabolism
D002843:Chromatin; D006657:Histones; D009707:Nucleosomes; D005557:Formaldehyde; D003851:Deoxyribonucleases; D008836:Micrococcal Nuclease