Structure of Chromatin at Deoxyribonucleic Acid Replication Forks: Nuclease Hypersensitivity Results from both Prenucleosomal Deoxyribonucleic Acid and an Immature Chromatin Structure

Relative to nonreplicating DNA in mature simian virus 40 (SV40) chromosomes, newly synthesized DNA in replicating SV40 chromosomes was found to be hypersensitive to the nonspecific endonucleases, micrococcal nuclease (MNase), DNase I, and DNase II. Nascent DNA, pulse labeled in either intact cells or nuclear extracts supplemented with cytosol, was digested about 5-fold faster and about 25% more extensively than uniformly labeled DNA in mature viral chromosomes. Pulse-chase experiments in vitro revealed a time-dependent chromatin maturation process that involved two distinct steps: (i) conversion of prenucleosomal DNA (PN-DNA) into immature nucleosomal oligomers and (ii) maturation of newly assembled chromatin into a structure with increased nuclease resistance. PN-DNA was hypersensitive to MNase, releasing short DNA fragments which were subsequently solubilized by the nuclease. However, when the nascent PN-DNA was specifically removed by digestion of replicating viral chromosomes with Escherichia coli exonuclease III (3′–5′) and phage T7 exonuclease (5′–3′), subsequent digestion of the remaining chromatin with MNase revealed the same degree of hypersensitivity observed prior to exonuclease treatment. Furthermore, newly assembled nucleosomal oligomers, isolated after a brief MNase digestion of replicating viral chromosomes, were also hypersensitive to MNase relative to oligomers isolated from mature chromosomes. Hybridization analysis of the DNA in these immature oligomers revealed that it originated from both sides of replication forks. Inhibition of DNA polymerase α by aphidicolin inhibited conversion of PN-DNA into nucleosomes but did not inhibit loss of nucleosomal hypersensitivity to MNase. In contrast, components in the soluble fraction of the subcellular system (“cytosol”) were required for both DNA replication and chromatin maturation. Analysis of the nucleoprotein products from a MNase digestion of replicating and mature SV40 chromosomes failed to detect a change in nucleosome structure that corresponded to the loss of nuclease hypersensitivity. However, the results presented demonstrate that both PN-DNA and newly assembled immature chromatin, present on both arms of SV40 replication forks, contribute to the commonly observed hypersensitivity of newly replicated chromatin to endonucleases.