Primase is required for helicase activity and helicase alters the specificity of primase in the enteropathogen Clostridium difficile.

DNA replication is an essential and conserved process in all domains of life and may serve as a target for the development of new antimicrobials. However, such developments are hindered by subtle mechanistic differences and limited understanding of DNA replication in pathogenic microorganisms. Clostridium difficile is the main cause of healthcare-associated diarrhoea and its DNA replication machinery is virtually uncharacterized. We identify and characterize the mechanistic details of the putative replicative helicase (CD3657), helicase-loader ATPase (CD3654) and primase (CD1454) of C. difficile, and reconstitute helicase and primase activities in vitro We demonstrate a direct and ATP-dependent interaction between the helicase loader and the helicase. Furthermore, we find that helicase activity is dependent on the presence of primase in vitro The inherent trinucleotide specificity of primase is determined by a single lysine residue and is similar to the primase of the extreme thermophile Aquifex aeolicus. However, the presence of helicase allows more efficient de novo synthesis of RNA primers from non-preferred trinucleotides. Thus, loader-helicase-primase interactions, which crucially mediate helicase loading and activation during DNA replication in all organisms, differ critically in C. difficile from that of the well-studied Gram-positive Bacillus subtilis model.

Cellular location and activity of Escherichia coli RecG proteins shed light on the function of its structurally unresolved C-terminus.

RecG is a DNA translocase encoded by most species of bacteria. The Escherichia coli protein targets branched DNA substrates and drives the unwinding and rewinding of DNA strands. Its ability to remodel replication forks and to genetically interact with PriA protein have led to the idea that it plays an important role in securing faithful genome duplication. Here we report that RecG co-localises with sites of DNA replication and identify conserved arginine and tryptophan residues near its C-terminus that are needed for this localisation. We establish that the extreme C-terminus, which is not resolved in the crystal structure, is vital for DNA unwinding but not for DNA binding. Substituting an alanine for a highly conserved tyrosine near the very end results in a substantial reduction in the ability to unwind replication fork and Holliday junction structures but has no effect on substrate affinity. Deleting or substituting the terminal alanine causes an even greater reduction in unwinding activity, which is somewhat surprising as this residue is not uniformly present in closely related RecG proteins. More significantly, the extreme C-terminal mutations have little effect on localisation. Mutations that do prevent localisation result in only a slight reduction in the capacity for DNA repair.

Modulation of DNA damage tolerance in Escherichia coli recG and ruv strains by mutations affecting PriB, the ribosome and RNA polymerase.

RecG is a DNA translocase that helps to maintain genomic integrity. Initial studies suggested a role in promoting recombination, a possibility consistent with synergism between recG and ruv null alleles and reinforced when the protein was shown to unwind Holliday junctions. In this article we describe novel suppressors of recG and show that the pathology seen without RecG is suppressed on reducing or eliminating PriB, a component of the PriA system for replisome assembly and replication restart. Suppression is conditional, depending on additional mutations that modify ribosomal subunit S6 or one of three subunits of RNA polymerase. The latter suppress phenotypes associated with deletion of priB, enabling the deletion to suppress recG. They include alleles likely to disrupt interactions with transcription anti-terminator, NusA. Deleting priB has a different effect in ruv strains. It provokes abortive recombination and compromises DNA repair in a manner consistent with PriB being required to limit exposure of recombinogenic ssDNA. This synergism is reduced by the RNA polymerase mutations identified. Taken together, the results reveal that RecG curbs a potentially negative effect of proteins that direct replication fork assembly at sites removed from the normal origin, a facility needed to resolve conflicts between replication and transcription.

Chromosomal replication initiation machinery of low-G+C-content Firmicutes.

Much of our knowledge of the initiation of DNA replication comes from studies in the gram-negative model organism Escherichia coli. However, the location and structure of the origin of replication within the E. coli genome and the identification and study of the proteins which constitute the E. coli initiation complex suggest that it might not be as universal as once thought. The archetypal low-G+C-content gram-positive Firmicutes initiate DNA replication via a unique primosomal machinery, quite distinct from that seen in E. coli, and an examination of oriC in the Firmicutes species Bacillus subtilis indicates that it might provide a better model for the ancestral bacterial origin of replication. Therefore, the study of replication initiation in organisms other than E. coli, such as B. subtilis, will greatly advance our knowledge and understanding of these processes as a whole. In this minireview, we highlight the structure-function relationships of the Firmicutes primosomal proteins, discuss the significance of their oriC architecture, and present a model for replication initiation at oriC.

The RdgC protein employs a novel mechanism involving a finger domain to bind to circular DNA.

The DNA-binding protein RdgC has been identified as an inhibitor of RecA-mediated homologous recombination in Escherichia coli. In Neisseria species, RdgC also has a role in virulence-associated antigenic variation. We have previously solved the crystal structure of the E. coli RdgC protein and shown it to form a toroidal dimer. In this study, we have conducted a mutational analysis of residues proposed to mediate interactions at the dimer interfaces. We demonstrate that destabilizing either interface has a serious effect on in vivo function, even though a stable complex with circular DNA was still observed. We conclude that tight binding is required for inhibition of RecA activity. We also investigated the role of the RdgC finger domain, and demonstrate that it plays a crucial role in the binding of circular DNA. Together, these data allow us to propose a model for how RdgC loads onto DNA. We discuss how RdgC might inhibit RecA-mediated strand exchange, and how RdgC might be displaced by other DNA metabolism enzymes such as polymerases and helicases.

Promoting and avoiding recombination: contrasting activities of the Escherichia coli RuvABC Holliday junction resolvase and RecG DNA translocase.

RuvABC and RecG are thought to provide alternative pathways for the late stages of recombination in Escherichia coli. Inactivation of both blocks the recovery of recombinants in genetic crosses. RuvABC resolves Holliday junctions, with RuvAB driving branch migration and RuvC catalyzing junction cleavage. RecG also drives branch migration, but no nuclease has been identified that might act with RecG to cleave junctions, apart from RusA, which is not normally expressed. We searched for an alternative nuclease using a synthetic lethality assay to screen for mutations causing inviability in the absence of RuvC, on the premise that a strain without any ability to cut junctions might be inviable. All the mutations identified mapped to polA, dam, or uvrD. None of these genes encodes a nuclease that cleaves Holliday junctions. Probing the reason for the inviability using the RusA Holliday junction resolvase provided strong evidence in each case that the RecG pathway is very ineffective at removing junctions and indicated that a nuclease component most probably does not exist. It also revealed new suppressors of recG, which were located to the ssb gene. Taken together with the results from the synthetic lethality assays, the properties of the mutant SSB proteins provide evidence that, rather than promoting recombination, a major function of RecG is to curb potentially pathological replication initiated via PriA protein at sites remote from oriC.

Is RecG a general guardian of the bacterial genome?

The RecG protein of Escherichia coli is a double-stranded DNA translocase that unwinds a variety of branched DNAs in vitro, including Holliday junctions, replication forks, D-loops and R-loops. Coupled with the reported pleiotropy of recG mutations, this broad range of potential targets has made it hard to pin down what the protein does in vivo, though roles in recombination and replication fork repair have been suggested. However, recent studies suggest that RecG provides a more general defence against pathological DNA replication. We have postulated that this is achieved through the ability of RecG to eliminate substrates that the replication restart protein, PriA, could otherwise exploit to re-replicate the chromosome. Without RecG, PriA triggers a cascade of events that interfere with the duplication and segregation of chromosomes. Here we review the studies that led us to this idea and to conclude that RecG may be both a specialist activity and a general guardian of the genome.

A soluble RecN homologue provides means for biochemical and genetic analysis of DNA double-strand break repair in Escherichia coli.

RecN is a highly conserved, SMC-like protein in bacteria. It plays an important role in the repair of DNA double-strand breaks and is therefore a key factor in maintaining genome integrity. The insolubility of Escherichia coli RecN has limited efforts to unravel its function. We overcame this limitation by replacing the resident coding sequence with that of Haemophilus influenzae RecN. The heterologous construct expresses Haemophilus RecN from the SOS-inducible E. coli promoter. The hybrid gene is fully functional, promoting survival after I-SceI induced DNA breakage, gamma irradiation or exposure to mitomycin C as effectively as the native gene, indicating that the repair activity is conserved between these two species. H. influenzae RecN is quite soluble, even when expressed at high levels, and is readily purified. Its analysis by ionisation-mass spectrometry, gel filtration and glutaraldehyde crosslinking indicates that it is probably a dimer under physiological conditions, although a higher multimer cannot be excluded. The purified protein displays a weak ATPase activity that is essential for its DNA repair function in vivo. However, no DNA-binding activity was detected, which contrasts with RecN from Bacillus subtilis. RecN proteins from Aquifex aeolicus and Bacteriodes fragilis also proved soluble. Neither binds DNA, but the Aquifex RecN has weak ATPase activity. Our findings support studies indicating that RecN, and the SOS response in general, behave differently in E. coli and B. subtilis. The hybrid recN reported provides new opportunities to study the genetics and biochemistry of how RecN operates in E. coli.

Archaeal Hel308 domain V couples DNA binding to ATP hydrolysis and positions DNA for unwinding over the helicase ratchet.

Hel308 and PolQ are paralogues with roles promoting genome stability in archaea and higher eukaryotes. The context in which they act is not clear, although Hel308 helicase from archaea may interact with abnormal replication forks. The atomic structure of archaeal Hel308 from Archaeoglobus fulgidus in complex with DNA was recently reported and has given insights into the mechanisms of superfamily-2 helicases generally. An intriguing aspect of the structure was the positioning of a C-terminal domain V relative to single-stranded DNA and to the helicase ratchet domain IV. We have mutagenised a triplet of arginine residues in domain V of archaeal Hel308 to assess the effects on DNA binding, unwinding, and ATPase activities. Our observations can now be interpreted in light of the atomic structure. We describe crucial roles for domain V as a brake on ATP hydrolysis by coupling it to binding single-stranded DNA and in positioning DNA relative to the helicase ratchet domain IV for efficient unwinding of forked DNA.

Ring structure of the Escherichia coli DNA-binding protein RdgC associated with recombination and replication fork repair.

The DNA-binding protein, RdgC, is associated with recombination and replication fork repair in Escherichia coli and with the virulence-associated, pilin antigenic variation mediated by RecA and other recombination proteins in Neisseria species. We solved the structure of the E. coli protein and refined it to 2.4A. RdgC crystallizes as a dimer with a head-to-head, tail-to-tail organization forming a ring with a 30 A diameter hole at the center. The protein fold is unique and reminiscent of a horseshoe with twin gates closing the open end. The central hole is lined with positively charged residues and provides a highly plausible DNA binding channel consistent with the nonspecific mode of binding detected in vitro and with the ability of RdgC to modulate RecA function in vivo.

Interactions of RadB, a DNA repair protein in archaea, with DNA and ATP.

The RecA family of recombinases (RecA, Rad51, RadA and UvsX) catalyse strand-exchange between homologous DNA molecules by utilising conserved DNA-binding modules and a common core ATPase domain. RadB was identified in archaea as a Rad51-like protein on the basis of conserved ATPase sequences. However, RadB does not catalyse strand exchange and does not turn over ATP efficiently. RadB does bind DNA, and here we report a triplet of residues (Lys-His-Arg) that is highly conserved at the RadB C terminus, and is crucial for DNA binding. This is consistent with the motif forming a "basic patch" of highly conserved residues identified in an atomic structure of RadB from Thermococcus kodakaraensis. As the triplet motif is conserved at the C terminus of XRCC2 also, a mammalian Rad51-paralogue, we present a phylogenetic analysis that clarifies the relationship between RadB, Rad51-paralogues and recombinases. We investigate interactions between RadB and ATP using genetics and biochemistry; ATP binding by RadB is needed to promote survival of Haloferax volcanii after UV irradiation, and ATP, but not other NTPs, induces pronounced conformational change in RadB. This is the first genetic analysis of radB, and establishes its importance for maintaining genome stability in archaea. ATP-induced conformational change in RadB may explain previous reports that RadB controls Holliday junction resolution by Hjc, depending on the presence or the absence of ATP.

DNA binding by the substrate specificity (wedge) domain of RecG helicase suggests a role in processivity.

RecG differs from most helicases acting on branched DNA in that it is thought to catalyze unwinding via translocation of a monomer on dsDNA, with a wedge domain facilitating strand separation. Conserved phenylalanines in the wedge are shown to be critical for DNA binding. When detached from the helicase domains, the wedge bound a Holliday junction with high affinity but failed to bind a replication fork structure. Further stabilizing contacts are identified in full-length RecG, which may explain fork binding. Detached from the wedge, the helicase region unwound junctions but had extremely low substrate affinity, arguing against the "classical inchworm" mode of translocation. We propose that the processivity of RecG on branched DNA substrates is dependent on the ability of the wedge to establish strong binding at the branch point. This keeps the helicase motor in contact with the substrate, enabling it to drive dsDNA translocation with high efficiency.

Conservation of RecG activity from pathogens to hyperthermophiles.

Maintaining the integrity of the genome is essential for the survival of all organisms. RecG helicase plays an important part in this process in Escherichia coli, promoting recombination and DNA repair, and providing ways to rescue stalled replication forks by way of a Holliday junction intermediate. We purified RecG proteins from three other species: two Gram-positive mesophiles, Bacillus subtilis and Streptococcus pneumoniae, and one extreme thermophile, Aquifex aeolicus. All three proteins bind and unwind replication fork and Holliday junction DNA molecules with efficiencies similar to the E. coli protein. Proteins from the Gram-positive species promote DNA repair in E. coli, indicating either that RecG acts alone or that any necessary protein-protein interactions are conserved. The S. pneumoniae RecG reduces plasmid copy number when expressed in E. coli, indicating that like the E. coli protein it unwinds plasmid R loop structures used to prime replication. This effect is not seen with B. subtilis RecG; the protein either lacks R loop unwinding activity or is compromised by having insufficient ATP. The A. aeolicus protein unwinds DNA well at 60 degrees C but is less efficient at 37 degrees C, explaining its inability to function in E. coli at this temperature. The N-terminal extension present in this protein was investigated and found to be dispensable for activity and thermo-stability. The results presented suggest that the role of RecG in DNA replication and repair is likely to be conserved throughout all bacteria, which underlines the importance of this protein in genome duplication and cell survival.

Interplay between DNA replication, recombination and repair based on the structure of RecG helicase.

Recent studies in Escherichia coli indicate that the interconversion of DNA replication fork and Holliday junction structures underpins chromosome duplication and helps secure faithful transmission of the genome from one generation to the next. It facilitates interplay between DNA replication, recombination and repair, and provides means to rescue replication forks stalled by lesions in or on the template DNA. Insight into how this interconversion may be catalysed has emerged from genetic, biochemical and structural studies of RecG protein, a member of superfamily 2 of DNA and RNA helicases. We describe how a single molecule of RecG might target a branched DNA structure and translocate a single duplex arm to drive branch migration of a Holliday junction, interconvert replication fork and Holliday junction structures and displace the invading strand from a D loop formed during recombination at a DNA end. We present genetic evidence suggesting how the latter activity may provide an efficient pathway for the repair of DNA double-strand breaks that avoids crossing over, thus facilitating chromosome segregation at cell division.

A model for dsDNA translocation revealed by a structural motif common to RecG and Mfd proteins.

RecG protein differs from other helicases analysed to atomic resolution in that it mediates strand separation via translocation on double-stranded (ds) rather than single-stranded (ss) DNA. We describe a highly conserved helical hairpin motif in RecG and show it to be important for helicase activity. It places two arginines (R609 and R630) in opposing positions within the component helices where they are stabilized by a network of hydrogen bonds involving a glutamate from helicase motif VI. We suggest that disruption of this feature, triggered by ATP hydrolysis, moves an adjacent loop structure in the dsDNA-binding channel and that a swinging arm motion of this loop drives translocation. Substitutions that reverse the charge at R609 or R630 reduce DNA unwinding and ATPase activities, and increase dsDNA binding, but do not affect branched DNA binding. Sequences forming the helical hairpin and loop structures are highly conserved in Mfd protein, a transcription-coupled DNA repair factor that also translocates on dsDNA. The possibility of type I restriction enzymes and chromatin-remodelling factors using similar structures to drive translocation on dsDNA is discussed.