3-DIMENSIONAL RECONSTRUCTIONS FROM CRYOELECTRON MICROSCOPY IMAGES REVEAL AN INTIMATE COMPLEX BETWEEN HELICASE DNAB AND ITS LOADING PARTNER DNAC

Background: DNA helicases play a fundamental role in all aspects of nu cleic acid metabolism and defects in these enzymes have been implicate d in a number of inherited human disorders. DnaB is the major replicat ive DNA helicase in Escherichia coli and has been used as a model syst em for studying the structure acid function of hexameric helicases, Th e native protein is a hexamer of identical subunits, which in solution forms a complex with six molecules of the loading protein DnaC. DnaB is delivered from this complex onto the DNA template, with the subsequ ent release of DnaC, We report here the structures of the DnaB helicas e hexamer and its complex with DnaC under a defined set of experimenta l conditions, as determined by three-dimensional cryoelectron microsco py, It was hoped that the structures would provide insight into the me chanisms of helicase activity. Results: The DnaB structure reveals tha t six DnaB monomers assemble as three asymmetric dimers to form a pola r, ring-like hexamer, The hexamer has two faces, one displaying threef old and the other sixfold symmetry. The six DnaC protomers bind tightl y to the sixfold face of the DnaB hexamer. This is the first report of a three-dimensional structure of a helicase obtained using cryoelectr on microscopy, and the first report of the structure of a helicase in complex with a loading protein. Conclusions: The structures of the Dna B helicase and its complex with DnaC reveal some interesting structura l features relevant to helicase function and to the assembly of the tw o-protein complex, The results presented here provide a basis for a mo re complete understanding of the structure and function of these impor tant proteins.