title: Structural basis for DNA strand separation by a hexameric replicative helicase.
abstract: Hexameric helicases are processive DNA unwinding machines but how they engage with a replication fork during unwinding is unknown. Using electron microscopy and single particle analysis we determined structures of the intact hexameric helicase E1 from papillomavirus and two complexes of E1 bound to a DNA replication fork end-labelled with protein tags. By labelling a DNA replication fork with streptavidin (dsDNA end) and Fab (5' ssDNA) we located the positions of these labels on the helicase surface, showing that at least 10 bp of dsDNA enter the E1 helicase via a side tunnel. In the currently accepted 'steric exclusion' model for dsDNA unwinding, the active 3' ssDNA strand is pulled through a central tunnel of the helicase motor domain as the dsDNA strands are wedged apart outside the protein assembly. Our structural observations together with nuclease footprinting assays indicate otherwise: strand separation is taking place inside E1 in a chamber above the helicase domain and the 5' passive ssDNA strands exits the assembly through a separate tunnel opposite to the dsDNA entry point. Our data therefore suggest an alternative to the current general model for DNA unwinding by hexameric helicases.
authors.affiliation: Department of Biological Sciences, Birkbeck College, Institute of Structural and Molecular Biology, Malet Street, London WC1E 7HX, UK.
authors.forename: Yuriy
authors.initials: Y
authors.lastname: Chaban
authors.affiliation: Academic Unit of Molecular Oncology, University of Sheffield Medical School, Beech Hill Road, Sheffield S10 2RX, UK.
authors.forename: Jonathan A
authors.initials: JA
authors.lastname: Stead
authors.affiliation: Department of Biological Sciences, Birkbeck College, Institute of Structural and Molecular Biology, Malet Street, London WC1E 7HX, UK.
authors.forename: Ksenia
authors.initials: K
authors.lastname: Ryzhenkova
authors.affiliation: Departament of Biolody, University of York, York YO10 5DD, UK.
authors.forename: Fiona
authors.initials: F
authors.lastname: Whelan
authors.affiliation: Department of Biological Sciences, Birkbeck College, Institute of Structural and Molecular Biology, Malet Street, London WC1E 7HX, UK.
authors.forename: Ekaterina P
authors.initials: EP
authors.lastname: Lamber
authors.affiliation: Departament of Biolody, University of York, York YO10 5DD, UK.
authors.forename: Alfred
authors.initials: A
authors.lastname: Antson
authors.affiliation: Academic Unit of Molecular Oncology, University of Sheffield Medical School, Beech Hill Road, Sheffield S10 2RX, UK c.m.sanders@sheffield.ac.uk.
authors.forename: Cyril M
authors.initials: CM
authors.lastname: Sanders
authors.affiliation: Department of Biological Sciences, Birkbeck College, Institute of Structural and Molecular Biology, Malet Street, London WC1E 7HX, UK e.orlova@mail.cryst.bbk.ac.uk.
authors.forename: Elena V
authors.initials: EV
authors.lastname: Orlova
mesh_terms: D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004265:DNA Helicases / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D008958:Models, Molecular; D027383:Papillomaviridae / Q000201:enzymology; D017434:Protein Structure, Tertiary
chemical_list: D004277:DNA, Single-Stranded; D004247:DNA; D004265:DNA Helicases