Physical Basis for the Loading of a Bacterial Replicative Helicase onto DNA

• Published in: Molecular cell Vol. 74; no. 1; pp. 173 - 184.e4
• Main Authors: Arias-Palomo, Ernesto, Puri, Neha, O’Shea Murray, Valerie L., Yan, Qianyun, Berger, James M.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 04-04-2019
• Subjects: Adenosine Triphosphate - metabolism; Binding Sites; Cryoelectron Microscopy; DNA Primase - genetics; DNA Primase - metabolism; DNA Replication; DNA, Bacterial - biosynthesis; DNA, Bacterial - chemistry; DNA, Bacterial - genetics; DnaB Helicases - chemistry; DnaB Helicases - genetics; DnaB Helicases - metabolism; Escherichia coli - enzymology; Escherichia coli - genetics; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Gene Expression Regulation, Bacterial; Hydrolysis; Models, Molecular; Nucleic Acid Conformation; Protein Binding; Protein Conformation; Structure-Activity Relationship
• ISSN: 1097-2765; 1097-4164
• DOI: 10.1016/j.molcel.2019.01.023

In cells, dedicated AAA+ ATPases deposit hexameric, ring-shaped helicases onto DNA to initiate chromosomal replication. To better understand the mechanisms by which helicase loading can occur, we used cryo-EM to determine sub-4-Å-resolution structures of the E. coli DnaB⋅DnaC helicase⋅loader complex with nucleotide in pre- and post-DNA engagement states. In the absence of DNA, six DnaC protomers latch onto and crack open a DnaB hexamer using an extended N-terminal domain, stabilizing this conformation through nucleotide-dependent ATPase interactions. Upon binding DNA, DnaC hydrolyzes ATP, allowing DnaB to isomerize into a topologically closed, pre-translocation state competent to bind primase. Our data show how DnaC opens the DnaB ring and represses the helicase prior to DNA binding and how DnaC ATPase activity is reciprocally regulated by DnaB and DNA. Comparative analyses reveal how the helicase loading mechanism of DnaC parallels and diverges from homologous AAA+ systems involved in DNA replication and transposition.
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•DnaC remodels intra- and inter-DnaB contacts to drive ring opening and DNA binding•DNA activates DnaC ATPase activity and both remodels and reseals the DnaB ring•Following DNA binding, DnaB adopts a translocation-competent conformation
Arias-Palomo et al. present the cryo-EM structures of a replicative bacterial helicase-loader complex (E. coli DnaBC) in pre- and post-loading states, revealing how the loader breaks the helicase ring to deposit it at the origin of replication and how ssDNA engagement closes and activates the helicase.