Putative Cooperative ATP-DnaA Binding to Double-Stranded DnaA Box and Single-Stranded DnaA-Trio Motif upon Helicobacter pylori Replication Initiation Complex Assembly.

oriC is a region of the bacterial chromosome at which the initiator protein DnaA interacts with specific sequences, leading to DNA unwinding and the initiation of chromosome replication. The general architecture of oriCs is universal; however, the structure of oriC and the mode of orisome assembly differ in distantly related bacteria. In this work, we characterized oriC of Helicobacter pylori, which consists of two DnaA box clusters and a DNA unwinding element (DUE); the latter can be subdivided into a GC-rich region, a DnaA-trio and an AT-rich region. We show that the DnaA-trio submodule is crucial for DNA unwinding, possibly because it enables proper DnaA oligomerization on ssDNA. However, we also observed the reverse effect: DNA unwinding, enabling subsequent DnaA-ssDNA oligomer formation-stabilized DnaA binding to box ts1. This suggests the interplay between DnaA binding to ssDNA and dsDNA upon DNA unwinding. Further investigation of the ts1 DnaA box revealed that this box, together with the newly identified c-ATP DnaA box in oriC1, constitute a new class of ATP-DnaA boxes. Indeed, in vitro ATP-DnaA unwinds H. pylori oriC more efficiently than ADP-DnaA. Our results expand the understanding of H. pylori orisome formation, indicating another regulatory pathway of H. pylori orisome assembly.

HP1021 is a redox switch protein identified in Helicobacter pylori.

Helicobacter pylori is a gram-negative, microaerophilic, pathogenic bacterium and a widespread colonizer of humans. H. pylori has developed mechanisms that enable it to overcome the harsh environment of the human stomach, including reactive oxygen species (ROS). Interestingly, up to now no typical regulator dedicated to the oxidative-stress response has been discovered. In this work, we reveal that the inhibitor of replication initiation HP1021 functions as a redox switch protein in H. pylori and plays an important role in response to oxidative stress of the gastric pathogen. Each of the two predicted HP1021 domains contains three cysteine residues. We show that the cysteine residues of HP1021 are sensitive to oxidation both in vitro and in vivo, and we demonstrate that HP1021 DNA-binding activity to oriC depends on the redox state of the protein. Moreover, Zn2+ modulates HP1021 affinity towards oriC template DNA. Transcription analysis of selected H. pylori genes by RT-qPCR indicated that HP1021 is directly involved in the oxygen-dependent control of H. pylori fecA3 and gluP genes, which are implicated in response to oxidative stress. In conclusion, HP1021 is a redox switch protein and could be a target for H. pylori control strategies.

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

The main roles of the DnaA protein are to bind the origin of chromosome replication (oriC), to unwind DNA and to provide a hub for the step-wise assembly of a replisome. DnaA is composed of four domains, with each playing a distinct functional role in the orisome assembly. Out of the four domains, the role of domain I is the least understood and appears to be the most species-specific. To better characterise Helicobacter pylori DnaA domain I, we have constructed a series of DnaA variants and studied their interactions with H. pylori bipartite oriC. We show that domain I is responsible for the stabilisation and organisation of DnaA-oriC complexes and provides cooperativity in DnaA-DNA interactions. Domain I mediates cross-interactions between oriC subcomplexes, which indicates that domain I is important for long-distance DnaA interactions and is essential for orisosme assembly on bipartite origins. HobA, which interacts with domain I, increases the DnaA binding to bipartite oriC; however, it does not stimulate but rather inhibits DNA unwinding. This suggests that HobA helps DnaA to bind oriC, but an unknown factor triggers DNA unwinding. Together, our results indicate that domain I self-interaction is important for the DnaA assembly on bipartite H. pylori oriC.