title: EM structure of a helicase-loader complex depicting a 6:2 binding sub-stoichiometry from Geobacillus kaustophilus HTA426.
abstract: During DNA replication, bacterial helicase is recruited as a complex in association with loader proteins to unwind the parental duplex. Previous structural studies have reported saturated 6:6 helicase-loader complexes with different conformations. However, structural information on the sub-stoichiometric conformations of these previously-documented helicase-loader complexes remains elusive. Here, with the aid of single particle electron-microscopy (EM) image reconstruction, we present the Geobacillus kaustophilus HTA426 helicase-loader (DnaC-DnaI) complex with a 6:2 binding stoichiometry in the presence of ATPγS. In the 19 Å resolution EM map, the undistorted and unopened helicase ring holds a robust loader density above the C-terminal RecA-like domain. Meanwhile, the path of the central DNA binding channel appears to be obstructed by the reconstructed loader density, implying its potential role as a checkpoint conformation to prevent the loading of immature complex onto DNA. Our data also reveals that the bound nucleotides and the consequently induced conformational changes in the helicase hexamer are essential for active association with loader proteins. These observations provide fundamental insights into the formation of the helicase-loader complex in bacteria that regulates the DNA replication process.
authors.affiliation: Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan.
authors.forename: Yen-Chen
authors.initials: YC
authors.lastname: Lin
authors.affiliation: Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan; Molecular Cell Biology, Taiwan International Graduate Program, Institute of Molecular Biology, Academia Sinica, and Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan.
authors.forename: Vankadari
authors.initials: V
authors.lastname: Naveen
authors.affiliation: Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan; Molecular Cell Biology, Taiwan International Graduate Program, Institute of Molecular Biology, Academia Sinica, and Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan. Electronic address: hsiao@gate.sinica.edu.tw.
authors.forename: Chwan-Deng
authors.initials: CD
authors.lastname: Hsiao
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry*; D001665:Binding Sites; D004265:DNA Helicases / Q000737:chemistry*; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry; D056506:Geobacillus / Q000201:enzymology*; D006868:Hydrolysis; D007091:Image Processing, Computer-Assisted; D008854:Microscopy, Electron; D009711:Nucleotides / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary
chemical_list: D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; C071702:DnaC protein, E coli; C448302:DnaI protein, Bacillus subtilis; D029968:Escherichia coli Proteins; D009711:Nucleotides; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D004265:DNA Helicases
keywords: DNA replication; Electron-microscopy structure; Helicase-loader complex; Primosome assembly; ssDNA loading