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

Pawel Jaworski,Christoph Weigel,Thorsten Mielke,Anna Zawilak-Pawlik,Malgorzata Nowaczyk-Cieszewska,Rafal Donczew,Dorota Zyla-Uklejewicz

doi:10.3390/ijms22126643

Abstract

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.

Highlights

Chromosomal DNA replication is an essential cellular process that is tightly regulated at the initiation step to ensure that it occurs once and only once per cell cycle [1,2,3]
We thoroughly characterized the oriC2 region of H. pylori, determining the roles of the motifs involved in DNA unwinding and stabilization, i.e., the GC-rich sequence, the DnaAtrio, and the AT-rich region
We revealed the putative interplay between DnaA binding to ssDNA and its interaction with DnaA box ts1 upon DNA unwinding

Summary

Introduction

Chromosomal DNA replication is an essential cellular process that is tightly regulated at the initiation step to ensure that it occurs once and only once per cell cycle [1,2,3]. Replication begins with the binding of the initiator protein DnaA to a specific chromosomal region termed oriC [4]. DnaA oligomerization leads to the assembly of a highly organized nucleoprotein complex that remodels the oriC structure and triggers duplex destabilization within an adjacent, helically-unstable AT-rich region termed the DNA unwinding element (DUE) [5,6,7]. The emergent replication bubble is subsequently stabilized by SSB and DnaA interactions and serves as the recruitment site for the enzymes responsible for strand separation and DNA synthesis [8,9,10,11]. The DnaA protein consists of four structural domains responsible for different but mutually dependent functions (see [3,12,13] and references therein).

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