Abstract
Bacteria, like all cells, must precisely duplicate their genomes before they divide. Regulation of this critical process focuses on forming a pre-replicative nucleoprotein complex, termed the orisome. Orisomes perform two essential mechanical tasks that configure the unique chromosomal replication origin, oriC to start a new round of chromosome replication: (1) unwinding origin DNA and (2) assisting with loading of the replicative DNA helicase on exposed single strands. In Escherichia coli, a necessary orisome component is the ATP-bound form of the bacterial initiator protein, DnaA. DnaA-ATP differs from DnaA-ADP in its ability to oligomerize into helical filaments, and in its ability to access a subset of low affinity recognition sites in the E. coli replication origin. The helical filaments have been proposed to play a role in both of the key mechanical tasks, but recent studies raise new questions about whether they are mandatory for orisome activity. It was recently shown that a version of E. coli oriC (oriCallADP), whose multiple low affinity DnaA recognition sites bind DnaA-ATP and DnaA-ADP similarly, was fully occupied and unwound by DnaA-ADP in vitro, and in vivo suppressed the lethality of DnaA mutants defective in ATP binding and ATP-specific oligomerization. However, despite their functional equivalency, orisomes assembled on oriCallADP were unable to trigger chromosome replication at the correct cell cycle time and displayed a hyper-initiation phenotype. Here we present a new perspective on DnaA-ATP, and suggest that in E. coli, DnaA-ATP is not required for mechanical functions, but rather is needed for site recognition and occupation, so that initiation timing is coupled to DnaA-ATP levels. We also discuss how other bacterial types may utilize DnaA-ATP and DnaA-ADP, and whether the high diversity of replication origins in the bacterial world reflects different regulatory strategies for how DnaA-ATP is used to control orisome assembly.
Highlights
The molecular mechanism responsible for triggering new rounds of chromosome replication in bacteria is precisely regulated
In E. coli, the cellular level of DnaA-ATP fluctuates during the cell cycle (Kurokawa et al, 1999), and the reproducibility of initiation timing from one cell cycle to the is achieved by coupling orisome assembly to DnaA-ATP levels
This is accomplished via a set of arranged low affinity DnaA-ATP recognition sites in E. coli oriC that direct orisome assembly by guiding cooperative binding of the initiator (Zawilak-Pawlik et al, 2005; Rozgaja et al, 2011; described in more detail below)
Summary
The molecular mechanism responsible for triggering new rounds of chromosome replication in bacteria is precisely regulated. E. coli oriC contains eight less canonical DnaA binding sites, most of which were identified only after in vitro DnaA binding assays (Grimwade et al, 2000; Rozgaja et al, 2011) These cryptic sites deviate from the consensus R box sequence by 2 or more bp (Figure 1), which disrupts some base-specific contacts (Figure 1). Six of the lower affinity sites (τ2, I1, I2, I3, C2, and C3) preferentially bind DnaA-ATP (McGarry et al, 2004; Kawakami et al, 2005; Grimwade et al, 2018), and occupation of these sites requires physiological levels of ATP (0.5–5 mM) (Saxena et al, 2013), as well as interactions between a critical arginine (R285) in DnaA’s domain III and the bound ATP of an adjacent DnaA molecule (Kawakami et al, 2005)(discussed further below) While it is not known why these six sites prefer DnaA-ATP, it is probable that conformational differences between DnaAATP and DnaA-ADP play a role. The amino acids involved in ATP/ADP binding and hydrolysis are located in a central
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