DESCRIPTION Recent findings suggest that the binding of the linker histone H1 to DNA may be more specific than previously believed, involving recognition of either certain nucleotide sequences or certain structural features of DNA (e.g. supercoils, 4-way junctions). The foreign collaborator recently discovered two plasmid restriction fragments that exhibit unusually high affinity for H1, based on results obtained with a simple gel shift electrophoresis assay. Preliminary studies of these H1 "hypersites" suggest that DNA curvature as well as specific sequences play a role in the high affinity binding. The proposed collaboration includes experiments which will test the hypothesis that the hypersite-containing DNA fragments form loops which place high-affinity sequences at or near a DNA cross-over. Initial experiments will verify the requirement for DNA curvature in H1-binding DNA fragments. Loop formation will be evaluated indirectly by gel electrophoresis and directly by scanning force microscopy. Two approaches will be used to determine if the binding of H1 to DNA hypersites is cooperative: first, by Western blotting to localize H1 in the gel shift assay, and second, by analytical ultracentrifugation. A third specific aim is to map the putative specific sequences involved in tight H1 binding; this will be attempted using a DNase protection assay. The final specific aim is to search for H1 hypersites in selected eukaryotic genome regions using the gel shift assay and to characterize these sites using the approach applied to the hypersite-containing plasmid fragments.