Essential bacterial helicases that counteract the toxicity of recombination proteins

PcrA, Rep and UvrD are three closely related bacterial helicases with a DExx signature. PcrA is encoded by Gram-positive bacteria and is essential for cell growth. Rep and UvrD are encoded by Gram-negative bacteria, and mutants lacking both helicases are also not viable. To understand the non-viability of the helicase mutants, we characterized spontaneous extragenic suppressors of a Bacillus subtilis pcrA null mutation. Here we report that one of these suppressors maps in recF and that previously isolated mutations in B.subtilis recF, recL, recO and recR, which belong to the same complementation group, all suppress the lethality of a pcrA mutation. Similarly, recF, recO or recR mutations suppress the lethality of the Escherichia coli rep uvrD double mutant. We conclude that RecFOR proteins are toxic in cells devoid of PcrA in Gram-positive bacteria, or Rep and UvrD in Gram-negative bacteria, and propose that the RecFOR proteins interfere with an essential cellular process, possibly replication, when DExx helicases PcrA, or Rep and UvrD are absent.     

The unstructured C-terminal extension of UvrD interacts with UvrB,but is dispensable for nucleotide excision repair

During nucleotide excision repair (NER) in bacteria the UvrC nuclease and the short oligonucleotide that contains the DNA lesion are removed from the post-incision complex by UvrD, a superfamily 1A helicase. Helicases are frequently regulated by interactions with partner proteins, and immunoprecipitation experiments have previously indicated that UvrD interacts with UvrB, a component of the post-incision complex. We examined this interaction using 2-hybrid analysis and surface plasmon resonance spectroscopy, and found that the N-terminal domain and the unstructured region at the C-terminus of UvrD interact with UvrB. We analysed the properties of a truncated UvrD protein that lacked the unstructured C-terminal region and found that it showed a diminished affinity for single-stranded DNA, but retained the ability to displace both UvrC and the lesion-containing oligonucleotide from a post-incision nucleotide excision repair complex. The interaction of the C-terminal region of UvrD with UvrB is therefore not an essential feature of the mechanism by which UvrD disassembles the post-incision complex during NER. In further experiments we showed that PcrA helicase from Bacillus stearothermophilus can also displace UvrC and the excised oligonucleotide from a post-incision NER complex, which supports the idea that PcrA performs a UvrD-like function during NER in Gram-positive organisms.     

PcrA is an essential DNA helicase of Bacillus subtilis fulfilling functions both in repair and rolling-circle replication

The only DNA helicase essential for Escherichia coli viability is DnaB, the chromosome replication fork helicase. In contrast, in Bacillus subtilis , in addition to the DnaB counterpart called DnaC, we have found a second essential DNA helicase, called PcrA. It is 40% identical to the Rep and UvrD DNA helicases of E. coli and 61% identical to the PcrA helicase of Staphylococcus aureus . This gene is located at 55° on the chromosome and belongs to a putative operon together with a ligase gene ( lig ) and two unknown genes named pcrB and yerH . As PcrA was essential for cell viability, conditional mutants were constructed. In such mutants, chromosomal DNA synthesis was slightly decreased upon PcrA depletion, and rolling-circle replication of the plasmid pT181 was inhibited. Analysis of the replication intermediates showed that leading-strand synthesis of pT181 was prevented upon PcrA depletion. To compare PcrA with Rep and UvrD directly, the protein was produced in rep and uvrD mutants of E. coli . PcrA suppressed the UV sensitivity defect of a uvrD mutant but not its mutator phenotype. Furthermore, it conferred a Rep⁻ phenotype on E. coli . Altogether, these results show that PcrA is an helicase used for plasmid rolling-circle replication and suggest that it is also involved in UV repair.     

Study of the ATP-binding site of helicase IV from Escherichia coli

Helicases contain conserved motifs involved in ATP/magnesium/nucleic acid binding and in the mechanisms coupling nucleotide hydrolysis to duplex unwinding. None of these motifs are located at the adenine-binding pocket of the protein. We show here that the superfamily I helicase, helicase IV from Escherichia coli, utilizes a conserved glutamine and conserved aromatic residue to interact with ATP. Other superfamily I helicases such as, UvrD/Rep/PcrA also possess these residues but in addition they interact with adenine via a conserved arginine, which is replaced by a serine in helicase IV. Mutation of this serine residue in helicase IV into histidine or methionine leads to proteins with unaffected ATPase and DNA-binding activities but with low helicase activity. This suggests that residues located at the adenine-binding pocket, in addition to be involved in ATP-binding, are important for efficient coupling between ATP hydrolysis and DNA unwinding.     

Regulation of E. coli Rep helicase activity by PriC

Stalled DNA replication forks can result in incompletely replicated genomes and cell death. DNA replication restart pathways have evolved to deal with repair of stalled forks and E. coli Rep helicase functions in this capacity. Rep and an accessory protein, PriC, assemble at a stalled replication fork to facilitate loading of other replication proteins. A Rep monomer is a rapid and processive single stranded (ss) DNA translocase but needs to be activated to function as a helicase. Activation of Rep in vitro requires self-assembly to form a dimer, removal of its auto-inhibitory 2B sub-domain, or interactions with an accessory protein. Rep helicase activity has been shown to be stimulated by PriC, although the mechanism of activation is not clear. Using stopped flow kinetics, analytical sedimentation and single molecule fluorescence methods, we show that a PriC dimer activates the Rep monomer helicase and can also stimulate the Rep dimer helicase. We show that PriC can self-assemble to form dimers and tetramers and that Rep and PriC interact in the absence of DNA. We further show that PriC serves as a Rep processivity factor, presumably co-translocating with Rep during DNA unwinding. Activation is specific for Rep since PriC does not activate the UvrD helicase. Interaction of PriC with the C-terminal acidic tip of the ssDNA binding protein, SSB, eliminates Rep activation by stabilizing the PriC monomer. This suggests a likely mechanism for Rep activation by PriC at a stalled replication fork.     

PcrA/UvrD/Rep DNA helicases in bacterial genomes

Helicases, which utilize the energy liberated by the hydrolysis of nucleotides to unwind nucleic acids, are involved in many aspects of nucleic acid metabolism. Various DNA helicases from the PcrA/UvrD/Rep subfamily are essential for the survival of different pathogenic bacteria and we have recently shown that they can be inhibited with small synthetic molecules. Altogether this suggests that these enzymes are potential new drug targets. Since little is known about the presence of these enzymes in bacterial genomes, 99 bacterial genomes were analyzed in the present study. This analysis reveals which and how many of these enzymes are found in bacteria, but more important, it identifies several of these enzymes as potential drug target candidates. In addition, this work identifies several proteins, called here PURL, that have a high homology with the PcrA/UvrD/Rep proteins and that may form an additional group in this helicase subfamily.     

Bacillus stearothermophilus PcrA monomer is a single-stranded DNA translocase but not a processive helicase in vitro

Structural studies of the Bacillus stearothermophilus PcrA protein along with biochemical studies of the single-stranded (ss) DNA translocation activity of PcrA monomers have led to the suggestion that a PcrA monomer possesses processive helicase activity in vitro. Yet definitive studies testing whether the PcrA monomer possesses processive helicase activity have not been performed. Here we show, using single turnover kinetic methods, that monomers of PcrA are able to translocate along ssDNA, in the 3' to 5' direction, rapidly and processively, whereas these same monomers display no detectable helicase activity under the same solution conditions in vitro. The PcrA monomer ssDNA translocation activity, although necessary, is not sufficient for processive helicase activity, and thus the translocase and helicase activities of PcrA are separable. These results also suggest that the helicase activity of PcrA needs to be activated either by self-assembly or through interactions with accessory proteins. This same behavior is displayed by both the Escherichia coli Rep and UvrD monomers. Hence, all three of these SF1 enzymes are ssDNA translocases as monomers but do not display processive helicase activity in vitro unless activated. The fact that the translocase and helicase activities are separable suggests that each activity may be used for different functions in vivo.     

Localization of an accessory helicase at the replisome is critical in sustaining efficient genome duplication

Genome duplication requires accessory helicases to displace proteins ahead of advancing replication forks. Escherichia coli contains three helicases, Rep, UvrD and DinG, that might promote replication of protein-bound DNA. One of these helicases, Rep, also interacts with the replicative helicase DnaB. We demonstrate that Rep is the only putative accessory helicase whose absence results in an increased chromosome duplication time. We show also that the interaction between Rep and DnaB is required for Rep to maintain rapid genome duplication. Furthermore, this Rep-DnaB interaction is critical in minimizing the need for both recombinational processing of blocked replication forks and replisome reassembly, indicating that colocalization of Rep and DnaB minimizes stalling and subsequent inactivation of replication forks. These data indicate that E. coli contains only one helicase that acts as an accessory motor at the fork in wild-type cells, that such an activity is critical for the maintenance of rapid genome duplication and that colocalization with the replisome is crucial for this function. Given that the only other characterized accessory motor, Saccharomyces cerevisiae Rrm3p, associates physically with the replisome, our demonstration of the functional importance of such an association indicates that colocalization may be a conserved feature of accessory replicative motors.     

Characterisation of Bacillus stearothermophilus PcrA helicase: evidence against an active rolling mechanism.

PcrA from Bacillus stearothermophilus is a DNA helicase for which, despite the availability of a crystal structure, there is very little biochemical information. We show that the enzyme has a broad nucleotide specificity, even being able to hydrolyse ethenonucleotides, and is able to couple the hydrolysis to unwinding of DNA substrates. In common with the Escherichia coli helicases Rep and UvrD, PcrA is a 3'-5' helicase but at high protein concentrations it can also displace a substrate with a 5' tail. However, in contrast to Rep and UvrD, we do not see any evidence for dimerisation of the protein even in the presence of DNA. The enzyme shows a specificity for the DNA substrate in gel mobility assays, with the preferred substrate being one with both single and double stranded regions of DNA. We propose that these data, together with existing structural evidence, support an inchworm rather than a rolling model for 3'-5' helicase activity.     

The UvrD helicase and its modulation by the mismatch repair protein MutL

UvrD is a superfamily I DNA helicase with well documented roles in excision repair and methyl-directed mismatch repair (MMR) in addition to poorly understood roles in replication and recombination. The MutL protein is a homodimeric DNA-stimulated ATPase that plays a central role in MMR in Escherichia coli. This protein has been characterized as the master regulator of mismatch repair since it interacts with and modulates the activity of several other proteins involved in the mismatch repair pathway including MutS, MutH and UvrD. Here we present a brief summary of recent studies directed toward arriving at a better understanding of the interaction between MutL and UvrD, and the impact of this interaction on the activity of UvrD and its role in mismatch repair.     

Regulation of UvrD Helicase Activity by MutL

Escherichia coli UvrD is a superfamily 1 helicase/translocase involved in multiple DNA metabolic processes including methyl-directed mismatch DNA repair. Although a UvrD monomer can translocate along single-stranded DNA, a UvrD dimer is needed for processive helicase activity in vitro. E. coli MutL, a regulatory protein involved in methyl-directed mismatch repair, stimulates UvrD helicase activity; however, the mechanism is not well understood. Using single-molecule fluorescence and ensemble approaches, we find that a single MutL dimer can activate latent UvrD monomer helicase activity. However, we also find that MutL stimulates UvrD dimer helicase activity. We further find that MutL enhances the DNA-unwinding processivity of UvrD. Hence, MutL acts as a processivity factor by binding to and presumably moving along with UvrD to facilitate DNA unwinding.     

E. coli Rep oligomers are required to initiate DNA unwinding in vitro.

E. coli Rep protein is a 3' to 5' SF1 superfamily DNA helicase which is monomeric in the absence of DNA, but can dimerize upon binding either single-stranded or duplex DNA. A variety of biochemical studies have led to proposals that Rep dimerization is important for its helicase activity; however, recent structural studies of Bacillus stearothermophilus PcrA have led to suggestions that SF1 helicases, such as E. coli Rep and E. coli UvrD, function as monomeric helicases. We have examined the question of whether Rep oligomerization is important for its DNA helicase activity using pre-steady state stopped-flow and chemical quenched-flow kinetic studies of Rep-catalyzed DNA unwinding. The results from four independent experiments demonstrate that Rep oligomerization is required for initiation of DNA helicase activity in vitro. No DNA unwinding is observed when only a Rep monomer is bound to the DNA substrate, even when fluorescent DNA substrates are used that can detect partial unwinding of the first few base-pairs at the ss-ds-DNA junction. In fact, under these conditions, ATP hydrolysis causes dissociation of the Rep monomer from the DNA, rather than DNA unwinding. These studies demonstrate that wild-type Rep monomers are unable to initiate DNA unwinding in vitro, and that oligomerization is required.     

Helicase structure and mechanism

Structural information on helicase proteins has expanded recently beyond the DNA helicases Rep and PcrA, and the hepatitis C virus RNA helicase to include UvrB, members of the DEA(D/H)-box RNA helicase family, examples of DnaB-related helicases and RuvB. The expanding database of structures has clarified the structural 'theme and variations' that relate the different helicase families. Furthermore, information is emerging on the functions of the conserved helicase motifs and their participation in the mechanisms by which these proteins catalyze the remodeling of DNA and RNA in ATP-dependent activities.     

The beta-propeller protein YxaL increases the processivity of the PcrA helicase

The DNA helicase PcrA is found in gram-positive bacteria and belongs to the superfamily 1 (SF1) of helicases, together with Rep and UvrD helicases from Escherichia coli. These helicases have been extensively studied in vitro and their mode of unwinding are well characterised. However, little is known about the putative cellular partners of such helicases. To identify PcrA-interacting factors, PcrA was used as a bait in a genome-wide yeast two-hybrid screen of a Bacillus subtilis library. Three proteins with unknown functions - YxaL, YwhK and YerB - were found to interact specifically with PcrA. The yxaL gene was cloned, the product was overexpressed and purified, and its effect on the PcrA activity was investigated in vitro. YxaL enhanced the processivity of the PcrA helicase. A comparison of the amino acid sequence of YxaL with other proteins from data banks suggests that YxaL belongs to a family of proteins with a repeated domain, which adopt a typical three-dimensional structure designated as a "beta-propeller". This raises the possibility that YxaL acts as a connector protein between PcrA and another cellular component.     

Comparisons between the structures of HCV and Rep helicases reveal structural similarities between SF1 and SF2 super-families of helicases.

Three helicase structures have been determined recently: that of the DNA helicase PcrA, that of the hepatitis C virus RNA helicase, and that of the Escherichia coli DNA helicase Rep. PcrA and Rep belong to the same super-family of helicases (SF1) and are structurally very similar. In contrast, the HCV helicase belongs to a different super-family of helicases, SF2, and shows little sequence homology with the PcrA/Rep helicases. Yet, the HCV helicase is structurally similar to Rep/PcrA, suggesting preservation of structural scaffolds and relationships between helicase motifs across these two super-families. The comparison study presented here also reveals the existence of a new helicase motif in the SF1 family of helicases.     

UvrD controls the access of recombination proteins to blocked replication forks

Blocked replication forks often need to be processed by recombination proteins prior to replication restart. In Escherichia coli, the UvrD repair helicase was recently shown to act at inactivated replication forks, where it counteracts a deleterious action of RecA. Using two mutants affected for different subunits of the polymerase III holoenzyme (Pol IIIh), we show here that the anti-RecA action of UvrD at blocked forks reflects two different activities of this enzyme. A defective UvrD mutant is able to antagonize RecA in cells affected for the Pol IIIh catalytic subunit DnaE. In this mutant, RecA action at blocked forks specifically requires the protein RarA (MgsA). We propose that UvrD prevents RecA binding, possibly by counteracting RarA. In contrast, at forks affected for the Pol IIIh clamp (DnaN), RarA is not required for RecA binding and the ATPase function of UvrD is essential to counteract RecA, supporting the idea that UvrD removes RecA from DNA. UvrD action on RecA is conserved in evolution as it can be performed in E. coli by the UvrD homologue from Bacillus subtilis, PcrA.     

A Conserved Helicase Processivity Factor Is Needed for Conjugation and Replication of an Integrative and Conjugative Element

Integrative and conjugative elements (ICEs) are agents of horizontal gene transfer and have major roles in evolution and acquisition of new traits, including antibiotic resistances. ICEs are found integrated in a host chromosome and can excise and transfer to recipient bacteria via conjugation. Conjugation involves nicking of the ICE origin of transfer (oriT) by the ICE–encoded relaxase and transfer of the nicked single strand of ICE DNA. For ICEBs1 of Bacillus subtilis, nicking of oriT by the ICEBs1 relaxase NicK also initiates rolling circle replication. This autonomous replication of ICEBs1 is critical for stability of the excised element in growing cells. We found a conserved and previously uncharacterized ICE gene that is required for conjugation and replication of ICEBs1. Our results indicate that this gene, helP (formerly ydcP), encodes a helicase processivity factor that enables the host-encoded helicase PcrA to unwind the double-stranded ICEBs1 DNA. HelP was required for both conjugation and replication of ICEBs1, and HelP and NicK were the only ICEBs1 proteins needed for replication from ICEBs1 oriT. Using chromatin immunoprecipitation, we measured association of HelP, NicK, PcrA, and the host-encoded single-strand DNA binding protein Ssb with ICEBs1. We found that NicK was required for association of HelP and PcrA with ICEBs1 DNA. HelP was required for association of PcrA and Ssb with ICEBs1 regions distal, but not proximal, to oriT, indicating that PcrA needs HelP to progress beyond nicked oriT and unwind ICEBs1. In vitro, HelP directly stimulated the helicase activity of the PcrA homologue UvrD. Our findings demonstrate that HelP is a helicase processivity factor needed for efficient unwinding of ICEBs1 for conjugation and replication. Homologues of HelP and PcrA-type helicases are encoded on many known and putative ICEs. We propose that these factors are essential for ICE conjugation, replication, and genetic stability.     

RepD-mediated recruitment of PcrA helicase at the Staphylococcus aureus pC221 plasmid replication origin,oriD

Plasmid encoded replication initiation (Rep) proteins recruit host helicases to plasmid replication origins. Previously, we showed that RepD recruits directionally the PcrA helicase to the pC221 oriD, remains associated with it, and increases its processivity during plasmid unwinding. Here we show that RepD forms a complex extending upstream and downstream of the core oriD. Binding of RepD causes remodelling of a region upstream from the core oriD forming a ‘landing pad’ for the PcrA. PcrA is recruited by this extended RepD–DNA complex via an interaction with RepD at this upstream site. PcrA appears to have weak affinity for this region even in the absence of RepD. Upon binding of ADPNP (non-hydrolysable analogue of ATP), by PcrA, a conformational rearrangement of the RepD–PcrA–ATP initiation complex confines it strictly within the boundaries of the core oriD. We conclude that RepD-mediated recruitment of PcrA at oriD is a three step process. First, an extended RepD–oriD complex includes a region upstream from the core oriD; second, the PcrA is recruited to this upstream region and thirdly upon ATP-binding PcrA relocates within the core oriD.