We plan to continue our studies into the mechanism of histone deposition during DNA replication in SV40 infected and transformed cells. We will further characterize the sites in the genome where the deposition of newly synthesized H2A, H2B occurs. We will utilize newly developed procedures of immunoprecipitation of ubiquinated histone complexes (crosslinked octamers) to determine whether the exchange phenomenon relates to the potential instability of a ubiquitin moiety within a nucleosome. We will use newly developed procedures of nick translation of nuclei with biotinylated nucleotide to isolate active genes on avidin-agarose. We will continue our studies into the exchange phenomenon using density labeled amino acids (15N, 13C, 2H) to label histones, crosslink the histones within the nucleosome into an octameric complex with formaldehyde and analyze the density of the complex. Because of the reversibility of this crosslink, the individual histones can be charaterized within the density fractionated octamers. We will try to perturb the exchange process by varying tempature (cells that grow between 4 and 37 degrees Centigrade) and in treatment with metabolic inhibitors such as actinomycin D. We will continue our studies into the role of superhelical density in regulating histone deposition. Histone deposition on new DNA is extensively inhibited in cells treated with the intercalating agent, chloroquin. This is shown by crosslinking whole cells with formaldehyde, isolating the chromatin and fractionating on density gradients. We will determine the superhelical density and continuity of this new DNA. We will test whether a potential lethal effect of anticancer drugs (those involved in protein mediated strand breakage) is through the inhibition of histone deposition on newly replicated DNA. We will utilize in vitro deposition procedures with polyglutamic acid:histone and DNA (supercoiled and relaxed) to measure rates of histone deposition (newly synthesized and old) and to measure the effect of nucleosome structure on the interaction of transcription factor, ICP4, with targe DNA sequences. These combined studies will provide a clearer understanding into the role of histones in the regulation of replication and transcription.