title: Structural basis for replication origin unwinding by an initiator primase of plasmid ColE2-P9: duplex DNA unwinding by a single protein.
abstract: Duplex DNA is generally unwound by protein oligomers prior to replication. The Rep protein of plasmid ColE2-P9 (34 kDa) is an essential initiator for plasmid DNA replication. This protein binds the replication origin (Ori) in a sequence-specific manner as a monomer and unwinds DNA. Here we present the crystal structure of the DNA-binding domain of Rep (E2Rep-DBD) in complex with Ori DNA. The structure unveils the basis for Ori-specific recognition by the E2Rep-DBD and also reveals that it unwinds DNA by the concerted actions of its three contiguous structural modules. The structure also shows that the functionally unknown PriCT domain, which forms a compact module, plays a central role in DNA unwinding. The conservation of the PriCT domain in the C termini of some archaeo-eukaryotic primases indicates that it probably plays a similar role in these proteins. Thus, this is the first report providing the structural basis for the functional importance of the conserved PriCT domain and also reveals a novel mechanism for DNA unwinding by a single protein.
authors.affiliation: From the Structural Biology Center, National Institute of Genetics, Yata 1111, Mishima, Shizuoka 411-8540, Japan, hitou@nig.ac.jp.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Itou
authors.affiliation: the Department of Cell Genetics, National Institute of Genetics, Yata 1111, Mishima, Shizuoka 411-8540, Japan, and.
authors.forename: Masaru
authors.initials: M
authors.lastname: Yagura
authors.affiliation: From the Structural Biology Center, National Institute of Genetics, Yata 1111, Mishima, Shizuoka 411-8540, Japan.
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Shirakihara
authors.affiliation: the Department of Biology, Faculty of Science, Shinshu University, Asahi 3-1-1, Matsumoto 390-8621, Japan.
authors.forename: Tateo
authors.initials: T
authors.lastname: Itoh
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D004265:DNA Helicases / Q000737:chemistry* / Q000378:metabolism; D019915:DNA Primase / Q000737:chemistry* / Q000378:metabolism; D004269:DNA, Bacterial / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D018741:Replication Origin; D015534:Trans-Activators / Q000737:chemistry* / Q000378:metabolism
chemical_list: D004269:DNA, Bacterial; D015534:Trans-Activators; C045264:replication initiator protein; D019915:DNA Primase; D004265:DNA Helicases
keywords: Crystal Structure; DNA Replication; DNA Replication Initiator; DNA Unwinding; Plasmid; PriCT Domain; Protein-DNA Interaction; X-ray Crystallography