The intrinsic ATPase activity of Mycobacterium tuberculosis DnaA promotes rapid oligomerization of DnaA on oriC

Abstract

SummaryOligomerization of the initiator protein, DnaA, on the origin of replication ( oriC ) is crucial for initiation of DNA replication . Studies in Escherichia coli (Gramnegative) have revealed that binding of DnaA to ATP, but not hydrolysis of ATP, is sufficient to promote DnaA binding, oligomerization and DNA strand separation. To begin understanding the initial events involved in the initiation of DNA replication in Mycobacterium tuberculosis (Gram-positive), we investigated interactions of M. tuberculosis DnaA (DnaA TB ) with oriC using surface plasmon resonance in the presence of ATP and ADP. We provide evidence that, in contrast to what is observed in E. coli , ATPase activity of DnaA TB promoted rapid oligomerization on oriC. In support, we found that a recombinant mutant DnaA TB proficient in binding to ATP, but deficient in ATPase activity, did not oligomerize as rapidly. The corresponding mutation in the dnaA gene of M. tuberculosis resulted in non-viability, presumably due to a defect in oriC -DnaA interactions. Dimethy sulphate (DMS) footprinting experiments revealed that DnaA TB bound to DnaA boxes similarly with ATP or ADP. DnaA TB binding to individual DnaA boxes revealed that rapid oligomerization on oriC is triggered only after the initial interaction of DnaA with individual DnaA boxes. We propose that ATPase activity enables the DnaA protomers on oriC to rapidly form oligomeric complexes competent for replication initiation.

Mycobacterium tuberculosis Origin of Replication and the Promoter for Immunodominant Secreted Antigen 85B Are the Targets of MtrA, the Essential Response Regulator

“…3C: F2, F3, F4, and F5). Such dispersed contacts across all of oriC, including the dnaA 3Ј-and dnaN 5Ј-coding DNA, are more typical of DnaA protein binding to multiple DnaA-boxes that span bacterial oriCs, including Mtb oriC (42). It is possible that the toe prints are not true MtrA-binding sites but rather could be due to the consequence of the formation of large MtrA-oriC nucleoprotein complexes.…”

“…S5) (41). Although detailed molecular details on how the initiation of DNA replication in Mtb occurs are unknown, recent studies reveal that the initial interactions of DnaA with DnaA-boxes in the presence of ATP are necessary for a rapid oligomerization of DnaA at oriC and the formation of the DnaA-oriC initiation complex competent for initiation (42). This initial step is followed by unwinding of oriC at the AT-rich sequences, which can occur in the absence of helicases and other replication proteins (41).…”

Facilitation of Dissociation Reaction of Nucleotides Bound to Mycobacterium tuberculosis DnaA

“…Presumably, the interaction of DnaA TB with phospholipids results in a decrease in the affinity of DnaA for ATP, oriC or both. Other results suggest that ADP-bound form of DnaA is not competent for binding and rapid oligomerization on oriC (13). Together, these results suggest that phospholipids regulate nucleotide bound state of DnaA and play important regulatory roles in M. tuberculosis oriC replication (12).…”

“…M. tuberculosis oriC is 550-bp long (10) and recombinant DnaA TB protein purified under denaturing conditions has been shown to associate with adenine nucleotides and oriC (12). DnaA TB specifically recognizes DnaA boxes and dimethylsulfate footprinting revealed the presence of nine DnaA-boxes that bear little or no sequence similarity to the five DnaA boxes of E. coli oriC (13). Acidic phospholipids have been shown to modulate DnaA TB interactions with adenine nucleotide and oriC stabilizes DnaA TB -ATP interactions and promotes dissociation of DnaA TB -ADP complexes (12).…”

SanG, a transcriptional activator, controls nikkomycin biosynthesis through binding to the sanN–sanO intergenic region in Streptomyces ansochromogenes

“…Some transcriptional activators with the ATPase domain oligomerize into ATPase-active rings that use the energy from ATP hydrolysis to physically remodel transcriptionally closed complexes in bacteria (Madiraju et al, 2006). As SanG without ATP/GTP is easy to aggregate and ATP/GTP did enhance the affinity of SanG for the target DNA, it is possible that ATP/GTP could stabilize the conformation of SanG.…”