A role for nonessential domain II of initiator protein, DnaA, in replication control.

REPLICATION initiation in bacteria is controlled by a number of factors that regulate the activity of the the AAA+ DnaA protein (reviewed in Messer 2002; Leonard and Grimwade 2005; Kaguni 2006; Katayama 2008), the functional and structural equivalent of eukaryotic Cdc6/Orc proteins. DnaA binds to several high-affinity binding sites, known as DnaA boxes, near the origin of replication, oriC, in Escherichia coli. In the presence of ATP, DnaA binding extends to several other sites by cooperative interactions, ultimately leading to the melting of an AT-rich region in oriC. The replicative helicase, DnaB, escorted by DnaC, is then recruited to this site, through interactions with the DnaA protein. Once the helicase is assembled on DNA, other factors including the DNA polymerase holoenzyme, processivity clamp, and primase bind to establish bidirectional replication forks emanating from oriC. A number of regulatory systems impinge on replication initiation via DnaA. One of these involves SeqA, a protein that binds cooperatively to GATC sites found in abundance near the origin. SeqA's binding is stronger when such sites are hemi-methylated by DNA adenine methylase (Dam), a situation that occurs transiently after these sequences are replicated. The binding of SeqA “sequesters” the origin (hence its name) and prevents it from accessing Dam and becoming fully methylated for up to one-third of the cell cycle (Campbell and Kleckner 1990; Lu et al. 1994; von Freiesleben et al. 2000). This sequestration establishes an “eclipse” period, a time at which binding of DnaA and reinitiation is actively prevented. In seqA mutants, cells initiate replication more frequently and more asynchronously than wild-type cells. A second system controls initiation capacity by altering the levels of ATP-bound DnaA protein. A protein homologous to DnaA (Hda, for homolog of DnaA) binds the processivity clamp, β, and DnaA, promoting hydrolysis of ATP (Kato and Katayama 2001). Mutants in hda are somewhat inviable and show over-replication, particularly when DnaA levels are elevated (Kato and Katayama 2001; Camara et al. 2005; Riber et al. 2006; Fujimitsu et al. 2008). In addition to these negative regulators, the DiaA protein positively regulates replication initiation. Mutants in diaA were isolated as suppressors of mutants in dnaA that over-initiate replication. By itself, loss of diaA is not lethal but modestly reduces replication initiation frequency and average DNA content per cell and alters the timing and synchrony of initiation (Ishida et al. 2004). DiaA forms a tetramer and directly interacts with multiple DnaA molecules and in vitro recruits DnaA to sites in oriC to stimulate open complex formation (Keyamura et al. 2007). It has been proposed that DnaA cooperative binding, especially to low-affinity sites dependent on the ATP-bound form of DnaA, may be promoted by DiaA. We became interested in SeqA while studying factors that promoted survival to chronic exposure to low levels of replication inhibitors (Sutera and Lovett 2006). Mutants in seqA and dam were sensitive to such agents, such as hydroxyurea and azidothymidine; this sensitivity was exacerbated under fast-growth conditions during which E. coli has multiple ongoing replication cycles. The sensitivity of seqA mutants to fork damage could be suppressed by two mutations in dnaA that reduced replication initiation efficiency. This study concluded that convergence of an unrestrained replication fork onto the site of previous damage was the basis of this sensitivity. Another mutant similarly sensitive to fork inhibitors was in the conserved GTPase, obgE (Foti et al. 2005). Hypomorphic alleles of obgE caused by C-terminal insertion of a Tn5-EZ transposon or mutation in the GTPase motif caused sensitivity to replication inhibitors. Moreover, these obgE alleles caused more inviability in combination with recA and recB, mutations that block double-strand break repair, especially when confronted with agents that slow or block DNA replication fork progression. We concluded that forks are more vulnerable to breakage or collapse in the obgE mutants. We also noted effects of obgE on replication initiation: in minimal medium, cells defective in obgE or overexpressing obgE had asynchronously initiated more replication forks than wild-type cells, as deduced by flow cytometry. Combining mutations in seqA with obgE caused synergistic effects on cell viability and DNA damage sensitivity (Foti et al. 2005). A double mutant in seqA and obgE formed extremely small colonies on rich medium and was much more sensitive, relative to either single mutant, to DNA damage. The phenotype of seqA obgE double mutants was unstable, and we observed the formation of large-colony suppressor variants that arose spontaneously. In this study, we characterize these suppressor mutations and show them to be caused by a single nonlethal deletion in domain II of the replication initiator protein DnaA. The phenotypes caused by this allele are consistent with reduction of replication initiation, properties that are shared by the loss of the positive regulatory protein DiaA. This work therefore establishes a role for domain II in the regulation of replication initiation, potentially in conjunction with DiaA.