Three-Dimensional Structure of N-Terminal Domain of DnaB Helicase and Helicase-Primase Interactions in Helicobacter pylori

Tara Kashav,S Arif Abdulrehman,Azat Gabdoulkhakov,Ramgopal Nitharwal,Suman Kumar Dhar,Wolfram Saenger,Samudrala Gourinath,Niyaz Ahmed

doi:10.1371/journal.pone.0007515

Tara Kashav, S Arif Abdulrehman + Show 6 more

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https://doi.org/10.1371/journal.pone.0007515

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Journal: PloS one	Publication Date: Oct 20, 2009
Citations: 44	License type: CC BY 4.0

Affiliation: Jawaharlal Nehru University, Freie Universität Berlin

Abstract

Replication initiation is a crucial step in genome duplication and homohexameric DnaB helicase plays a central role in the replication initiation process by unwinding the duplex DNA and interacting with several other proteins during the process of replication. N-terminal domain of DnaB is critical for helicase activity and for DnaG primase interactions. We present here the crystal structure of the N-terminal domain (NTD) of H. pylori DnaB (HpDnaB) helicase at 2.2 Å resolution and compare the structural differences among helicases and correlate with the functional differences. The structural details of NTD suggest that the linker region between NTD and C-terminal helicase domain plays a vital role in accurate assembly of NTD dimers. The sequence analysis of the linker regions from several helicases reveals that they should form four helix bundles. We also report the characterization of H. pylori DnaG primase and study the helicase-primase interactions, where HpDnaG primase stimulates DNA unwinding activity of HpDnaB suggesting presence of helicase-primase cohort at the replication fork. The protein-protein interaction study of C-terminal domain of primase and different deletion constructs of helicase suggests that linker is essential for proper conformation of NTD to interact strongly with HpDnaG. The surface charge distribution on the primase binding surface of NTDs of various helicases suggests that DnaB-DnaG interaction and stability of the complex is most probably charge dependent. Structure of the linker and helicase-primase interactions indicate that HpDnaB differs greatly from E.coli DnaB despite both belong to gram negative bacteria.

Highlights

Helicobacter pylori (H. pylori) is a gram-negative, microaerophilic spiral shaped bacterium that infects more than 50% of the human population globally and is responsible for causing chronic gastritis, peptic ulcer and gastric cancer [1]
To understand the roles played by N-terminal domain (NTD) in helicase function, the importance of the linker region and helicase primase interactions, we present here the structure of NTD of H. pylori DnaB (HpDnaB) at 2.2 Aresolution and compare it with other NTD structures
The best structure solution was obtained with recently released structure of NTD of Mycobacterium tuberculosis helicase (NtMtbDnaB, Protein Data Bank (PDB) iD: 2r5u), with two molecules in one asymmetric unit

Summary

Introduction

Helicobacter pylori (H. pylori) is a gram-negative, microaerophilic spiral shaped bacterium that infects more than 50% of the human population globally and is responsible for causing chronic gastritis, peptic ulcer and gastric cancer [1]. In E. coli, DnaB helicase plays a central role; which displays proteinprotein interactions right from the beginning to the end of replication process. The loading partner DnaC recruits DnaB as DnaB6-DnaC6 complex at oriC site and plays an important role during replisomal complex formation [9,10]. Bioinformatics studies shows the absence of a DnaC homolog in the H. pylori genome [2,11] and yeast two-hybrid studies shows that H. pylori DnaB (HpDnaB) does not interact with HpDnaA [12]. Our biochemical studies earlier had confirmed that HpDnaB is able to complement the DnaC function and loads itself to the oriC site to form the pre-replication complex [13]

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