The long-term objective of the applicant's research program is to elucidate the mechanisms of somatic chromatin biosynthesis, and to understand the propagation of epigenetic information to progeny cells. To this end, the present proposal focuses on studying histone-DNA interactions during chromatin replication and assembly in vitro. Experiments will be performed using cytoplasmic and nuclear exercise from somatic cells and sources of replication and assembly factors. The assembly substrate will be histone-depleted, nascent nuclear DNA< as well as purified DNA plasmids. In the initial phase, the appropriate DNA structure, histone modification state, and reaction conditions for in vitro assembly will be determined, based on the criteria of increased nuclease resistance, generation of a nucleosomal ladder, change in plasmid superhelical density, and electrophoretic migration of DNA protein complexes. Next, the topoisomerase requirements for the assembly of newly replicated DNA in vitro will be examine,d using camptothecin and VM-26 (specific inhibitors of eukaryotic DNA topoisomerases I and II, respectively). Once the parameters of assembly have been established, the influence of core histone acetylation on the deposition of histone H1 will be investigated, and the effects of H1 on assembly in vitro examined. As an integral part of these studies, the acetylation level of nascent nucleosomes containing segregated parental histones will be measured, using i) an antiserum that specifically recognizes the acetylated form of histone H4, and ii) a chemically cleavable biotinylated nucleotide. The [acetylated]H4-antiserum will also be used to identify possible nucleosome """"""""assembly factors"""""""" and histone escort complexes, by immunopredipitating radiolabeled cell extracts. Using the same technique, the ability of histones and DNA to form pre-nucleosomal assembly intermediates in partial reactions will also be tested.
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