Role of LrpC from Bacillus subtilis in DNA transactions during DNA repair and recombination

Bacillus subtilis LrpC is a sequence-independent DNA-binding and DNA-bending protein, which binds both single-stranded (ss) and double-stranded (ds) DNA and facilitates the formation of higher order protein–DNA complexes in vitro. LrpC binds at different sites within the same DNA molecule promoting intramolecular ligation. When bound to separate molecules, it promotes intermolecular ligation, and joint molecule formation between a circular ssDNA and a homologous ssDNA-tailed linear dsDNA. LrpC binding showed a higher affinity for 4-way (Holliday) junctions in their open conformation, when compared with curved dsDNA. Consistent with these biochemical activities, an lrpC null mutant strain rendered cells sensitive to DNA damaging agents such as methyl methanesulfonate and 4-nitroquinoline-1-oxide, and showed a segregation defect. These findings collectively suggest that LrpC may be involved in DNA transactions during DNA repair and recombination.     

Biochemical analysis of the substrate specificity and sequence preference of endonuclease IV from bacteriophage T4, a dC-specific endonuclease implicated in restriction of dC-substituted T4 DNA synthesis

Endonuclease IV encoded by denB of bacteriophage T4 is implicated in restriction of deoxycytidine (dC)-containing DNA in the host Escherichia coli. The enzyme was synthesized with the use of a wheat germ cell-free protein synthesis system, given a lethal effect of its expression in E.coli cells, and was purified to homogeneity. The purified enzyme showed high activity with single-stranded (ss) DNA and denatured dC-substituted T4 genomic double-stranded (ds) DNA but exhibited no activity with dsDNA, ssRNA or denatured T4 genomic dsDNA containing glucosylated deoxyhydroxymethylcytidine. Characterization of Endo IV activity revealed that the enzyme catalyzed specific endonucleolytic cleavage of the 5′ phosphodiester bond of dC in ssDNA with an efficiency markedly dependent on the surrounding nucleotide sequence. The enzyme preferentially targeted 5′-dTdCdA-3′ but tolerated various combinations of individual nucleotides flanking this trinucleotide sequence. These results suggest that Endo IV preferentially recognizes short nucleotide sequences containing 5′-dTdCdA-3′, which likely accounts for the limited digestion of ssDNA by the enzyme and may be responsible in part for the indispensability of a deficiency in denB for stable synthesis of dC-substituted T4 genomic DNA.     

RecR forms a ring-like tetramer that encircles dsDNA by forming a complex with RecF

In the RecFOR pathway, the RecF and RecR proteins form a complex that binds to DNA and exerts multiple functions, including directing the loading of RecA onto single-stranded (ss) DNA regions near double-stranded (ds) DNA–ssDNA junctions and preventing it from forming a filament beyond the ssDNA region. However, neither the structure of the RecFR complex nor its DNA-binding mechanism was previously identified. Here, size-exclusion chromatography and small-angle X-ray scattering data indicate that Thermus thermophilus (tt) RecR binds to ttRecF to form a globular structure consisting of four ttRecR and two ttRecF monomers. In addition, a low resolution model shows a cavity in the central part of the complex, suggesting that ttRecR forms a ring-like tetramer inside the ttRecFR complex. Mutant ttRecR proteins lacking the N- or C-terminal interfaces that are required for tetramer formation are unable to form a complex with ttRecF. Furthermore, a ttRecFR complex containing the DNA-binding deficient ttRecR K23E/R27E double mutant, which contains mutations lying inside the ring, exhibits significantly reduced dsDNA binding. Thus, we propose that the ring-like ttRecR tetramer has a key role in tethering the ttRecFR complex onto dsDNA and that the ring structure may function as a clamp protein.     

Torsional regulation of hRPA-induced unwinding of double-stranded DNA

All cellular single-stranded (ss) DNA is rapidly bound and stabilized by single stranded DNA-binding proteins (SSBs). Replication protein A, the main eukaryotic SSB, is able to unwind double-stranded (ds) DNA by binding and stabilizing transiently forming bubbles of ssDNA. Here, we study the dynamics of human RPA (hRPA) activity on topologically constrained dsDNA with single-molecule magnetic tweezers. We find that the hRPA unwinding rate is exponentially dependent on torsion present in the DNA. The unwinding reaction is self-limiting, ultimately removing the driving torsional stress. The process can easily be reverted: release of tension or the application of a rewinding torque leads to protein dissociation and helix rewinding. Based on the force and salt dependence of the in vitro kinetics we anticipate that the unwinding reaction occurs frequently in vivo. We propose that the hRPA unwinding reaction serves to protect and stabilize the dsDNA when it is structurally destabilized by mechanical stresses.     

Bacillus subtilis bacteriophage SPP1 hexameric DNA helicase,G40P,interacts with forked DNA

SPP1-encoded replicative DNA helicase gene 40 product (G40P) is an essential product for phage replication. Hexameric G40P, in the presence of AMP-PNP, preferentially binds unstructured single-stranded (ss)DNA in a sequence-independent manner. The efficiency of ssDNA binding, nucleotide hydrolysis and the unwinding activity of G40P are affected in a different manner by different nucleotide cofactors. Nuclease protection studies suggest that G40P protects the 5′ tail of a forked molecule, and the duplex region at the junction against exonuclease attack. G40P does not protect the 3′ tail of a forked molecule from exonuclease attack. By using electron microscopy we confirm that the ssDNA transverses the centre of the hexameric ring. Our results show that hexameric G40P DNA helicase encircles the 5′ tail, interacts with the duplex DNA at the ss–double-stranded DNA junction and excludes the 3′ tail of the forked DNA.     

Single molecule analysis of Thermus thermophilus SSB protein dynamics on single-stranded DNA

Single-stranded (ss) DNA binding (SSB) proteins play central roles in DNA replication, recombination and repair in all organisms. We previously showed that Escherichia coli (Eco) SSB, a homotetrameric bacterial SSB, undergoes not only rapid ssDNA-binding mode transitions but also one-dimensional diffusion (or migration) while remaining bound to ssDNA. Whereas the majority of bacterial SSB family members function as homotetramers, dimeric SSB proteins were recently discovered in a distinct bacterial lineage of extremophiles, the Thermus–Deinococcus group. Here we show, using single-molecule fluorescence resonance energy transfer (FRET), that homodimeric bacterial SSB from Thermus thermophilus (Tth) is able to diffuse spontaneously along ssDNA over a wide range of salt concentrations (20–500 mM NaCl), and that TthSSB diffusion can help transiently melt the DNA hairpin structures. Furthermore, we show that two TthSSB molecules undergo transitions among different DNA-binding modes while remaining bound to ssDNA. Our results extend our previous observations on homotetrameric SSBs to homodimeric SSBs, indicating that the dynamic features may be shared among different types of SSB proteins. These dynamic features of SSBs may facilitate SSB redistribution and removal on/from ssDNA, and help recruit other SSB-interacting proteins onto ssDNA for subsequent DNA processing in DNA replication, recombination and repair.     

Investigations on DNA intercalation and surface binding by SYBR Green I, its structure determination and methodological implications

The detection of double-stranded (ds) DNA by SYBR Green I (SG) is important in many molecular biology methods including gel electrophoresis, dsDNA quantification in solution and real-time PCR. Biophysical studies at defined dye/base pair ratios (dbprs) were used to determine the structure–property relationships that affect methods applying SG. These studies revealed the occurrence of intercalation, followed by surface binding at dbprs above ~0.15. Only the latter led to a significant increase in fluorescence. Studies with poly(dA) · poly(dT) and poly(dG) · poly(dC) homopolymers showed sequence-specific binding of SG. Also, salts had a marked impact on SG fluorescence. We also noted binding of SG to single-stranded (ss) DNA, although SG/ssDNA fluorescence was at least ~11-fold lower than with dsDNA. To perform these studies, we determined the structure of SG by mass spectrometry and NMR analysis to be [2-[N-(3-dimethylaminopropyl)-N-propylamino]-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenyl-quinolinium]. For comparison, the structure of PicoGreen (PG) was also determined and is [2-[N-bis-(3-dimethylaminopropyl)-amino]-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenyl-quinolinium]⁺. These structure–property relationships help in the design of methods that use SG, in particular dsDNA quantification in solution and real-time PCR.     

Anti-CRISPR AcrIF9 functions by inducing the CRISPR–Cas complex to bind DNA non-specifically

Phages and other mobile genetic elements express anti-CRISPR proteins (Acrs) to protect their genomes from destruction by CRISPR–Cas systems. Acrs usually block the ability of CRISPR–Cas systems to bind or cleave their nucleic acid substrates. Here, we investigate an unusual Acr, AcrIF9, that induces a gain-of-function to a type I-F CRISPR–Cas (Csy) complex, causing it to bind strongly to DNA that lacks both a PAM sequence and sequence complementarity. We show that specific and non-specific dsDNA compete for the same site on the Csy:AcrIF9 complex with rapid exchange, but specific ssDNA appears to still bind through complementarity to the CRISPR RNA. Induction of non-specific DNA-binding is a shared property of diverse AcrIF9 homologues. Substitution of a conserved positively charged surface on AcrIF9 abrogated non-specific dsDNA-binding of the Csy:AcrIF9 complex, but specific dsDNA binding was maintained. AcrIF9 mutants with impaired non-specific dsDNA binding activity in vitro displayed a reduced ability to inhibit CRISPR–Cas activity in vivo. We conclude that misdirecting the CRISPR–Cas complex to bind non-specific DNA is a key component of the inhibitory mechanism of AcrIF9. This inhibitory mechanism is distinct from a previously characterized anti-CRISPR, AcrIF1, that sterically blocks DNA-binding, even though AcrIF1and AcrIF9 bind to the same site on the Csy complex.     

The N-terminus of the human RecQL4 helicase is a homeodomain-like DNA interaction motif

The RecQL4 helicase is involved in the maintenance of genome integrity and DNA replication. Mutations in the human RecQL4 gene cause the Rothmund–Thomson, RAPADILINO and Baller–Gerold syndromes. Mouse models and experiments in human and Xenopus have proven the N-terminal part of RecQL4 to be vital for cell growth. We have identified the first 54 amino acids of RecQL4 (RecQL4_N54) as the minimum interaction region with human TopBP1. The solution structure of RecQL4_N54 was determined by heteronuclear liquid–state nuclear magnetic resonance (NMR) spectroscopy (PDB 2KMU; backbone root-mean-square deviation 0.73 Å). Despite low-sequence homology, the well-defined structure carries an overall helical fold similar to homeodomain DNA-binding proteins but lacks their archetypical, minor groove-binding N-terminal extension. Sequence comparison indicates that this N-terminal homeodomain-like fold is a common hallmark of metazoan RecQL4 and yeast Sld2 DNA replication initiation factors. RecQL4_N54 binds DNA without noticeable sequence specificity yet with apparent preference for branched over double-stranded (ds) or single-stranded (ss) DNA. NMR chemical shift perturbation observed upon titration with Y-shaped, ssDNA and dsDNA shows a major contribution of helix α3 to DNA binding, and additional arginine side chain interactions for the ss and Y-shaped DNA.     

Bacillus subtilis RecN binds and protects 3'-single-stranded DNA extensions in the presence of ATP

Bacillus subtilis RecN appears to be an early detector of breaks in double-stranded DNA. In vivo, RecN forms discrete nucleoid-associated structures and in vitro exhibits Mg²⁺-dependent single-stranded (ss) DNA binding and ssDNA-dependent ATPase activities. In the presence of ATP or ADP, RecN assembles to form large networks with ssDNA molecules (designated complexes CII and CIII) that involve ATP binding and requires a 3′-OH at the end of ssDNA molecule. Addition of dATP–RecA complexes dissociates RecN from these networks, but this is not observed following addition of an ssDNA binding protein. Apparently, ATP modulates the RecN–ssDNA complex for binding to ssDNA extensions and, in vivo, RecN–ATP bound to 3′-ssDNA might sequester ssDNA ends within complexes that protect the ssDNA while the RecA accessory proteins recruit RecA. With the association of RecA to ssDNA, RecN would dissociate from the DNA end facilitating the subsequent steps in DNA repair.     

Recognition and processing of double-stranded DNA by ExoX,a distributive 3′–5′ exonuclease

Members of the DnaQ superfamily are major 3′–5′ exonucleases that degrade either only single-stranded DNA (ssDNA) or both ssDNA and double-stranded DNA (dsDNA). However, the mechanism by which dsDNA is recognized and digested remains unclear. Exonuclease X (ExoX) is a distributive DnaQ exonuclease that cleaves both ssDNA and dsDNA substrates. Here, we report the crystal structures of Escherichia coli ExoX in complex with three different dsDNA substrates: 3′ overhanging dsDNA, blunt-ended dsDNA and 3′ recessed mismatch-containing dsDNA. In these structures, ExoX binds to dsDNA via both a conserved substrate strand-interacting site and a previously uncharacterized complementary strand-interacting motif. When ExoX complexes with blunt-ended dsDNA or 5′ overhanging dsDNA, a ‘wedge’ composed of Leu12 and Gln13 penetrates between the first two base pairs to break the 3′ terminal base pair and facilitates precise feeding of the 3′ terminus of the substrate strand into the ExoX cleavage active site. Site-directed mutagenesis showed that the complementary strand-binding site and the wedge of ExoX are dsDNA specific. Together with the results of structural comparisons, our data support a mechanism by which normal and mismatched dsDNA are recognized and digested by E. coli ExoX. The crystal structures also provide insight into the structural framework of the different substrate specificities of the DnaQ family members.     

SOS repair can be about as effective for single-stranded DNA as for double-stranded DNA and even more so.

As ordinarily measured, the SOS repair of damaged DNA by Weigle reactivation appears to be more effective for double-stranded (ds) than for single-stranded (ss) DNA bacteriophages. A complicating feature, which is usually not considered, is the possibility of DNA-protein cross-linking of ssDNA to the viral capsid, which would conceivably be an extraneous source of nonreactivable lesions. This idea is supported in studies of phage S13 by the observation that photoreactivation more than doubles when naked ssDNA is substituted for encapsidated ssDNA as the UV target. The same effect was observed for Weigle reactivation; there was little, if any, difference in the reactivation of ssDNA and dsDNA when naked DNA was irradiated. Moreover, in a uvrA mutant, ssDNA actually had the advantage; Weigle reactivation was then more than twice as effective for ssDNA as for dsDNA. It is also shown that when a suitable measure of Weigle mutagenesis is used, there is no convincing evidence that dsDNA is mutagenized more effectively than ssDNA.     

Visualizing RAD51-mediated joint molecules: implications for recombination mechanism and the effect of sequence heterology

The defining event in homologous recombination is the exchange of base-paired partners between a single-stranded (ss) DNA and a homologous duplex driven by recombinase proteins, such as human RAD51. To understand the mechanism of this essential genome maintenance event, we analyzed the structure of RAD51–DNA complexes representing strand exchange intermediates at nanometer resolution by scanning force microscopy. Joint molecules were formed between substrates with a defined ssDNA segment and homologous region on a double-stranded (ds) partner. We discovered and quantified several notable architectural features of RAD51 joint molecules. Each end of the RAD51-bound joints had a distinct structure. Using linear substrates, a 10-nt region of mispaired bases blocked extension of joint molecules in all examples observed, whereas 4 nt of heterology only partially blocked joint molecule extension. Joint molecules, including 10 nt of heterology, had paired DNA on either side of the heterologous substitution, indicating that pairing could initiate from the free 3′end of ssDNA or from a region adjacent to the ss–ds junction. RAD51 filaments covering joint ss–dsDNA regions were more stable to disassembly than filaments covering dsDNA. We discuss how distinct structural features of RAD51-bound DNA joints can play important roles as recognition sites for proteins that facilitate and control strand exchange.     

A new structural insight into XPA–DNA interactions

XPA (xeroderma pigmentosum group A) protein is an essential factor for NER (nucleotide excision repair) which is believed to be involved in DNA damage recognition/verification, NER factor recruiting and stabilization of repair intermediates. Past studies on the structure of XPA have focused primarily on XPA interaction with damaged DNA. However, how XPA interacts with other DNA structures remains unknown though recent evidence suggest that these structures could be important for its roles in both NER and non-NER activities. Previously, we reported that XPA recognizes undamaged DNA ds/ssDNA (double-strand/single-strandDNA) junctions with a binding affinity much higher than its ability to bind bulky DNA damage. To understand how this interaction occurs biochemically we implemented a structural determination of the interaction using a MS-based protein footprinting method and limited proteolysis. By monitoring surface accessibility of XPA lysines to NHS-biotin modification in the free protein and the DNA junction-bound complex we show that XPA physically interacts with the DNA junctions via two lysines, K168 and K179, located in the previously known XPA(98–219) DBD (DNA-binding domain). Importantly, we also uncovered new lysine residues, outside of the known DBD, involved in the binding. We found that residues K221, K222, K224 and K236 in the C-terminal domain are involved in DNA binding. Limited proteolysis analysis of XPA–DNA interactions further confirmed this observation. Structural modelling with these data suggests a clamp-like DBD for the XPA binding to ds/ssDNA junctions. Our results provide a novel structure-function view of XPA–DNA junction interactions.     

Mechanistic studies on bleomycin-mediated DNA damage: multiple binding modes can result in double-stranded DNA cleavage

The bleomycins (BLMs) are a family of natural glycopeptides used clinically as antitumor agents. In the presence of required cofactors (Fe²⁺ and O₂), BLM causes both single-stranded (ss) and double-stranded (ds) DNA damage with the latter thought to be the major source of cytotoxicity. Previous biochemical and structural studies have demonstrated that BLM can mediate ss cleavage through multiple binding modes. However, our studies have suggested that ds cleavage occurs by partial intercalation of BLM's bithiazole tail 3′ to the first cleavage site that facilitates its re-activation and re-organization to the second strand without dissociation from the DNA where the second cleavage event occurs. To test this model, a BLM A5 analog (CD-BLM) with β-cyclodextrin attached to its terminal amine was synthesized. This attachment presumably precludes binding via intercalation. Cleavage studies measuring ss:ds ratios by two independent methods were carried out. Studies using [³²P]-hairpin technology harboring a single ds cleavage site reveal a ss:ds ratio of 6.7 ± 1.2:1 for CD-BLM and 3.4:1 and 3.1 ± 0.3:1 for BLM A2 and A5, respectively. In contrast with BLM A5 and A2, however, CD-BLM mediates ds-DNA cleavage through cooperative binding of a second CD-BLM molecule to effect cleavage on the second strand. Studies using the supercoiled plasmid relaxation assay revealed a ss:ds ratio of 2.8:1 for CD-BLM in comparison with 7.3:1 and 5.8:1, for BLM A2 and A5, respectively. This result in conjunction with the hairpin results suggest that multiple binding modes of a single BLM can lead to ds-DNA cleavage and that ds cleavage can occur using one or two BLM molecules. The significance of the current study to understanding BLM's action in vivo is discussed.     

Bacillus subtilis tau subunit of DNA polymerase III interacts with bacteriophage SPP1 replicative DNA helicase G40P

Genetic evidence suggests that the Bacillus subtilis dnaX gene only encodes for the τ subunit of both DNA polymerases III (Pol IIIs). The B.subtilis full-length protein and their mutant derivatives τ(373– 563) (lacking the N-terminal, domains I–III or amino acid residues 1–372) and τ(1–372) (lacking the C-terminal region or amino acids 373–563) have been purified. The τ protein forms tetramers, τ(373– 563) forms dimers, whereas τ(1–372), depending on the ionic strength, forms trimers or tetramers in solution. In the absence of single-stranded (ss) DNA and a nucleotide cofactor, τ interacts with the SPP1 hexameric replicative G40P DNA helicase in solution or with G40P-ATP bound to ssDNA, with a 1:1 stoichiometry. G40P(109–442), lacking the N-terminal amino acid residues 1–108, interacts with the C-terminal moiety of τ. The data indicate that the interaction of G40P with the τ subunit of Pol III, is relevant for the loading of the Pol IIIs into the SPP1 G38P-promoted open complex.     

Efficient target cleavage by Type V Cas12a effectors programmed with split CRISPR RNA

CRISPR RNAs (crRNAs) that direct target DNA cleavage by Type V Cas12a nucleases consist of constant repeat-derived 5′-scaffold moiety and variable 3′-spacer moieties. Here, we demonstrate that removal of most of the 20-nucleotide scaffold has only a slight effect on in vitro target DNA cleavage by a Cas12a ortholog from Acidaminococcus sp. (AsCas12a). In fact, residual cleavage was observed even in the presence of a 20-nucleotide crRNA spacer moiety only. crRNAs split into separate scaffold and spacer RNAs catalyzed highly specific and efficient cleavage of target DNA by AsCas12a in vitro and in lysates of human cells. In addition to dsDNA target cleavage, AsCas12a programmed with split crRNAs also catalyzed specific ssDNA target cleavage and non-specific ssDNA degradation (collateral activity). V-A effector nucleases from Francisella novicida (FnCas12a) and Lachnospiraceae bacterium (LbCas12a) were also functional with split crRNAs. Thus, the ability of V-A effectors to use split crRNAs appears to be a general property. Though higher concentrations of split crRNA components are needed to achieve efficient target cleavage, split crRNAs open new lines of inquiry into the mechanisms of target recognition and cleavage and may stimulate further development of single-tube multiplex and/or parallel diagnostic tests based on Cas12a nucleases.     

Helicobacter pylori DprA alleviates restriction barrier for incoming DNA

Helicobacter pylori is a Gram-negative bacterium that colonizes human stomach and causes gastric inflammation. The species is naturally competent and displays remarkable diversity. The presence of a large number of restriction–modification (R–M) systems in this bacterium creates a barrier against natural transformation by foreign DNA. Yet, mechanisms that protect incoming double-stranded DNA (dsDNA) from restriction enzymes are not well understood. A DNA-binding protein, DNA Processing Protein A (DprA) has been shown to facilitate natural transformation of several Gram-positive and Gram-negative bacteria by protecting incoming single-stranded DNA (ssDNA) and promoting RecA loading on it. However, in this study, we report that H. pylori DprA (HpDprA) binds not only ssDNA but also dsDNA thereby conferring protection to both from various exonucleases and Type II restriction enzymes. Here, we observed a stimulatory role of HpDprA in DNA methylation through physical interaction with methyltransferases. Thus, HpDprA displayed dual functional interaction with H. pylori R–M systems by not only inhibiting the restriction enzymes but also stimulating methyltransferases. These results indicate that HpDprA could be one of the factors that modulate the R–M barrier during inter-strain natural transformation in H. pylori.     

RecR-mediated Modulation of RecF Dimer Specificity for Single- and Double-stranded DNA

RecF pathway proteins play an important role in the restart of stalled replication and DNA repair in prokaryotes. Following DNA damage, RecF, RecR, and RecO initiate homologous recombination (HR) by loading of the RecA recombinase on single-stranded (ss) DNA, protected by ssDNA-binding protein. The specific role of RecF in this process is not well understood. Previous studies have proposed that RecF directs the RecOR complex to boundaries of damaged DNA regions by recognizing single-stranded/double-stranded (ss/ds) DNA junctions. RecF belongs to ABC-type ATPases, which function through an ATP-dependent dimerization. Here, we demonstrate that the RecF of Deinococcus radiodurans interacts with DNA as an ATP-dependent dimer, and that the DNA binding and ATPase activity of RecF depend on both the structure of DNA substrate, and the presence of RecR. We found that RecR interacts as a tetramer with the RecF dimer. RecR increases the RecF affinity to dsDNA without stimulating ATP hydrolysis but destabilizes RecF binding to ssDNA and dimerization, likely due to increasing the ATPase rate. The DNA-dependent binding of RecR to the RecF-DNA complex occurs through specific protein-protein interactions without significant contributions from RecR-DNA interactions. Finally, RecF neither alone nor in complex with RecR preferentially binds to the ss/dsDNA junction. Our data suggest that the specificity of the RecFOR complex toward the boundaries of DNA damaged regions may result from a network of protein-protein and DNA-protein interactions, rather than a simple recognition of the ss/dsDNA junction by RecF.Homologous recombination (HR)² is one of the primary mechanisms by which cells repair dsDNA breaks (DSBs) and ssDNA gaps (SSGs), and is important for restart of stalled DNA replication (1). HR is initiated when RecA-like recombinases bind to ssDNA forming an extended nucleoprotein filament, referred to as a presynaptic complex (2). The potential for genetic rearrangements dictates that HR initiation is tightly regulated at multiple levels (1). During replication, the ssDNA-binding protein (SSB) protects transiently unwound DNA chains, preventing interactions with recombinases. Following DNA damage, recombination mediator proteins (RMPs) initiate HR by facilitating the formation of the recombinase filaments with ssDNA, while removing SSB (3, 4). Mutations in human proteins involved in HR initiation are linked to cancer predisposition, chromosome instability, UV sensitivity, and premature aging diseases (4–8). To date, little is known about the mechanism by which RMPs regulate the formation of the recombinase filaments on the SSB-protected ssDNA.In Escherichia coli, there are two major recombination pathways, RecBCD and RecF (9, 10). A helicase/nuclease RecBCD complex processes DSBs and recruits RecA on ssDNA in a sequence-specific manner (11–13). The principle players in the RecF pathway are the RecF, RecO, and RecR proteins, which form an epistatic group that is important for SSG repair, for restart of stalled DNA replication, and under specific conditions, can also process DSBs (14–20). Homologs of RecF, -O, and -R are present in the majority of known bacteria (21), including Deinococcus radiodurans, extremely radiation-resistant bacteria that lacks the RecBCD pathway, yet is capable of repairing thousands of DSBs (22, 23). In addition, the sequence or functional homologs of RecF pathway proteins are involved in similar pathways in eukaryotes that include among others WRN, BLM, RAD52, and BRCA2 proteins (4–8).The involvement of all three RecF, -O, and -R proteins in HR initiation is well documented by genetic and cellular approaches (18, 24–30), yet their biochemical functions in the initiation process remain unclear, particularly with respect to RecF. RecO and RecR proteins are sufficient to promote formation of the RecA filament on SSB-bound ssDNA in vitro (27). The UV-sensitive phenotype of recF mutants can be suppressed by RecOR overexpression, suggesting that RecF may direct the RMP complex to DNA-damaged regions where HR initiation is required (31). In agreement with this hypothesis, RecF dramatically increases the efficiency of the RecA loading at ds/ssDNA junctions with a 3′ ssDNA extension under specific conditions (32). RecF and RecR proteins also prevent the RecA filaments from extending into dsDNA regions adjacent to SSGs (33). These data suggest that RecF may directly recognize an ss/dsDNA junction structure (34). However, DNA binding experiments have not provided clear evidence to support such a hypothesis (11).The targeting promoted by RecF may also occur through more complex processes. RecF shares a high structural similarity with the head domain of Rad50, an ABC-type ATPase that recognizes DSBs and initiates repair in archaea and eukaryotes (35). All known ABC-type ATPases function as oligomeric complexes in which a sequence of inter- and intra-molecular interactions is triggered by the ATP-dependent dimerization and the dimer-dependent ATP hydrolysis (36–39). RecF is also an ATP-dependent DNA-binding protein and a weak DNA-dependent ATPase (11, 40). RecF forms an ATP-dependent dimer and all three conserved motifs (Walker A, Walker B, and “signature”) of RecF are important for ATP-dependent dimerization, ATP hydrolysis, and functional resistance to DNA damage (35). Thus, RecF may function in recombination initiation through a complex pathway of protein-protein and DNA-protein interactions regulated by ATP-dependent RecF dimerization.In this report, we present a detailed characterization of the RecF dimerization, and its role in the RecF interaction with various DNA substrates, with RecR, and in ATP hydrolysis. Our data outline the following key findings. First, RecF interacts with DNA as a dimer. Second, neither RecF alone nor the RecFR complex preferentially binds the ss/dsDNA junction. Finally, RecR changes the ATPase activity and the DNA binding of RecF by destabilizing the interaction with ssDNA, and greatly enhancing the interaction with dsDNA. Our results suggest that the specificity of RecF for the boundaries of SSGs is likely to result from a sequence of protein-protein interaction events rather than a simple RecF ss/dsDNA binding, underlining a highly regulated mechanism of the HR initiation by the RecFOR proteins.