RESUMO
Bidirectional DNA replication from a chromosome origin requires the asymmetric loading of two helicases, one for each replisome. Our understanding of the molecular mechanisms underpinning helicase loading at bacterial chromosome origins is incomplete. Here we report both positive and negative mechanisms for directing helicase recruitment in the model organism Bacillus subtilis. Systematic characterization of the essential initiation protein DnaD revealed distinct protein interfaces required for homo-oligomerization, interaction with the master initiator protein DnaA, and interaction with the helicase co-loader protein DnaB. Informed by these properties of DnaD, we went on to find that the developmentally expressed repressor of DNA replication initiation, SirA, blocks the interaction between DnaD and DnaA, thereby restricting helicase recruitment from the origin during sporulation to inhibit further initiation events. These results advance our understanding of the mechanisms underpinning DNA replication initiation in B. subtilis, as well as guiding the search for essential cellular activities to target for antimicrobial drug design.
Assuntos
Bacillus subtilis , Proteínas de Bactérias , DNA Helicases , Esporos Bacterianos , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Bacillus subtilis/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Replicação do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , DnaB Helicases/genética , DnaB Helicases/metabolismo , Origem de Replicação , Esporos Bacterianos/metabolismoRESUMO
Genome replication is a fundamental biological activity shared by all organisms. Chromosomal replication proceeds bidirectionally from origins, requiring the loading of two helicases, one for each replisome. However, the molecular mechanisms underpinning helicase loading at bacterial chromosome origins (oriC) are unclear. Here we investigated the essential DNA replication initiation protein DnaD in the model organism Bacillus subtilis. A set of DnaD residues required for ssDNA binding was identified, and photo-crosslinking revealed that this ssDNA binding region interacts preferentially with one strand of oriC. Biochemical and genetic data support the model that DnaD recognizes a new single-stranded DNA (ssDNA) motif located in oriC, the DnaD Recognition Element (DRE). Considered with single particle cryo-electron microscopy (cryo-EM) imaging of DnaD, we propose that the location of the DRE within oriC orchestrates strand-specific recruitment of helicase during DNA replication initiation. These findings significantly advance our mechanistic understanding of bidirectional replication from a bacterial chromosome origin.
Assuntos
Bacillus subtilis , Proteínas de Bactérias , Proteínas de Ligação a DNA , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cromossomos Bacterianos/genética , Cromossomos Bacterianos/metabolismo , Microscopia Crioeletrônica , DNA Helicases/genética , DNA Helicases/metabolismo , Replicação do DNA , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Origem de ReplicaçãoRESUMO
The homotetrameric DnaD protein is essential in low G+C content gram positive bacteria and is involved in replication initiation at oriC and re-start of collapsed replication forks. It interacts with the ubiquitously conserved bacterial master replication initiation protein DnaA at the oriC but structural and functional details of this interaction are lacking, thus contributing to our incomplete understanding of the molecular details that underpin replication initiation in bacteria. DnaD comprises N-terminal (DDBH1) and C-terminal (DDBH2) domains, with contradicting bacterial two-hybrid and yeast two-hybrid studies suggesting that either the former or the latter interact with DnaA, respectively. Using Nuclear Magnetic Resonance (NMR) we showed that both DDBH1 and DDBH2 interact with the N-terminal domain I of DnaA and studied the DDBH2 interaction in structural detail. We revealed two families of conformations for the DDBH2-DnaA domain I complex and showed that the DnaA-interaction patch of DnaD is distinct from the DNA-interaction patch, suggesting that DnaD can bind simultaneously DNA and DnaA. Using sensitive single-molecule FRET techniques we revealed that DnaD remodels DnaA-DNA filaments consistent with stretching and/or untwisting. Furthermore, the DNA binding activity of DnaD is redundant for this filament remodelling. This in turn suggests that DnaA and DnaD are working collaboratively in the oriC to locally melt the DNA duplex during replication initiation.
Assuntos
Proteínas de Bactérias/genética , Replicação do DNA/genética , Proteínas de Ligação a DNA/genética , Origem de Replicação/genética , Bacillus subtilis/genética , Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , DnaB Helicases/química , DnaB Helicases/genética , Espectroscopia de Ressonância Magnética , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexo de Reconhecimento de Origem/genética , Ligação Proteica/genética , Domínios Proteicos/genética , Relação Estrutura-AtividadeRESUMO
SUMMARY: JEPETTO (Java Enrichment of Pathways Extended To TOpology) is a Cytoscape 3.x plugin performing integrative human gene set analysis. It identifies functional associations between genes and known cellular pathways, and processes using protein interaction networks and topological analysis. The plugin integrates information from three separate web servers we published previously, specializing in enrichment analysis, pathways expansion and topological matching. This integration substantially simplifies the analysis of user gene sets and the interpretation of the results. We demonstrate the utility of the JEPETTO plugin on a set of misregulated genes associated with Alzheimer's disease. AVAILABILITY: Source code and binaries are freely available for download at http://apps.cytoscape.org/apps/jepetto, implemented in Java and multi-platform. Installable directly via Cytoscape plugin manager. Released under the GNU General Public Licence.
Assuntos
Software , Doença de Alzheimer/genética , Regulação da Expressão Gênica , Humanos , Mapas de Interação de ProteínasRESUMO
Genome duplication is essential for the proliferation of cellular life and this process is generally initiated by dedicated replication proteins at chromosome origins. In bacteria, DNA replication is initiated by the ubiquitous DnaA protein, which assembles into an oligomeric complex at the chromosome origin (oriC) that engages both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) to promote DNA duplex opening. However, the mechanism of DnaA specifically opening a replication origin was unknown. Here we show that Bacillus subtilis DnaAATP assembles into a continuous oligomer at the site of DNA melting, extending from a dsDNA anchor to engage a single DNA strand. Within this complex, two nucleobases of each ssDNA binding motif (DnaA-trio) are captured within a dinucleotide binding pocket created by adjacent DnaA proteins. These results provide a molecular basis for DnaA specifically engaging the conserved sequence elements within the bacterial chromosome origin basal unwinding system (BUS).
Assuntos
Replicação do DNA , Proteínas de Ligação a DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas de Bactérias/metabolismo , Origem de Replicação , Bactérias/genética , DNA , DNA de Cadeia Simples/genética , DNA Bacteriano/metabolismo , Cromossomos Bacterianos/genética , Cromossomos Bacterianos/metabolismoRESUMO
Research over the last two decades has revealed that bacterial genomes are highly organized and that bacteria have sophisticated mechanisms in place to ensure their correct replication and segregation into progeny cells. Here we discuss techniques that can be used with live bacterial cells to analyze DNA replisome dynamics, double-strand chromosome breaks, and restart of repaired replication forks.
Assuntos
Bacillus subtilis , Quebra Cromossômica , Bacillus subtilis/genética , Cromossomos Bacterianos/genética , Replicação do DNA , HumanosRESUMO
As DNA sequencing and synthesis become cheaper and more easily accessible, the scale and complexity of biological engineering projects is set to grow. Yet, although there is an accelerating convergence between biotechnology and digital technology, a deficit in software and laboratory techniques diminishes the ability to make biotechnology more agile, reproducible, and transparent while, at the same time, limiting the security and safety of synthetic biology constructs. To partially address some of these problems, this paper presents an approach for physically linking engineered cells to their digital footprint-we called it digital twinning. This enables the tracking of the entire engineering history of a cell line in a specialized version control system for collaborative strain engineering via simple barcoding protocols.