Biochemical and electron microscopic image analysis of the hexameric E1 helicase

DNA replication initiator proteins bind site specifically to origin sites a nd in most cases participate in the early steps of unwinding the duplex. Th e papillomavirus preinitiation complex that assembles on the origin of repl ication is composed of proteins E1 and the activator protein E2, E2 is an a ncillary factor that increases the affinity of E1 for the ori site through cooperative binding. Here we show that duplex DNA affects E1 (in the absenc e of E2) to assemble into an active hexameric structure. As a 10-base oligo nucleotide can also induce this oligomerization, it seems likely that DNA b inding allosterically induces a conformation that enhances hexamers, E1 ass embles as a bi-lobed, presumably double hexameric structure on duplex DNA a nd can initiate bi-directional unwinding from an ori site. The DNA takes an apparent straight path through the double hexamers. Image analysis of E1 h exameric rings shows that the structures are heterogeneous and have either a 6- or 3-fold symmetry. The rings are about 40-50 Angstrom thick and 125 A ngstrom in diameter. The density of the central cavity appears to be a vari able and we speculate that a plugged center may represent a conformational flexibility of a subdomain of the monomer, to date unreported for other hex americ helicases.