DNA replication initiation inBacillus subtilis: structural and functional characterization of the essential DnaA–DnaD interaction

Abstract

The homotetrameric DnaD protein is essential in low G+C content gram positive bacteria and is involved in replication initiation at oriC and re-start of collapsed replication forks. It interacts with the ubiquitously conserved bacterial master replication initiation protein DnaA at the oriC but structural and functional details of this interaction are lacking, thus contributing to our incomplete understanding of the molecular details that underpin replication initiation in bacteria. DnaD comprises N-terminal (DDBH1) and C-terminal (DDBH2) domains, with contradicting bacterial two-hybrid and yeast two-hybrid studies suggesting that either the former or the latter interact with DnaA, respectively. Using Nuclear Magnetic Resonance (NMR) we showed that both DDBH1 and DDBH2 interact with the N-terminal domain I of DnaA and studied the DDBH2 interaction in structural detail. We revealed two families of conformations for the DDBH2-DnaA domain I complex and showed that the DnaA-interaction patch of DnaD is distinct from the DNA-interaction patch, suggesting that DnaD can bind simultaneously DNA and DnaA. Using sensitive single-molecule FRET techniques we revealed that DnaD remodels DnaA–DNA filaments consistent with stretching and/or untwisting. Furthermore, the DNA binding activity of DnaD is redundant for this filament remodelling. This in turn suggests that DnaA and DnaD are working collaboratively in the oriC to locally melt the DNA duplex during replication initiation.

Identification of a basal system for unwinding a bacterial chromosome origin

“…Interestingly, electron microscopy shows that HobA promotes DnaA-dependent bridging of two separate DNA molecules (Zawilak-Pawlik et al, 2007). In B. subtilis, the tetrameric replication initiation protein DnaD (Huang et al, 2008;Schneider et al, 2008) binds to domain I of DnaA (Matthews & Simmons, 2018;Martin et al, 2019), making it an attractive candidate to facilitate DnaA oligomerization ( Fig EV5iv). Such multimeric intermediates would likely be relatively flexible complexes, which is consistent with the observation that the DnaA-box at the distal incC subregion can function in either orientation and at great distances away from the DnaA-trios.…”

Identification of the Unwinding Region in the Clostridioides difficile Chromosomal Origin of Replication

“…Nevertheless, some residues (P45, F48) appear to be conserved in most of the selected organisms ( Figure 1A ), though no functional role for these residues is known. Potentially, these residues might be involved in protein-protein interactions or DnaA oligomerization, as these functions have been mapped to Domain I of DnaA ( Weigel et al, 1999 ; Abe et al, 2007 ; Natrajan et al, 2009 ; Jameson et al, 2014 ; Kim et al, 2017 ; Zawilak-Pawlik et al, 2017 ; Martin et al, 2018 ; Matthews and Simmons, 2019 ; Nowaczyk-Cieszewska et al, 2020 ).…”

“…In B. subtilis , DnaD, DnaB, and DnaI helicase loader proteins associate sequentially with the origin region resulting in the recruitment of the DnaC helicase protein ( Marsin et al, 2001 ; Velten et al, 2003 ; Smits et al, 2010 ; Jameson and Wilkinson, 2017 ). In B. subtilis , DnaD binds to DnaA and it is postulated that this affects the stability of the DnaA filament and consequently the unwinding of the oriC ( Ishigo-Oka et al, 2001 ; Martin et al, 2018 ; Matthews and Simmons, 2019 ). B. subtilis DnaB protein also affects the DNA topology and has been shown to be important for recruiting oriC to the membrane ( Rokop et al, 2004 ; Zhang et al, 2005 ).…”

“…DnaA proteins generally comprise four domains ( Zawilak-Pawlik et al, 2017 ). Domain I is involved in protein-protein interactions and is responsible for DnaA oligomerization ( Weigel et al, 1999 ; Abe et al, 2007 ; Natrajan et al, 2009 ; Jameson et al, 2014 ; Kim et al, 2017 ; Zawilak-Pawlik et al, 2017 ; Martin et al, 2018 ; Matthews and Simmons, 2019 ; Nowaczyk-Cieszewska et al, 2020 ). Little is known about a specific function of Domain II and this domain may even be absent ( Erzberger et al, 2002 ).…”

The role of Helicobacter pylori DnaA domain I in orisome assembly on a bipartite origin of chromosome replication

“…In addition, the Bacillus subtilis domain III interacts with Soj and YabA (Scholefield, Errington, & Murray, ; Scholefield & Murray, ). The E. coli domain I interacts with DiaA, HU, L2, Dps and YfdR (Chodavarapu, Felczak, & Kaguni, ; Chodavarapu, Felczak, Felczak, Yaniv, & Kaguni, ; Chodavarapu, Gomez, Gomez, Vicente, & Kaguni, ; Ishida et al, ; Noguchi & Katayama, ), while the B. subtilis DnaA domain I interacts with SirA and the helicase co‐loader DnaD (Martin et al, ; Matthews & Simmons, ; Jameson et al, ; Rahn‐Lee, Merrikh, Grossman, & Losick, ).…”