Biology subjects

Soultanas, P.

Publications and source records attributed to Soultanas, P..

7 recordsLinked to original sources

bioRxiv2022

Human HelQ and DNA polymerase δ interact to halt DNA synthesis and stimulate DNA single-strand annealing

DNA strand breaks can be repaired by base-pairing with unbroken homologous DNA, forming a template for new DNA synthesis that patches over the break site. In eukaryotes multiple DNA break repair pathways utilize DNA polymerase {delta} (Pol {delta}) to synthesise new DNA from the available 3OH at the strand break. Here we show that DNA synthesis by human Pol {delta} is halted by the HelQ DNA repair protein directly targeting isolated Pol {delta} or Pol {delta} in complex with PCNA and RPA. The mechanism is independent of DNA binding by HelQ or Pol {delta}, maps to a region of HelQ that also modulates RPA, and requires multiple Pol {delta} subunits. Interaction of HelQ with the POLD3 subunit of Pol {delta} stimulated DNA single-strand annealing activity of HelQ. The data implicates HelQ in preventing genetic instability by restraining DNA synthesis in multiple DNA break repair pathways.

biochemistry↗

bioRxiv2022

The DNA replication initiation protein DnaD is recruited to a specific strand of the Bacillus subtilis chromosome origin

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. The molecular mechanisms for 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 (DnaD Recognition Element, "DRE"). Considered with cryo-electron microscopy (cryo-EM) imaging of full length DnaD, we propose that the location of the DRE within the oriC orchestrates strand-specific recruitment of helicase to achieve bidirectional DNA replication. These findings significantly advance our mechanistic understanding of bidirectional replication from a bacterial chromosome origin.

molecular biology↗

bioRxiv2022

SirA inhibits the essential DnaA:DnaD interaction to block helicase recruitment during Bacillus subtilis sporulation

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 with DnaA, thereby inhibiting helicase recruitment to the origin during sporulation. 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.

molecular biology↗

bioRxiv2021

Positive and negative control of helicase recruitment at a bacterial chromosome origin

The mechanisms responsible for helicase loading during the initiation of chromosome replication in bacteria are unclear. Here we report both a positive and a negative mechanism for directing helicase recruitment in the model organism Bacillus subtilis. Systematic mutagenesis of the essential replication initiation gene dnaD and characterization of DnaD variants revealed protein interfaces required for interacting with the master initiator DnaA and with a specific single-stranded DNA (ssDNA) sequence located in the chromosome origin (DnaD Recognition Element, "DRE"). We propose that the location of the DRE within the replication origin orchestrates recruitment of helicase to achieve bidirectional DNA replication. We also report that the developmentally expressed repressor of DNA replication initiation, SirA, acts by blocking the interaction of DnaD with DnaA, thereby inhibiting helicase recruitment to the origin. These findings significantly advance our mechanistic understanding of helicase recruitment and regulation during bacterial DNA replication initiation. Because DnaD is essential for the viability of clinically relevant Gram-positive pathogens, DnaD is an attractive target for drug development.

microbiology↗

bioRxiv2020

Glycolytic pyruvate kinase moonlighting activities in DNA replication initiation and elongation

SUMMARYCells have evolved a metabolic control of DNA replication to respond to a wide range of nutritional conditions. Accumulating data suggest that this poorly understood control depends, at least in part, on Central Carbon Metabolism (CCM). In Bacillus subtilis, the glycolytic pyruvate kinase (PykA) is intricately linked to replication. This 585 amino-acid-long enzyme comprises a catalytic (Cat) domain that binds to phosphoenolpyruvate (PEP) and ADP to produce pyruvate and ATP, and a C-terminal domain of unknown function. Interestingly, the C-terminal domain termed PEPut interacts with Cat and is homologous a domain that, in other metabolic enzymes, are phosphorylated at a conserved TSH motif at the expense of PEP and ATP to drive sugar import and catalytic or regulatory activities. To gain insights into the role of PykA in replication, DNA synthesis was analyzed in various Cat and PEPut mutants grown in a medium where the metabolic activity of PykA is dispensable for growth. Measurements of replication parameters (ori/ter ratio, C period and fork speed) and of the pyruvate kinase activity showed that PykA mutants exhibit replication defects resulting from side chain modifications in the PykA protein rather than from a reduction of its metabolic activity. Interestingly, Cat and PEPut have distinct commitments in replication: while Cat impacts positively and negatively replication fork speed, PEPut stimulates initiation through a process depending on Cat-PEPut interaction and growth conditions. Residues binding to PEP and ADP in Cat, stabilizing the Cat-PEPut interaction and belonging to the TSH motif of PEPut were found important for the commitment of PykA in replication. In vitro, PykA affects the activities of replication enzymes (the polymerase DnaE, helicase DnaC and primase DnaG) essential for initiation and elongation and genetically linked to pykA. Our results thus connect replication initiation and elongation to CCM metabolites (PEP, ATP and ADP), critical Cat and PEPut residues and to multiple links between PykA and the replication enzymes DnaE, DnaC and DnaG. We propose that PykA is endowed with a moonlighting activity that senses the concentration of signaling metabolites and interacts with replication enzymes to convey information on the cellular metabolic state to the replication machinery and adjust replication initiation and elongation to metabolism. This defines a new type of replication regulator proposed to be part of the metabolic control that gates replication in the cell cycle.Competing Interest StatementThe authors have declared no competing interest.View Full Text

microbiology↗

bioRxiv2019

Interaction of HelQ helicase with RPA modulates RPA-DNA binding and stimulates HelQ to unwind DNA through a protein roadblock.

Cells reactivate compromised DNA replication forks using enzymes that include DNA helicases for separating DNA strands and remodelling protein-DNA complexes. HelQ helicase promotes replication-coupled DNA repair in mammals in a network of interactions with other proteins. We report newly identified HelQ helicase activities, when acting alone and when interacting with RPA. HelQ helicase was strongly inhibited by a DNA-protein barrier (BamHIE111A), and by an abasic site in the translocating DNA strand. Interaction of HelQ with RPA activated DNA unwinding through the protein barrier, but not through the abasic site. Activation was lost when RPA was replaced with bacterial SSB or DNA binding-defective RPA, RPAARO1. We observed stable HelQ-RPA-DNA ternary complex formation, and present evidence that an intrinsically disordered N-terminal region of HelQ (N-HelQ) interacts with RPA, destabilising RPA-DNA binding. Additionally, SEC-MALS showed that HelQ multimers are converted into catalytically active dimers when ATP-Mg2+ is bound. HelQ and RPA are proposed to jointly promote replication fork recovery by helicase-catalysed displacement of DNA-bound proteins, after HelQ gains access to ssDNA through its N-terminal domain interaction with RPA.

biochemistry↗

bioRxiv2018

DNA replication initiation in Bacillus subtilis; Structural and functional characterisation of the essential DnaA-DnaD interaction

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 show that both DDBH1 and DDBH2 interact with the N-terminal domain I of DnaA and studied the DDBH2 interaction in structural detail by NMR. 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.

biochemistry↗