scholarly journals Structural insights into DNA replication initiation in Helicobacter pylori

2014 ◽  
Vol 70 (a1) ◽  
pp. C1632-C1632
Author(s):  
Alexandre Bazin ◽  
Mickaël Cherrier ◽  
Laurent Terradot
Keyword(s):  
Helicobacter Pylori ◽  
Dna Replication ◽  
Bacterial Genome ◽  
Coli Strain ◽  
Replication Initiation ◽  
Replicative Helicase ◽  
Dna Replication Initiation ◽  
Terminal Domain ◽  
Helicase Dnab ◽  
H Pylori

In Gram-negative bacteria, opening of DNA double strand during replication is performed by the replicative helicase DnaB. This protein allows for replication fork elongation by unwinding DNA and interacting with DnaG primase. DnaB is composed of two domains: an N-terminal domain (NTD) and a C-terminal domain (CTD) connected by a flexible linker. The protein forms two-tiered hexamers composed of a NTD-ring and a CTD-ring. In Escherichia coli, the initiator protein DnaA binds to the origin of replication oriC and induces the opening of a AT-rich region. The replicative helicase DnaB is then loaded onto single stranded DNA by interacting with DnaA and with the AAA+ helicase loader DnaC. However, AAA+ loaders are absent in 80% of the bacterial genome, raising the question of how helicases are loaded in these bacteria [1]. In the genome of human pathogen Helicobacter pylori, no AAA+ loader has been identified. Moreover H. pylori DnaB (HpDnaB) has the ability to support replication of an otherwise unviable E. coli strain that bears a defective copy of DnaC by complementation [2]. In order to better understand the properties of HpDnaB we have first shown that HpDnaB forms double hexamers by negative stain electron microscopy [3]. Then, we have then solved the crystal structure of HpDnaB at a resolution of 6.7Å by X-ray crystallography with Rfree/Rfactor of 0.29/0.25. The structure reveals that the protein adopts a new dodecameric arrangement generated by crystallographic three fold symmetry. When compared to hexameric DnaBs, the hexamer of HpDnaB displays an original combination of NTD-ring and CTD-ring symmetries, intermediate between apo and ADP-bound structure. Biochemistry studies of HpDnaB interaction with HpDnaG-CTD and ssDNA provides mechanistic insights into the initial steps of DNA replication in H. pylori. Our results offer an alternative solution of helicase loading and DNA replication initiation in H. pylori and possibly other bacteria that do not employ helicase loaders.

scholarly journals DpiA Binding to the Replication Origin of Escherichia coli Plasmids and Chromosomes Destabilizes Plasmid Inheritance and Induces the Bacterial SOS Response

2003 ◽  
Vol 185 (20) ◽  
pp. 6025-6031 ◽  
Author(s):  
Christine Miller ◽  
Hanne Ingmer ◽  
Line Elnif Thomsen ◽  
Kirsten Skarstad ◽  
Stanley N. Cohen
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Replication Origin ◽  
Sos Response ◽  
Dna Helicase ◽  
Replication Initiation ◽  
Chromosomal Dna ◽  
E Coli ◽  
Dna Replication Initiation ◽  
Helicase Dnab

ABSTRACT The dpiA and dpiB genes of Escherichia coli, which are orthologs of genes that regulate citrate uptake and utilization in Klebsiella pneumoniae, comprise a two-component signal transduction system that can modulate the replication of and destabilize the inheritance of pSC101 and certain other plasmids. Here we show that perturbed replication and inheritance result from binding of the effector protein DpiA to A+T-rich replication origin sequences that resemble those in the K. pneumoniae promoter region targeted by the DpiA ortholog, CitB. Consistent with its ability to bind to A+T-rich origin sequences, overproduction of DpiA induced the SOS response in E. coli, suggesting that chromosomal DNA replication is affected. Bacteria that overexpressed DpiA showed an increased amount of DNA per cell and increased cell size—both also characteristic of the SOS response. Concurrent overexpression of the DNA replication initiation protein, DnaA, or the DNA helicase, DnaB—both of which act at A+T-rich replication origin sequences in the E. coli chromosome and DpiA-targeted plasmids—reversed SOS induction as well as plasmid destabilization by DpiA. Our finding that physical and functional interactions between DpiA and sites of replication initiation modulate DNA replication and plasmid inheritance suggests a mechanism by which environmental stimuli transmitted by these gene products can regulate chromosomal and plasmid dynamics.

A Novel Fluorescence-Based Screen for Inhibitors of the Initiation of DNA Replication in Bacteria

2019 ◽  
Vol 16 (3) ◽  
pp. 272-277 ◽  
Author(s):  
Rasmus N. Klitgaard ◽  
Anders Løbner-Olesen
Keyword(s):  
Dna Replication ◽  
High Throughput ◽  
Cyclic Peptide ◽  
Replication Initiation ◽  
False Positive Result ◽  
Over Expression ◽  
Initiator Protein ◽  
Dna Replication Initiation ◽  
Cellular Processes ◽  
Initiation Of Dna Replication

Background:One of many strategies to overcome antibiotic resistance is the discovery of compounds targeting cellular processes, which have not yet been exploited.Materials and Methods:Using various genetic tools, we constructed a novel high throughput, cellbased, fluorescence screen for inhibitors of chromosome replication initiation in bacteria.Results:The screen was validated by expression of an intra-cellular cyclic peptide interfering with the initiator protein DnaA and by over-expression of the negative initiation regulator SeqA. We also demonstrated that neither tetracycline nor ciprofloxacin triggers a false positive result. Finally, 400 extracts isolated mainly from filamentous actinomycetes were subjected to the screen.Conclusion:We concluded that the presented screen is applicable for identifying putative inhibitors of DNA replication initiation in a high throughput setup.

scholarly journals Serodiagnosis and Bacterial Genome of Helicobacter pylori Infection

Toxins ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 467
Author(s):  
Aina Ichihara ◽  
Hinako Ojima ◽  
Kazuyoshi Gotoh ◽  
Osamu Matsushita ◽  
Susumu Take ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Antibody Titer ◽  
Bacterial Genome ◽  
Serum Antibody ◽  
Gene Mutations ◽  
Bacterial Genomes ◽  
Western Blots ◽  
A Genome ◽  
Vaca Gene ◽  
H Pylori

The infection caused by Helicobacter pylori is associated with several diseases, including gastric cancer. Several methods for the diagnosis of H. pylori infection exist, including endoscopy, the urea breath test, and the fecal antigen test, which is the serum antibody titer test that is often used since it is a simple and highly sensitive test. In this context, this study aims to find the association between different antibody reactivities and the organization of bacterial genomes. Next-generation sequences were performed to determine the genome sequences of four strains of antigens with different reactivity. The search was performed on the common genes, with the homology analysis conducted using a genome ring and dot plot analysis. The two antigens of the highly reactive strains showed a high gene homology, and Western blots for CagA and VacA also showed high expression levels of proteins. In the poorly responsive antigen strains, it was found that the inversion occurred around the vacA gene in the genome. The structure of bacterial genomes might contribute to the poor reactivity exhibited by the antibodies of patients. In the future, an accurate serodiagnosis could be performed by using a strain with few gene mutations of the antigen used for the antibody titer test of H. pylori.

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

2021 ◽  
Vol 22 (12) ◽  
pp. 6643
Author(s):  
Pawel Jaworski ◽  
Dorota Zyla-Uklejewicz ◽  
Malgorzata Nowaczyk-Cieszewska ◽  
Rafal Donczew ◽  
Thorsten Mielke ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Replication Initiation ◽  
Dna Unwinding ◽  
Rich Region ◽  
Initiator Protein ◽  
New Class ◽  
H Pylori ◽  
General Architecture ◽  
Dnaa Box

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.

scholarly journals Structural insights into the interaction of helicase and primase in Mycobacterium tuberculosis

2018 ◽  
Vol 475 (21) ◽  
pp. 3493-3509 ◽  
Author(s):  
Dhakaram Pangeni Sharma ◽  
Ramachandran Vijayan ◽  
Syed Arif Abdul Rehman ◽  
Samudrala Gourinath
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Replication Initiation ◽  
Binding Studies ◽  
Functional Conservation ◽  
Terminal Domain ◽  
Binding Domains ◽  
Initiation System ◽  
Helical Hairpin ◽  
Helicase Dnab ◽  
Species Specific

The helicase–primase interaction is an essential event in DNA replication and is mediated by the highly variable C-terminal domain of primase (DnaG) and N-terminal domain of helicase (DnaB). To understand the functional conservation despite the low sequence homology of the DnaB-binding domains of DnaGs of eubacteria, we determined the crystal structure of the helicase-binding domain of DnaG from Mycobacterium tuberculosis (MtDnaG-CTD) and did so to a resolution of 1.58 Å. We observed the overall structure of MtDnaG-CTD to consist of two subdomains, the N-terminal globular region (GR) and the C-terminal helical hairpin region (HHR), connected by a small loop. Despite differences in some of its helices, the globular region was found to have broadly similar arrangements across the species, whereas the helical hairpins showed different orientations. To gain insights into the crucial helicase–primase interaction in M. tuberculosis, a complex was modeled using the MtDnaG-CTD and MtDnaB-NTD crystal structures. Two nonconserved hydrophobic residues (Ile605 and Phe615) of MtDnaG were identified as potential key residues interacting with MtDnaB. Biosensor-binding studies showed a significant decrease in the binding affinity of MtDnaB-NTD with the Ile605Ala mutant of MtDnaG-CTD compared with native MtDnaG-CTD. The loop, connecting the two helices of the HHR, was concluded to be largely responsible for the stability of the DnaB–DnaG complex. Also, MtDnaB-NTD showed micromolar affinity with DnaG-CTDs from Escherichia coli and Helicobacter pylori and unstable binding with DnaG-CTD from Vibrio cholerae. The interacting domains of both DnaG and DnaB demonstrate the species-specific evolution of the replication initiation system.

scholarly journals Archaeal Orc1 protein interacts with T-rich single-stranded DNA

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Katarzyna Wegrzyn ◽  
Igor Konieczny
Keyword(s):  
Dna Replication ◽  
Replication Initiation ◽  
Single Stranded Dna ◽  
Dna Replication Initiation ◽  
Double Stranded Dna ◽  
Nucleoprotein Complexes ◽  
Eukaryotic Organisms ◽  
Replication Initiation Proteins ◽  
Hydrolysis Of ◽  
Replication Activity

Abstract Objective The ability to form nucleoprotein complexes is a fundamental activity of DNA replication initiation proteins. They bind within or nearby the region of replication origin what results in melting of a double-stranded DNA (dsDNA) and formation of single-stranded DNA (ssDNA) region where the replication machinery can assemble. For prokaryotic initiators it was shown that they interact with the formed ssDNA and that this interaction is required for the replication activity. The ability to interact with ssDNA was also shown for Saccharomyces cerevisiae replication initiation protein complex ORC. For Archaea, which combine features of both prokaryotic and eukaryotic organisms, there was no evidence whether DNA replication initiators can interact with ssDNA. We address this issue in this study. Results Using purified Orc1 protein from Aeropyrum pernix (ApOrc1) we analyzed its ability to interact with ssDNA containing sequence of an AT-rich region of the A. pernix origin Ori1 as well as with homopolymers of thymidine (polyT) and adenosine (polyA). The Bio-layer interferometry, surface plasmon resonance and microscale thermophoresis showed that the ApOrc1 can interact with ssDNA and it binds preferentially to T-rich ssDNA. The hydrolysis of ATP is not required for this interaction.

scholarly journals DNA Content and Nucleoid Distribution in Methanothermobacter thermautotrophicus

2005 ◽  
Vol 187 (5) ◽  
pp. 1856-1858 ◽  
Author(s):  
Alan I. Majerník ◽  
Magnus Lundgren ◽  
Paul McDermott ◽  
Rolf Bernander ◽  
James P. J. Chong
Keyword(s):  
Flow Cytometry ◽  
Dna Replication ◽  
Chromosome Segregation ◽  
Dna Content ◽  
Replication Initiation ◽  
Epifluorescence Microscopy ◽  
Single Chromosome ◽  
Dna Replication Initiation ◽  
Methanothermobacter Thermautotrophicus ◽  
Multiple Cells

ABSTRACT Flow cytometry and epifluorescence microscopy results for the euryarchaeon Methanothermobacter thermautotrophicus were consistent with filaments containing multiple cells. Filaments of one to four cells contained two to eight nucleoids. Single chromosome-containing cells were not observed. Filaments containing multiple genome copies displayed synchronous DNA replication initiation. Chromosome segregation occurred during replication or rapidly after replication termination.

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