Journal: Molecular Microbiology
Article Title: Loading mechanisms of ring helicases at replication origins
Figure Lengend Snippet: A. Helicase loader-mediated loading in A. aeolicus . A schematic diagram showing directional helicase loader-mediated and DnaA-mediated loading mechanisms on the 5′ and 3′ strands, respectively, based upon the A. aeolicus system ( Duderstadt et al ., 2011 ). The DnaA filament is shown to wrap double-stranded DNA around the outside with flexible helicase-interacting domains (domain I) projecting out from the filament. The filament extends into the 5′ strand of the melted DUE but in this case with the ssDNA in the interior of the filament. The helicase loader (green) forms a continuous heterofilament with the ATP-end of the DnaA-ssDNA filament, by docking the arginine from its Box VII into the ATP binding site of the DnaA, and delivers the helicase (brown) in the correct orientation onto the 5′ strand. The interactions of the flexible N-terminal DnaA domains with the helicase deliver it onto the 3′ strand in the opposite direction. A side-view of the Bacillus stearothermophilus DnaB helicase is shown with the characteristic two-tier (N-terminal and C-terminal tiers) ring structure of bacterial helicases ( Bailey et al ., 2007 ). B. Organization of the E. coli oriC . The relative positions of DnaA binding sites (R, I and τ sites), NAP ( N ucleoid A ssociated P roteins) binding sites for IHF and FIS and the DUE are indicated. Binding of IHF between the R1 and R5 sites sharply bends the DNA, as indicated in the inset showing the crystal structure of IHF binding and bending double-stranded DNA. C. DnaA-mediated helicase loading in E. coli . A schematic model showing how two ring helicases are directionally loaded onto the E. coli oriC ( Ozaki and Katayama, 2012 ). The DUE is cooperatively melted via binding of DnaA and IHF and the first helicase is loaded onto the bottom (A-rich) strand directionally. Subsequent translocation forward (in the 5′–3′ direction) melts a larger segment of the duplex allowing loading of the second helicase in the top (T-rich) strand and in the opposite direction. In both cases loading is mediated by the flexible N-terminal helicase-interacting domains of DnaA projecting out of the filament. For the sake of simplicity no helicase loader is depicted but it may participate indirectly in the process by binding onto the C-terminal tier of the helicase ring forcing opening of the ring for loading to proceed.
Article Snippet: Structural distortions imposed on the DNA double helix and a squeeze-pump ATP-driven action of the two head-to-head hexameric helicases pulling the DNA in opposite directions force melting of the double helix (reviewed in ).
Techniques: Binding Assay, Immunohistofluorescence, Translocation Assay