Crystal structure and mutational study of RecOR provide insight into its mode of DNA binding

The crystal structure of the complex formed between Deinococcus radiodurans RecR and RecO (drRecOR) has been determined. In accordance with previous biochemical characterisation, the drRecOR complex displays a RecR:RecO molecular ratio of 2:1. The biologically relevant drRecOR entity consists of a heterohexamer in the form of two drRecO molecules positioned on either side of the tetrameric ring of drRecR, with their OB (oligonucleotide/oligosaccharide-binding) domains pointing towards the interior of the ring. Mutagenesis studies validated the protein-protein interactions observed in the crystal structure and allowed mapping of the residues in the drRecOR complex required for DNA binding. Furthermore, the preferred DNA substrate of drRecOR was identified as being 3'-overhanging DNA, as encountered at ssDNA-dsDNA junctions. Together these results suggest a possible mechanism for drRecOR recognition of stalled replication forks.     

Identification of the RecR Toprim domain as the binding site for both RecF and RecO. A role of RecR in RecFOR assembly at double-stranded DNA-single-stranded DNA junctions

The RecR protein forms complexes with RecF or RecO that direct the specific loading of RecA onto gapped DNA. However, the binding sites of RecF and RecO on RecR have yet to be identified. In this study, a Thermus thermophilus RecR dimer model was constructed by NMR analysis and homology modeling. NMR titration analysis suggested that the hairpin region of the helix-hairpin-helix motif in the cavity of the RecR dimer is a binding site for double-stranded DNA (dsDNA) and that the acidic cluster region of the Toprim domain is a RecO binding site. Mutations of Glu-84, Asp-88, and Glu-144 residues comprising that acidic cluster were generated. The E144A and E84A mutations decreased the binding affinity for RecO, but the D88A did not. Interestingly, the binding ability to RecF was abolished by E144A, suggesting that the region surrounding the RecR Glu-144 residue could be a binding site not only for RecO but also for RecF. Furthermore, RecR and RecF formed a 4:2 heterohexamer in solution that was unaffected by adding RecO, indicating a preference by RecR for RecF over RecO. The RecFR complex is considered to be involved in the recognition of the dsDNA-ssDNA junction, whereas RecO binds single-stranded DNA (ssDNA) and ssDNA-binding protein. Thus, the RecR Toprim domain may contribute to the RecO interaction with RecFR complexes at the dsDNA-ssDNA junction site during recombinational DNA repair mediated by the RecFOR.     

An ‘open’ structure of the RecOR complex supports ssDNA binding within the core of the complex

Efficient DNA repair is critical for cell survival and the maintenance of genome integrity. The homologous recombination pathway is responsible for the repair of DNA double-strand breaks within cells. Initiation of this pathway in bacteria can be carried out by either the RecBCD or the RecFOR proteins. An important regulatory player within the RecFOR pathway is the RecOR complex that facilitates RecA loading onto DNA. Here we report new data regarding the assembly of Deinococcus radiodurans RecOR and its interaction with DNA, providing novel mechanistic insight into the mode of action of RecOR in homologous recombination. We present a higher resolution crystal structure of RecOR in an ‘open’ conformation in which the tetrameric RecR ring flanked by two RecO molecules is accessible for DNA binding. We show using small-angle neutron scattering and mutagenesis studies that DNA binding does indeed occur within the RecR ring. Binding of single-stranded DNA occurs without any major conformational changes of the RecOR complex while structural rearrangements are observed on double-stranded DNA binding. Finally, our molecular dynamics simulations, supported by our biochemical data, provide a detailed picture of the DNA binding motif of RecOR and reveal that single-stranded DNA is sandwiched between the two facing oligonucleotide binding domains of RecO within the RecR ring.     

The process of displacing the single-stranded DNA-binding protein from single-stranded DNA by RecO and RecR proteins

The regions of single-stranded (ss) DNA that result from DNA damage are immediately coated by the ssDNA-binding protein (SSB). RecF pathway proteins facilitate the displacement of SSB from ssDNA, allowing the RecA protein to form protein filaments on the ssDNA region, which facilitates the process of recombinational DNA repair. In this study, we examined the mechanism of SSB displacement from ssDNA using purified Thermus thermophilus RecF pathway proteins. To date, RecO and RecR are thought to act as the RecOR complex. However, our results indicate that RecO and RecR have distinct functions. We found that RecR binds both RecF and RecO, and that RecO binds RecR, SSB and ssDNA. The electron microscopic studies indicated that SSB is displaced from ssDNA by RecO. In addition, pull-down assays indicated that the displaced SSB still remains indirectly attached to ssDNA through its interaction with RecO in the RecO-ssDNA complex. In the presence of both SSB and RecO, the ssDNA-dependent ATPase activity of RecA was inhibited, but was restored by the addition of RecR. Interestingly, the interaction of RecR with RecO affected the ssDNA-binding properties of RecO. These results suggest a model of SSB displacement from the ssDNA by RecF pathway proteins.     

The RecOR proteins modulate RecA protein function at 5' ends of single-stranded DNA.

The Escherichia coli RecF, RecO and RecR pro teins have previously been implicated in bacterial recombinational DNA repair at DNA gaps. The RecOR-facilitated binding of RecA protein to single-stranded DNA (ssDNA) that is bound by single-stranded DNA-binding protein (SSB) is much faster if the ssDNA is linear, suggesting that a DNA end (rather than a gap) facilitates binding. In addition, the RecOR complex facilitates RecA protein-mediated D-loop formation at the 5' ends of linear ssDNAs. RecR protein remains associated with the RecA filament and its continued presence is required to prevent filament disassembly. RecF protein competes with RecO protein for RecR protein association and its addition destabilizes RecAOR filaments. An enhanced function of the RecO and RecR proteins can thus be seen in vitro at the 5' ends of linear ssDNA that is not as evident in DNA gaps. This function is countered by the RecF/RecO competition for association with the RecR protein.     

Ring-shaped architecture of RecR: implications for its role in homologous recombinational DNA repair

RecR, together with RecF and RecO, facilitates RecA loading in the RecF pathway of homologous recombinational DNA repair in procaryotes. The human Rad52 protein is a functional counterpart of RecFOR. We present here the crystal structure of RecR from Deinococcus radiodurans (DR RecR). A monomer of DR RecR has a two-domain structure: the N-terminal domain with a helix-hairpin-helix (HhH) motif and the C-terminal domain with a Cys4 zinc-finger motif, a Toprim domain and a Walker B motif. Four such monomers form a ring-shaped tetramer of 222 symmetry with a central hole of 30-35 angstroms diameter. In the crystal, two tetramers are concatenated, implying that the RecR tetramer is capable of opening and closing. We also show that DR RecR binds to both dsDNA and ssDNA, and that its HhH motif is essential for DNA binding.     

Mechanism of RecO recruitment to DNA by single-stranded DNA binding protein

RecO is a recombination mediator protein (RMP) important for homologous recombination, replication repair and DNA annealing in bacteria. In all pathways, the single-stranded (ss) DNA binding protein, SSB, plays an inhibitory role by protecting ssDNA from annealing and recombinase binding. Conversely, SSB may stimulate each reaction through direct interaction with RecO. We present a crystal structure of Escherichia coli RecO bound to the conserved SSB C-terminus (SSB-Ct). SSB-Ct binds the hydrophobic pocket of RecO in a conformation similar to that observed in the ExoI/SSB-Ct complex. Hydrophobic interactions facilitate binding of SSB-Ct to RecO and RecO/RecR complex in both low and moderate ionic strength solutions. In contrast, RecO interaction with DNA is inhibited by an elevated salt concentration. The SSB mutant lacking SSB-Ct also inhibits RecO-mediated DNA annealing activity in a salt-dependent manner. Neither RecO nor RecOR dissociates SSB from ssDNA. Therefore, in E. coli, SSB recruits RMPs to ssDNA through SSB-Ct, and RMPs are likely to alter the conformation of SSB-bound ssDNA without SSB dissociation to initiate annealing or recombination. Intriguingly, Deinococcus radiodurans RecO does not bind SSB-Ct and weakly interacts with the peptide in the presence of RecR, suggesting the diverse mechanisms of DNA repair pathways mediated by RecO in different organisms.     

A novel structure of DNA repair protein RecO from Deinococcus radiodurans

Recovery of arrested replication requires coordinated action of DNA repair, replication, and recombination machineries. Bacterial RecO protein is a member of RecF recombination repair pathway important for replication recovery. RecO possesses two distinct activities in vitro, closely resembling those of eukaryotic protein Rad52: DNA annealing and RecA-mediated DNA recombination. Here we present the crystal structure of the RecO protein from the extremely radiation resistant bacteria Deinococcus radiodurans (DrRecO) and characterize its DNA binding and strand annealing properties. The RecO structure is totally different from the Rad52 structure. DrRecO is comprised of three structural domains: an N-terminal domain which adopts an OB-fold, a novel alpha-helical domain, and an unusual zinc-binding domain. Sequence alignments suggest that the multidomain architecture is conserved between RecO proteins from other bacterial species and is suitable to elucidate sites of protein-protein and DNA-protein interactions necessary for RecO functions during the replication recovery and DNA repair.     

Allosteric effects of SSB C-terminal tail on assembly of E. coli RecOR proteins

Escherichia coli RecO is a recombination mediator protein that functions in the RecF pathway of homologous recombination, in concert with RecR, and interacts with E. coli single stranded (ss) DNA binding (SSB) protein via the last 9 amino acids of the C-terminal tails (SSB-Ct). Structures of the E. coli RecR and RecOR complexes are unavailable; however, crystal structures from other organisms show differences in RecR oligomeric state and RecO stoichiometry. We report analytical ultracentrifugation studies of E. coli RecR assembly and its interaction with RecO for a range of solution conditions using both sedimentation velocity and equilibrium approaches. We find that RecR exists in a pH-dependent dimer-tetramer equilibrium that explains the different assembly states reported in previous studies. RecO binds with positive cooperativity to a RecR tetramer, forming both RecR₄O and RecR₄O₂ complexes. We find no evidence of a stable RecO complex with RecR dimers. However, binding of RecO to SSB-Ct peptides elicits an allosteric effect, eliminating the positive cooperativity and shifting the equilibrium to favor a RecR₄O complex. These studies suggest a mechanism for how SSB binding to RecO influences the distribution of RecOR complexes to facilitate loading of RecA onto SSB coated ssDNA to initiate homologous recombination.     

Regulation of bacterial RecA protein function

The RecA protein is a recombinase functioning in recombinational DNA repair in bacteria. RecA is regulated at many levels. The expression of the recA gene is regulated within the SOS response. The activity of the RecA protein itself is autoregulated by its own C-terminus. RecA is also regulated by the action of other proteins. To date, these include the RecF, RecO, RecR, DinI, RecX, RdgC, PsiB, and UvrD proteins. The SSB protein also indirectly affects RecA function by competing for ssDNA binding sites. The RecO and RecR, and possibly the RecF proteins, all facilitate RecA loading onto SSB-coated ssDNA. The RecX protein blocks RecA filament extension, and may have other effects on RecA activity. The DinI protein stabilizes RecA filaments. The RdgC protein binds to dsDNA and blocks RecA access to dsDNA. The PsiB protein, encoded by F plasmids, is uncharacterized, but may inhibit RecA in some manner. The UvrD helicase removes RecA filaments from RecA. All of these proteins function in a network that determines where and how RecA functions. Additional regulatory proteins may remain to be discovered. The elaborate regulatory pattern is likely to be reprised for RecA homologues in archaeans and eukaryotes.     

RecFOR and RecOR as distinct RecA loading pathways

The molecular role of the RecF protein in loading RecA protein onto single-stranded DNA (ssDNA)-binding protein-coated ssDNA has been obscured by the facility with which the RecO and RecR proteins alone perform this function. We now show that RecFOR and RecOR define distinct RecA loading functions that operate optimally in different contexts. RecFOR, but not RecOR, is most effective when RecF(R) is bound near an ssDNA/double-stranded (dsDNA) junction. However, RecF(R) has no enhanced binding affinity for such a junction. RecO and RecR proteins are both required under all conditions in which the RecFOR pathway operates. The RecOR pathway is uniquely distinguished by a required interaction between RecO protein and the ssDNA binding protein C terminus. The RecOR pathway is more efficient for RecA loading onto ssDNA when no proximal dsDNA is available. A merger of new and published results leads to a new model for RecFOR function.     

Deinococcus radiodurans DR1088 is a novel RecF‐interacting protein that stimulates single‐stranded DNA annealing

RecF, together with the recombination mediators RecO and RecR, is required in the RecFOR homologous recombination repair pathway in bacteria. In this study, a recF‐dr1088 operon, which is highly conserved in the Deinococcus‐Thermus phylum, was identified in Deinococcus radiodurans. Interaction between DRRecF and DR1088 was confirmed by yeast two‐hybrid and pull‐down assays. DR1088 exhibited some RecO‐like biochemical properties including single/double‐stranded DNA binding activity, ssDNA binding protein (SSB) replacement ability and ssDNA (with or without SSB) annealing activity. However, unlike other recombination proteins, dr1088 is essential for cell viability. These results indicate that DR1088 might play a role in DNA replication and DNA repair processes.     

Regulation of Bacterial RecA Protein Function

ABSTRACT

The RecA protein is a recombinase functioning in recombinational DNA repair in bacteria. RecA is regulated at many levels. The expression of the recA gene is regulated within the SOS response. The activity of the RecA protein itself is autoregulated by its own C-terminus. RecA is also regulated by the action of other proteins. To date, these include the RecF, RecO, RecR, DinI, RecX, RdgC, PsiB, and UvrD proteins. The SSB protein also indirectly affects RecA function by competing for ssDNA binding sites. The RecO and RecR, and possibly the RecF proteins, all facilitate RecA loading onto SSB-coated ssDNA. The RecX protein blocks RecA filament extension, and may have other effects on RecA activity. The DinI protein stabilizes RecA filaments. The RdgC protein binds to dsDNA and blocks RecA access to dsDNA. The PsiB protein, encoded by F plasmids, is uncharacterized, but may inhibit RecA in some manner. The UvrD helicase removes RecA filaments from RecA. All of these proteins function in a network that determines where and how RecA functions. Additional regulatory proteins may remain to be discovered. The elaborate regulatory pattern is likely to be reprised for RecA homologues in archaeans and eukaryotes.     

RecOR complex including RecR N-N dimer and RecO monomer displays a high affinity for ssDNA

RecR is an important recombination mediator protein in the RecFOR pathway. RecR together with RecO and RecF facilitates RecA nucleoprotein filament formation and homologous pairing. Structural and biochemical studies of Thermoanaerobacter tengcongensis RecR (TTERecR) and its series mutants revealed that TTERecR uses the N-N dimer as a basic functional unit to interact with TTERecO monomer. Two TTERecR N-N dimers form a ring-shaped tetramer via an interaction between their C-terminal regions. The tetramer is a result of crystallization only. Hydrophobic interactions between the entire helix-hairpin-helix domains within the N-terminal regions of two TTERecR monomers are necessary for formation of a RecR functional N-N dimer. The TTERecR N-N dimer conformation also affects formation of a hydrophobic patch, which creates a binding site for TTERecO in the TTERecR Toprim domain. In addition, we demonstrate that TTERecR does not bind single-stranded DNA (ssDNA) and binds double-stranded DNA very weakly, whereas TTERecOR complex can stably bind DNA, with a higher affinity for ssDNA than double-stranded DNA. Based on these results, we propose an interaction model for the RecOR:ssDNA complex.     

RecO acts with RecF and RecR to protect and maintain replication forks blocked by UV-induced DNA damage in Escherichia coli

In Escherichia coli, recF and recR are required to stabilize and maintain replication forks arrested by UV-induced DNA damage. In the absence of RecF, replication fails to recover, and the nascent lagging strand of the arrested replication fork is extensively degraded by the RecQ helicase and RecJ nuclease. recO mutants are epistatic with recF and recR with respect to recombination and survival assays after DNA damage. In this study, we show that RecO functions with RecF and RecR to protect the nascent lagging strand of arrested replication forks after UV-irradiation. In the absence of RecO, the nascent DNA at arrested replication forks is extensively degraded and replication fails to recover. The extent of nascent DNA degradation is equivalent in single, double, or triple mutants of recF, recO, or recR, and the degradation is dependent upon RecJ and RecQ functions. Because RecF has been shown to protect the nascent lagging strand from degradation, these observations indicate that RecR and RecO function with RecF to protect the same nascent strand of the arrested replication fork and are likely to act at a common point during the recovery process. We discuss these results in relation to the biochemical and cellular properties of RecF, RecO, and RecR and their potential role in loading RecA filaments to maintain the replication fork structure after the arrest of replication by UV-induced DNA damage.     

RecQ and RecJ process blocked replication forks prior to the resumption of replication in UV-irradiated Escherichia coli

The accurate recovery of replication following DNA damage and repair is critical for the maintenance of genomic integrity. In Escherichia coli, the recovery of replication following UV-induced DNA damage is dependent upon several proteins in the recF pathway, including RecF, RecO, and RecR. Two other recF pathway proteins, the RecQ helicase and the RecJ exonuclease, have been shown to affect the sites and frequencies at which illegitimate rearrangements occur following UV-induced DNA damage, suggesting that they also may function during the recovery of replication. We show here that RecQ and RecJ process the nascent DNA at blocked replication forks prior to the resumption of DNA synthesis. The processing involves selective degradation of the nascent lagging DNA strand and it requires both RecQ and RecJ. We suggest that this processing may serve to lengthen the substrate that can be recognized and stabilized by the RecA protein at the replication fork, thereby helping to ensure the accurate recovery of replication after the obstructing lesion has been repaired. Received: 1 June 1999 / Accepted: 28 July 1999     

A dual role for mycobacterial RecO in RecA-dependent homologous recombination and RecA-independent single-strand annealing

Mycobacteria have two genetically distinct pathways for the homology-directed repair of DNA double-strand breaks: homologous recombination (HR) and single-strand annealing (SSA). HR is abolished by deletion of RecA and reduced in the absence of the AdnAB helicase/nuclease. By contrast, SSA is RecA-independent and requires RecBCD. Here we examine the function of RecO in mycobacterial DNA recombination and repair. Loss of RecO elicits hypersensitivity to DNA damaging agents similar to that caused by deletion of RecA. We show that RecO participates in RecA-dependent HR in a pathway parallel to the AdnAB pathway. We also find that RecO plays a role in the RecA-independent SSA pathway. The mycobacterial RecO protein displays a zinc-dependent DNA binding activity in vitro and accelerates the annealing of SSB-coated single-stranded DNA. These findings establish a role for RecO in two pathways of mycobacterial DNA double-strand break repair and suggest an in vivo function for the DNA annealing activity of RecO proteins, thereby underscoring their similarity to eukaryal Rad52.     

Unveiling novel RecO distant orthologues involved in homologous recombination

The generation of a RecA filament on single-stranded DNA is a critical step in homologous recombination. Two main pathways leading to the formation of the nucleofilament have been identified in bacteria, based on the protein complexes mediating RecA loading: RecBCD (AddAB) and RecFOR. Many bacterial species seem to lack some of the components involved in these complexes. The current annotation of the Helicobacter pylori genome suggests that this highly diverse bacterial pathogen has a reduced set of recombination mediator proteins. While it is now clear that homologous recombination plays a critical role in generating H. pylori diversity by allowing genomic DNA rearrangements and integration through transformation of exogenous DNA into the chromosome, no complete mediator complex is deduced from the sequence of its genome. Here we show by bioinformatics analysis the presence of a RecO remote orthologue that allowed the identification of a new set of RecO proteins present in all bacterial species where a RecR but not RecO was previously identified. HpRecO shares less than 15% identity with previously characterized homologues. Genetic dissection of recombination pathways shows that this novel RecO and the remote RecB homologue present in H. pylori are functional in repair and in RecA-dependent intrachromosomal recombination, defining two initiation pathways with little overlap. We found, however, that neither RecOR nor RecB contributes to transformation, suggesting the presence of a third, specialized, RecA-dependent pathway responsible for the integration of transforming DNA into the chromosome of this naturally competent bacteria. These results provide insight into the mechanisms that this successful pathogen uses to generate genetic diversity and adapt to changing environments and new hosts.     

RecFOR proteins load RecA protein onto gapped DNA to accelerate DNA strand exchange: a universal step of recombinational repair

Genetic evidence suggests that the RecF, RecO, and RecR (RecFOR) proteins participate in a common step of DNA recombination and repair, yet the biochemical event requiring collaboration of all three proteins is unknown. Here, we show that the concerted action of the RecFOR complex directs the loading of RecA protein specifically onto gapped DNA that is coated with single-stranded DNA binding (SSB) protein, thereby accelerating DNA strand exchange. The RecFOR complex recognizes the junction between the ssDNA and dsDNA regions and requires a base-paired 5' terminus at the junction. Thus, the RecFOR complex is a structure-specific mediator that targets recombinational repair to ssDNA-dsDNA junctions. This reaction reconstitutes the initial steps of recombinational gapped DNA repair and uncovers an event also common to the repair of ssDNA-tailed intermediates of dsDNA-break repair. We propose that the behavior of the RecFOR proteins is mimicked by functional counterparts that exist in all organisms.     

SSB protein limits RecOR binding onto single-stranded DNA

The RecO and RecR proteins form a complex that promotes the nucleation of RecA protein filaments onto SSB protein-coated single-stranded DNA (ssDNA). However, even when RecO and RecR proteins are provided at optimal concentrations, the loading of RecA protein is surprisingly slow, typically proceeding with a lag of 10 min or more. The rate-limiting step in RecOR-promoted RecA nucleation is the binding of RecOR protein to ssDNA, which is inhibited by SSB protein despite the documented interaction between RecO and SSB. Full activity of RecOR is seen only when RecOR is preincubated with ssDNA prior to the addition of SSB. The slow binding of RecOR to SSB-coated ssDNA involves the C terminus of SSB. When an SSB variant that lacks the C-terminal 8 amino acids is used, the capacity of RecOR to facilitate RecA loading onto the ssDNA is largely abolished. The results are used in an expanded model for RecOR action.