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.

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.

Loading a ring: structure of the Bacillus subtilis DnaB protein, a co-loader of the replicative helicase.

Loading of the ring-shaped replicative helicase is a critical step in the initiation of DNA replication. Bacillus subtilis has adopted a two-protein strategy to load its hexameric replicative helicase: DnaB and DnaI interact with the helicase and mediate its delivery onto DNA. We present here the 3D electron microscopy structure of the DnaB protein, along with a detailed analysis of both its oligomeric state and its domain organization. DnaB is organized as an asymmetric tetramer that is comprised of two stacked components, one arranged as a closed collar and the other as an open sigma shape. Intriguingly, the 3D map of DnaB exhibits an overall architecture similar to the structure of the Escherichia coli gamma-complex, the loader of the ring-shaped processivity factor. We propose a model whereby each DnaB monomer participates in both stacked components of the tetramer and displays a different overall shape. This asymmetric quaternary organization could be a general feature of ring loaders.

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.

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.

Restart of DNA replication in Gram-positive bacteria: functional characterisation of the Bacillus subtilis PriA initiator.

The PriA protein was identified in Escherichia coli as a factor involved in the replication of extrachromosomal elements such as bacteriophage phiX174 and plasmid pBR322. Recent data show that PriA plays an important role in chromosomal replication, by promoting reassembly of the replication machinery during reinitiation of inactivated forks. A gene encoding a product 32% identical to the E.coli PriA protein has been identified in Bacillus subtilis. To characterise this protein, designated PriA(Bs), we constructed priA(Bs) mutants. These mutants are poorly viable, filamentous and sensitive to rich medium and UV irradiation. Replication of pAMbeta1-type plasmids, which is initiated through the formation of a D-loop structure, and the activity of the primosome assembly site ssiA of plasmid pAMbeta1 are strongly affected in the mutants. The purified PriA(Bs) protein binds preferentially to the active strand of ssiA, even in the presence of B.subtilis SSB protein (SSB(Bs)). PriA(Bs) also binds stably and specifically to an artificial D-loop structure in vitro. These data show that PriA(Bs) recognises two specific substrates, ssiA and D-loops, and suggest that it triggers primosome assembly on them. PriA(Bs) also displays a single-stranded DNA-dependent ATPase activity, which is reduced in the presence of SSB(Bs), unless the ssiA sequence is present on the ssDNA substrate. Finally, PriA(Bs) is shown to be an active helicase. Altogether, these results demonstrate a clear functional identity between PriA(Ec) and PriA(Bs). However, PriA(Bs) does not complement an E.coli priA null mutant strain. This host specificity may be due to the divergence between the proteins composing the E.coli and B.subtilis PriA-dependent primosomes.

Domain structure of the HSC70 cochaperone, HIP.

The domain structure of the HSC70-interacting protein (HIP), a 43-kDa cytoplasmic cochaperone involved in the regulation of HSC70 chaperone activity and the maturation of progesterone receptor, has been probed by limited proteolysis and biophysical and biochemical approaches. HIP proteolysis by thrombin and chymotrypsin generates essentially two fragments, an NH2-terminal fragment of 25 kDa (N25) and a COOH-terminal fragment of 18 kDa (C18) that appear to be well folded and stable as indicated by circular dichroism and recombinant expression in Escherichia coli. NH2-terminal amino acid sequencing of the respective fragments indicates that both proteases cleave HIP within a predicted alpha-helix following the tetratricopeptide repeat (TPR) region, despite their different specificities and the presence of several potential cleavage sites scattered throughout the sequence, thus suggesting that this region is particularly accessible and may constitute a linker between two structural domains. After size exclusion chromatography, N25 and C18 elute as two distinct and homogeneous species having a Stokes radius of 49 and 24 A, respectively. Equilibrium sedimentation and sedimentation velocity indicate that N25 is a stable dimer, whereas C18 is monomeric in solution, with sedimentation coefficients of 3.2 and 2.3 S and f/f(o) values of 1.5 and 1.1 for N25 and C18, respectively, indicating that the N25 is elongated whereas C18 is globular in shape. Both domains are able to bind to the ATPase domain of HSC70 and inhibit rhodanese aggregation. Moreover, their effects appear to be additive when used in combination, suggesting a cooperation of these domains in the full-length protein not only for HSC70 binding but also for chaperone activity. Altogether, these results indicate that HIP is made of two structural and functional domains, an NH2-terminal 25-kDa domain, responsible for the dimerization and the overall asymmetry of the molecule, and a COOH-terminal 18-kDa globular domain, both involved in HSC70 and unfolded protein binding.