DnaC, the indispensable companion of DnaB helicase, controls the accessibility of DnaB helicase by primase

Abstract

Former studies relying on hydrogen/deuterium exchange analysis suggest that DnaC bound to DnaB alters the conformation of the N-terminal domain (NTD) of DnaB to impair the ability of this DNA helicase to interact with primase. Supporting this idea, the work described herein based on biosensor experiments and enzyme-linked immunosorbent assays shows that the DnaB-DnaC complex binds poorly to primase in comparison with DnaB alone. Using a structural model of DnaB complexed with the C-terminal domain of primase, we found that Ile-85 is located at the interface in the NTD of DnaB that contacts primase. An alanine substitution for Ile-85 specifically interfered with this interaction and impeded DnaB function in DNA replication, but not its activity as a DNA helicase or its ability to bind to ssDNA. By comparison, substitutions of Asn for Ile-136 (I136N) and Thr for Ile-142 (I142T) in a subdomain previously named the helical hairpin in the NTD of DnaB altered the conformation of the helical hairpin and/or compromised its pairwise arrangement with the companion subdomain in each brace of protomers of the DnaB hexamer. In contrast with the I85A mutant, the latter were defective in DNA replication due to impaired binding to both ssDNA and primase. In view of these findings, we propose that DnaC controls the ability of DnaB to interact with primase by modifying the conformation of the NTD of DnaB.

Mechanisms of opening and closing of the bacterial replicative helicase

“…Structural analysis of the G. stearothermophilus hexameric helicase bound to the G. subtilis DnaI loading factor and the DnaG primase revealed a closed helicase ring; this finding provides support for a potential ring-making role for DnaI (Liu et al, 2013). This work, and that of many other groups (Kobori and Kornberg, 1982; Mallory et al, 1990; Learn et al, 1997; Stephens and McMacken, 1997; Davey et al, 2002; Davey and O'Donnell, 2003; Strycharska et al, 2013; Chodavarapu et al, 2015; Felczak et al, 2017), has established that, although both DnaC and λP are ring-breaking helicase loaders, they operate via mechanisms that are similar, but also different. Both are essential proteins (Wechsler, 1975; Echols and Murialdo, 1978).…”

DnaB helicase is recruited to the replication initiation complex via binding of DnaA domain I to the lateral surface of the DnaB N-terminal domain

“…1A), which is called the ssDUE recruitment mechanism and is a prerequisite for the successive loading of the replicative helicase DnaB onto the ssDNA region (1,(8)(9)(10)(11)(12). The DnaC loader is an AAA1 ATPase that forms a stable complex with DnaB and promotes the ssDNA interaction and conformational change of DnaB, making it competent for loading onto ssDNA (4,(13)(14)(15)(16)(17).…”

“…DnaC monomers bind DnaB, yielding a stable complex that facilitates a conformational change in DnaB that enables it to encircle ssDNA, thereby activating the loading of DnaB onto ssDNA (13,14,16,17). When DnaB is loaded onto ssDNA, DnaC is dissociated, a process that is stimulated by DnaG binding (14,15,40). Loaded DnaB progresses on the ssDNA strand in a 59-to-39 direction and unwinds the forward dsDNA (35,36).…”

The Macromolecular Machines that Duplicate the Escherichia coli Chromosome as Targets for Drug Discovery

“…DnaC does not act independently but must form a stable complex with DnaB (as the DnaB-DnaC complex) at the stage of replication initiation at oriC . Several independent studies strongly suggest that a site near the N-terminus of DnaC interacts with a specific surface in the C-terminal region of DnaB [ 181 , 182 , 183 , 184 , 185 ]. Formation of the DnaB-DnaC complex, which does not require a nucleoside di- or triphosphate bound to either protein, leads to as many as six DnaC molecules bound per DnaB hexamer [ 176 , 177 , 183 ].…”

“…The interaction of DnaC with ssDNA may help to load the open-ring form of DnaB when complexed to DnaC onto the region of oriC unwound by DnaA [ 152 ]. Following the loading of the DnaB-DnaC complex at oriC to form a macromolecular complex containing DnaA, DnaB and DnaC, primer formation by primase leads to the dissociation of DnaC from DnaB and its activation as a DNA helicase [ 166 , 185 , 189 ].…”