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

Abstract

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.Supplemental Information includes seven figures and six videos and can be found with this article online at https://doi.

A structural view of bacterial DNA replication

“…Observed by cryo‐EM, the E. coli DnaB 6 .DnaC 6 complex forms a three‐tiered assembly in which DnaC N‐terminal domain, binding to the DnaB‐CTD adopts a spiral configuration that produces a break in DnaB, allowing mounting onto ssDNA [Fig. (d)] . ATPase activity of DnaC is not required for ring opening or loading of DnaB onto ssDNA .…”

“…3(d)]. 44 ATPase activity of DnaC is not required for ring opening or loading of DnaB onto ssDNA. 45 Instead, DnaC bound to ATP appears to stabilize the open spiral conformation of the complex.…”

The molecular coupling between substrate recognition and ATP turnover in a AAA+ hexameric helicase loader

“…DnaC-type loaders help place DnaB hexamers on single-stranded DNA by opening the helicase ring and allowing a nucleic acid strand to enter laterally into the pore of the complex (Arias-Palomo et al, 2019). In the context of DnaB, six DnaC protomers form a cracked hexameric oligomer, generating composite ATPase active sites at five of the six subunit interfaces (Fig.…”

“…1B,C) (Arias-Palomo et al, 2013, 2019. The AAA+ fold of DnaC binds ssDNA directly, at a site distal from its ATP-binding locus (Arias-Palomo et al, 2019;Ioannou et al, 2006;Mott et al, 2008). Prior to binding DNA, six DnaC protomers latch onto a DnaB hexamer through their HBDs to promote helicase ring opening.…”

“…Prior to binding DNA, six DnaC protomers latch onto a DnaB hexamer through their HBDs to promote helicase ring opening. In this configuration, EM studies have shown that DnaB resides in an ADP-like state, and DnaC in an ATP-bound form (Arias-Palomo et al, 2019). DNA binding induces a large conformational rearrangement in the DnaB hexamer and compresses the DnaC ATPase ring, switching the ATPase status of the helicase and the loader into ATP and ADP-forms, respectively.…”