Patrice Polard

A Key Presynaptic Role in Transformation for a Widespread Bacterial Protein: DprA Conveys Incoming ssDNA to RecA

Natural transformation is a mechanism for genetic exchange in many bacterial genera. It proceeds through the uptake of exogenous DNA and subsequent homology-dependent integration into the genome. In Streptococcus pneumoniae, this integration requires the ubiquitous recombinase, RecA, and DprA, a protein of unknown function widely conserved in bacteria. To unravel the role of DprA, we have studied the properties of the purified S. pneumoniae protein and its Bacillus subtilis ortholog (Smf). We report that DprA and Smf bind cooperatively to single-stranded DNA (ssDNA) and that these proteins both self-interact and interact with RecA. We demonstrate that DprA-RecA-ssDNA filaments are produced and that these filaments catalyze the homology-dependent formation of joint molecules. Finally, we show that while the Escherichia coli ssDNA-binding protein SSB limits access of RecA to ssDNA, DprA lowers this barrier. We propose that DprA is a new member of the recombination-mediator protein family, dedicated to natural bacterial transformation.

A Two-Protein Strategy for the Functional Loading of a Cellular Replicative DNA Helicase

The delivery of a ring-shaped hexameric helicase onto DNA is a fundamental step of DNA replication, conserved in all cellular organisms. We report the biochemical characterization of the bacterial hexameric replicative helicase DnaC of Bacillus subtilis with that of the two replication initiation proteins DnaI and DnaB. We show that DnaI and DnaB interact physically and functionally with the DnaC helicase and mediate its functional delivery onto DNA. Thus, DnaB and DnaI form a pair of helicase loaders, revealing a two-protein strategy for the loading of a replicative helicase. We also present evidence that the DnaC helicase loading mechanism appears to be of the ring-assembly type, proceeding through the recruitment of DnaC monomers and their hexamerization around single-stranded DNA by the coordinated action of DnaI and DnaB.

Anticipating chromosomal replication fork arrest: SSB targets repair DNA helicases to active forks.

In bacteria, several salvage responses to DNA replication arrest culminate in reassembly of the replisome on inactivated forks to resume replication. The PriA DNA helicase is a prominent trigger of this replication restart process, preceded in many cases by a repair and/or remodeling of the arrested fork, which can be performed by many specific proteins. The mechanisms that target these rescue effectors to damaged forks in the cell are unknown. We report that the single‐stranded DNA binding (SSB) protein is the key factor that links PriA to active chromosomal replication forks in vivo. This targeting mechanism determines the efficiency by which PriA reaches its specific DNA‐binding site in vitro and directs replication restart in vivo. The RecG and RecQ DNA helicases, which are involved in intricate replication reactivation pathways, also associate with the chromosomal replication forks by similarly interacting with SSB. These results identify SSB as a platform for linking a ‘repair toolbox’ with active replication forks, providing a first line of rescue responses to accidental arrest.

Transposase-induced excision and circularization of the bacterial insertion sequence IS911

We have investigated the role of three IS911‐specified proteins in transposition in vivo: the products of the upstream (OrfA) and downstream (OrfB) open reading frames, and a transframe protein (OrfAB) produced by −1 translational frameshifting between orfA and orfB. The production of OrfAB alone is shown to lead both to excision and to circularization of the element and to be sufficient for intermolecular transposition into a plasmid target. Simultaneous and independent production of OrfA is shown to stimulate OrfAB‐mediated intermolecular transposition while greatly reducing the appearance of transposon circles. We have not been able to detect a role for OrfB. Although under certain conditions, the vector plasmid undergoes precise resealing after IS911 excision, the data suggest that this is not normally the case and that the donor plasmid is not generally conserved. The use of IS911 derivatives carrying mutations in the terminal 2 bp suggested that circle formation represents a site‐specific intramolecular transposition event. We present a model which explains both intra‐ and intermolecular transposition events in terms of a single reaction mechanism of the ‘cut and paste’ type.

DnaB, DnaD and DnaI proteins are components of the Bacillus subtilis replication restart primosome

Phenotypes of Bacillus subtilis priA mutants suggest that they are deficient in the restart of stalled chromosomal replication forks. The presumed activity of PriA in the restart process is to promote the assembly of a multiprotein complex, the primosome, which functions to recruit the replication fork helicase onto the DNA. We have proposed previously that three proteins involved in the initiation of replication at oriC in B. subtilis, DnaB, DnaD and DnaI, are components of the PriA primosome in this bacterium. However, the involvement of these proteins in replication restart has not yet been studied. Here, we describe dnaB mutations that suppress the phenotypes of B. subtilis priA mutants. In a representative mutant, the DnaC helicase is loaded onto single‐stranded DNA in a PriA‐independent, DnaD‐ and DnaI‐dependent manner. These observations confirm that DnaB, DnaD and DnaI are primosomal proteins in B. subtilis. Moreover, their involvement in the suppression of priA phenotypes shows that they participate in replication fork restart in B. subtilis.

Functional interplay between the Bacillus subtilis DnaD and DnaB proteins essential for initiation and re‐initiation of DNA replication

Initiation and re‐initiation of chromosomal DNA replication in bacteria rely on divergent multiprotein assemblies, which direct the functional delivery of the replicative helicase on single‐stranded DNA (ssDNA) at specific sites. These two processes are triggered either at the single chromosomal origin oriC or at arrested forks by the conserved DnaA and PriA proteins respectively. In Bacillus subtilis, these two pathways further require the three essential proteins DnaB, DnaD and DnaI, restrictively encoded in Gram positive bacteria of low GC content. We have recently shown that DnaI and DnaB act as a pair of loaders of the DnaC replicative helicase. The role of DnaD appeared more enigmatic. It was previously shown to interact with DnaA and to display weak ssDNA binding activity. Here, we report that purified DnaD can interact physically with PriA and with DnaB. We show that the lethality of the temperature‐sensitive dnaD23 mutant can be suppressed by different DnaB point mutants, which were found to be identical to the suppressors of priA null mutants. The DnaD23 protein displays lower ssDNA binding activity than DnaD. Conversely, the DnaB75 protein, the main dnaD23 suppressor, has gained affinity for ssDNA. Finally, we observed that this interplay between DnaD and DnaB is crucial for their concerted interaction with SSB‐coated ssDNA, which is the expected substrate for the loading of the replicative helicase in vivo. Altogether, these results highlight the need for both DnaD and DnaB to interact individually and together with ssDNA during the early stages of initiation and re‐initiation of chromosomal DNA replication. They also point at a main structural role of DnaD in the multiprotein assemblies built during these two essential processes.

The bacterial condensin/cohesin‐like protein complex acts in DNA repair and regulation of gene expression

Structural maintenance of chromosome (SMC) and the SMC‐interacting kleisin protein families have key functions in the chromosome organization of most organisms. Here, we report that the Bacillus subtilis kleisin, ScpA, can form a ternary complex with the SMC and ScpB proteins in a yeast tri‐hybrid assay, supporting the notion of a bacterial cohesin/condensin‐like complex. Furthermore, ScpA interacts in two‐hybrid assays with the AddAB complex, essential for recombinational repair, with DegS, a two‐component sensor kinase, as well as with other potential transcription regulators. Point mutations in scpA allowing growth under conditions not permissive for the spcA null and not affecting chromosome condensation were isolated. Among these mutations, some affected DNA repair and gene regulation, thus separating ScpA functions in these two pathways from its functions in chromosome condensation and segregation. Some separation‐of‐function mutations in scpA caused a deficiency in the repair of mitomycin C DNA lesions that was suppressed by increasing the intracellular dosage of the interacting AddAB complex. Another mutation in scpA deregulated the expression of genes encoding degradative enzymes that are known to be controlled by the interacting DegS kinase. We propose that the SMC–ScpA–ScpB complex could: (i) recruit the AddAB helicase/nuclease to act in post‐replicative repair; and (ii) form a complex with the DegS sensor kinase that inhibits its kinase activity. Moreover, our results indicate that the role of cohesin and condensin complexes in DNA repair and gene regulation is evolutionary conserved.