Role of ATP hydrolysis in the DNA translocase activity of the bovine papillomavirus (BPV-1) E1 helicase

The E1 protein of bovine papillomavirus type-1 is the viral replication initiator protein and replicative helicase. Here we show that the C-terminal ~300 amino acids of E1, that share homology with members of helicase superfamily 3 (SF3), can act as an autonomous helicase. E1 is monomeric in the absence of ATP but assembles into hexamers in the presence of ATP, single-stranded DNA (ssDNA) or both. A 16 base sequence is the minimum for efficient hexamerization, although the complex protects ~30 bases from nuclease digestion, supporting the notion that the DNA is bound within the protein complex. In the absence of ATP, or in the presence of ADP or the non–hydrolysable ATP analogue AMP–PNP, the interaction with short ssDNA oligonucleotides is exceptionally tight (T_1/2 > 6 h). However, in the presence of ATP, the interaction with DNA is destabilized (T_1/2 ~60 s). These results suggest that during the ATP hydrolysis cycle an internal DNA-binding site oscillates from a high to a low-affinity state, while protein–protein interactions switch from low to high affinity. This reciprocal change in protein–protein and protein–DNA affinities could be part of a mechanism for tethering the protein to its substrate while unidirectional movement along DNA proceeds.     

Complexed crystal structure of replication restart primosome protein PriB reveals a novel single-stranded DNA-binding mode

PriB is a primosomal protein required for replication restart in Escherichia coli. PriB stimulates PriA helicase activity via interaction with single-stranded DNA (ssDNA), but the molecular details of this interaction remain unclear. Here, we report the crystal structure of PriB complexed with a 15 bases oligonucleotide (dT15) at 2.7 Å resolution. PriB shares structural similarity with the E.coli ssDNA-binding protein (EcoSSB). However, the structure of the PriB–dT15 complex reveals that PriB binds ssDNA differently. Results from filter-binding assays show that PriB–ssDNA interaction is salt-sensitive and cooperative. Mutational analysis suggests that the loop L₄₅ plays an important role in ssDNA binding. Based on the crystal structure and biochemical analyses, we propose a cooperative mechanism for the binding of PriB to ssDNA and a model for the assembly of the PriA–PriB–ssDNA complex. This report presents the first structure of a replication restart primosomal protein complexed with DNA, and a novel model that explains the interactions between a dimeric oligonucleotide-binding-fold protein and ssDNA.     

Plant transformation by Agrobacterium tumefaciens: modulation of single-stranded DNA-VirE2 complex assembly by VirE1

Agrobacterium tumefaciens infects plant cells by the transfer of DNA. A key factor in this process is the bacterial virulence protein VirE2, which associates stoichiometrically with the transported single-stranded (ss) DNA molecule (T-strand). As observed in vitro by transmission electron microscopy, VirE2-ssDNA readily forms an extended helical complex with a structure well suited to the tasks of DNA protection and nuclear import. Here we have elucidated the role of the specific molecular chaperone VirE1 in regulating VireE2-VirE2 and VirE2-ssDNA interactions. VirE2 alone formed functional filamentous aggregates capable of ssDNA binding. In contrast, co-expression with VirE1 yielded monodisperse VirE1-VirE2 complexes. Cooperative binding of VirE2 to ssDNA released VirE1, resulting in a controlled formation mechanism for the helical complex that is further promoted by macromolecular crowding. Based on this in vitro evidence, we suggest that the constrained volume of the VirB channel provides a natural site for the exchange of VirE2 binding from VirE1 to the T-strand.     

VirE2: a unique ssDNA-compacting molecular machine

The translocation of single-stranded DNA (ssDNA) across membranes of two cells is a fundamental biological process occurring in both bacterial conjugation and Agrobacterium pathogenesis. Whereas bacterial conjugation spreads antibiotic resistance, Agrobacterium facilitates efficient interkingdom transfer of ssDNA from its cytoplasm to the host plant cell nucleus. These processes rely on the Type IV secretion system (T4SS), an active multiprotein channel spanning the bacterial inner and outer membranes. T4SSs export specific proteins, among them relaxases, which covalently bind to the 5' end of the translocated ssDNA and mediate ssDNA export. In Agrobacterium tumefaciens, another exported protein—VirE2—enhances ssDNA transfer efficiency 2000-fold. VirE2 binds cooperatively to the transferred ssDNA (T-DNA) and forms a compact helical structure, mediating T-DNA import into the host cell nucleus. We demonstrated—using single-molecule techniques—that by cooperatively binding to ssDNA, VirE2 proteins act as a powerful molecular machine. VirE2 actively pulls ssDNA and is capable of working against 50-pN loads without the need for external energy sources. Combining biochemical and cell biology data, we suggest that, in vivo, VirE2 binding to ssDNA allows an efficient import and pulling of ssDNA into the host. These findings provide a new insight into the ssDNA translocation mechanism from the recipient cell perspective. Efficient translocation only relies on the presence of ssDNA binding proteins in the recipient cell that compacts ssDNA upon binding. This facilitated transfer could hence be a more general ssDNA import mechanism also occurring in bacterial conjugation and DNA uptake processes.     

The VirE1VirE2 complex of Agrobacterium tumefaciens interacts with single-stranded DNA and forms channels

The VirE2 protein is crucial for the transfer of single-stranded DNA (ssDNA) from Agrobacterium tumefaciens to the nucleus of the plant host cell because of its ssDNA binding activity, assistance in nuclear import and putative ssDNA channel activity. The native form of VirE2 in Agrobacterium's cytoplasm is in complex with its specific chaperone, VirE1. Here, we describe the ability of the VirE1VirE2 complex to both bind ssDNA and form channels. The affinity of the VirE1VirE2 complex for ssDNA is slightly reduced compared with VirE2, but the kinetics of binding to ssDNA are unaffected by the presence of VirE1. Upon binding of VirE1VirE2 to ssDNA, similar helical structures to those reported for the VirE2-ssDNA complex were observed by electron microscopy. The VirE1VirE2 complex can release VirE1 once the VirE2-ssDNA complexes assembled. VirE2 exhibits a low affinity for small unilamellar vesicles composed of bacterial lipids and a high affinity for lipid vesicles containing sterols and sphingolipids, typical components of animal and plant membranes. In contrast, the VirE1VirE2 complex associated similarly with all kind of lipids. Finally, black lipid membrane experiments revealed the ability of the VirE1VirE2 complex to form channels. However, the majority of the channels displayed a conductance that was a third of the conductance of VirE2 channels. Our results demonstrate that the binding of VirE1 to VirE2 does not inhibit VirE2 functions and that the effector-chaperone complex is multifunctional.     

Recognition of T-rich single-stranded DNA by the cold shock protein Bs-CspB in solution

Cold shock proteins (CSP) belong to the family of single-stranded nucleic acid binding proteins with OB-fold. CSP are believed to function as ‘RNA chaperones’ and during anti-termination. We determined the solution structure of Bs-CspB bound to the single-stranded DNA (ssDNA) fragment heptathymidine (dT₇) by NMR spectroscopy. Bs-CspB reveals an almost invariant conformation when bound to dT₇ with only minor reorientations in loop β1–β2 and β3–β4 and of few aromatic side chains involved in base stacking. Binding studies of protein variants and mutated ssDNA demonstrated that Bs-CspB associates with ssDNA at almost diffusion controlled rates and low sequence specificity consistent with its biological function. A variation of the ssDNA affinity is accomplished solely by changes of the dissociation rate. ¹⁵N NMR relaxation and H/D exchange experiments revealed that binding of dT₇ increases the stability of Bs-CspB and reduces the sub-nanosecond dynamics of the entire protein and especially of loop β3–β4.     

A rapid and sensitive high-throughput screening method to identify compounds targeting protein–nucleic acids interactions

DNA-binding and RNA-binding proteins are usually considered ‘undruggable’ partly due to the lack of an efficient method to identify inhibitors from existing small molecule repositories. Here we report a rapid and sensitive high-throughput screening approach to identify compounds targeting protein–nucleic acids interactions based on protein–DNA or protein–RNA interaction enzyme-linked immunosorbent assays (PDI-ELISA or PRI-ELISA). We validated the PDI-ELISA method using the mammalian high-mobility-group protein AT-hook 2 (HMGA2) as the protein of interest and netropsin as the inhibitor of HMGA2–DNA interactions. With this method we successfully identified several inhibitors and an activator for HMGA2–DNA interactions from a collection of 29 DNA-binding compounds. Guided by this screening excise, we showed that netropsin, the specific inhibitor of HMGA2–DNA interactions, strongly inhibited the differentiation of the mouse pre-adipocyte 3T3-L1 cells into adipocytes, most likely through a mechanism by which the inhibition is through preventing the binding of HMGA2 to the target DNA sequences. This method should be broadly applicable to identify compounds or proteins modulating many DNA-binding or RNA-binding proteins.     

The Agrobacterium tumefaciens Chaperone-Like Protein, VirE1, Interacts with VirE2 at Domains Required for Single-Stranded DNA Binding and Cooperative Interaction

Agrobacterium tumefaciens transfers single-stranded DNA (ssDNA) into plants. Efficient tumorigenesis requires VirE1-dependent export of ssDNA-binding (SSB) protein VirE2. VirE1 binds VirE2 domains involved in SSB and self-association, and VirE1 may facilitate VirE2 export by preventing VirE2 aggregation and the premature binding of VirE2 to ssDNA.     

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.     

Highly conserved amino acids in Pax and Ets proteins are required for DNA binding and ternary complex assembly

Combinatorial association of DNA-binding proteins on composite binding sites enhances their nucleotide sequence specificity and functional synergy. As a paradigm for these interactions, Pax-5 (BSAP) assembles ternary complexes with Ets proteins on the B cell-specific mb-1 promoter through interactions between their respective DNA-binding domains. Pax-5 recruits Ets-1 to bind the promoter, but not the closely related Ets protein SAP1a. Here we show that, while several different mutations increase binding of SAP1a to an optimized Ets binding site, only conversion of Val68 to an acidic amino acid facilitates ternary complex assembly with Pax-5 on the mb-1 promoter. This suggests that enhanced DNA binding by SAP1a is not sufficient for recruitment by Pax-5, but instead involves protein–protein interactions mediated by the acidic side chain. Recruitment of Ets proteins by Pax-5 requires Gln22 within the N-terminal β-hairpin motif of its paired domain. The β-hairpin also participates in recognition of a subset of Pax-5-binding sites. Thus, Pax-5 incorporates protein–protein interaction and DNA recognition functions in a single motif. The Caenorhabditis elegans Pax protein EGL-38 also binds specifically to the mb-1 promoter and recruits murine Ets-1 or the C.elegans Ets protein T08H4.3, but not the related LIN-1 protein. Together, our results define specific amino acid requirements for Pax–Ets ternary complex assembly and show that the mechanism is conserved between evolutionarily related proteins of diverse animal species. Moreover, the data suggest that interactions between Pax and Ets proteins are an important mechanism that regulates fundamental biological processes in worms and humans.     

Measurement of the salt-dependent stabilization of partially open DNA by Escherichia coli SSB protein

The rezipping force of two complementary DNA strands under tension has been measured in the presence of Escherichia coli single-stranded-binding proteins under salt conditions ranging from 10– to 400 mM NaCl. The effectiveness of the binding protein in preventing rezipping is strongly dependent on salt concentration and compared with the salt dependence in the absence of the protein. At concentrations less than 50 mM NaCl, the protein prevents complete rezipping of λ-phage on the 2-s timescale of the experiment, when the ssDNA is under tensions as low as 3.5 ± 1 pN. For salt concentrations greater than 200 mM NaCl, the protein inhibits rezipping but cannot block rezipping when the tension is reduced below 6 ± 1.8 pN. This change in effectiveness as a function of salt concentration may correspond to salt-dependent changes in binding modes that were previously observed in bulk assays.     

Crystal structure of DnaT84–153-dT10 ssDNA complex reveals a novel single-stranded DNA binding mode

DnaT is a primosomal protein that is required for the stalled replication fork restart in Escherichia coli. As an adapter, DnaT mediates the PriA-PriB-ssDNA ternary complex and the DnaB/C complex. However, the fundamental function of DnaT during PriA-dependent primosome assembly is still a black box. Here, we report the 2.83 Å DnaT^84–153-dT10 ssDNA complex structure, which reveals a novel three-helix bundle single-stranded DNA binding mode. Based on binding assays and negative-staining electron microscopy results, we found that DnaT can bind to phiX 174 ssDNA to form nucleoprotein filaments for the first time, which indicates that DnaT might function as a scaffold protein during the PriA-dependent primosome assembly. In combination with biochemical analysis, we propose a cooperative mechanism for the binding of DnaT to ssDNA and a possible model for the assembly of PriA-PriB-ssDNA-DnaT complex that sheds light on the function of DnaT during the primosome assembly and stalled replication fork restart. This report presents the first structure of the DnaT C-terminal complex with ssDNA and a novel model that explains the interactions between the three-helix bundle and ssDNA.     

Combining chromatin immunoprecipitation and DNA footprinting: a novel method to analyze protein–DNA interactions in vivo

A variety of methods are available to analyze protein–DNA interactions in vivo. Two of the most prominent of these methods are chromatin immunoprecipitation (ChIP) and in vivo footprinting. Both of these procedures have specific limitations. For example, the ChIP assay fails to document where exactly a protein binds in vivo. The precipitation of a specific segment of DNA with antibodies directed against DNA-binding proteins does not necessarily indicate that the protein directly interacts with a sequence in the precipitate but could rather reflect protein–protein interactions. Furthermore, the results of in vivo footprinting studies are inconclusive if a DNA sequence is analyzed that is bound by a specific protein in only a certain fraction of cells. Finally, in vivo footprinting does not indicate which protein is bound at a specific site. We have developed a new procedure that combines the ChIP assay and DMS footprinting techniques. Using this method we show here that antibodies specific for USF1 and NF-E2 precipitate the murine β-globin promoter in MEL cells. DMS footprinting analysis of the DNA precipitated with NF-E2 antibodies revealed a protection over a partial NF-E2-binding site in the β-globin downstream promoter region. We believe that this novel method will generally benefit investigators interested in analyzing protein–DNA interactions in vivo.     

Interactions between the archaeal Cdc6 and MCM proteins modulate their biochemical properties

The origin recognition complex, Cdc6 and the minichromosome maintenance (MCM) complex play essential roles in the initiation of eukaryotic DNA replication. Homologs of these proteins may play similar roles in archaeal replication initiation. While the interactions among the eukaryotic initiation proteins are well documented, the protein–protein interactions between the archaeal proteins have not yet been determined. Here, an extensive structural and functional analysis of the interactions between the Methanothermobacter thermautotrophicus MCM and the two Cdc6 proteins (Cdc6-1 and -2) identified in the organism is described. The main contact between Cdc6 and MCM occurs via the N-terminal portion of the MCM protein. It was found that Cdc6–MCM interaction, but not Cdc6–DNA binding, plays the predominant role in regulating MCM helicase activity. In addition, the data showed that the interactions with MCM modulate the autophosphorylation of Cdc6-1 and -2. The results also suggest that MCM and DNA may compete for Cdc6-1 protein binding. The implications of these observations for the initiation of archaeal DNA replication are discussed.     

Protein–DNA binding: complexities and multi-protein codes

Binding of proteins to particular DNA sites across the genome is a primary determinant of specificity in genome maintenance and gene regulation. DNA-binding specificity is encoded at multiple levels, from the detailed biophysical interactions between proteins and DNA, to the assembly of multi-protein complexes. At each level, variation in the mechanisms used to achieve specificity has led to difficulties in constructing and applying simple models of DNA binding. We review the complexities in protein–DNA binding found at multiple levels and discuss how they confound the idea of simple recognition codes. We discuss the impact of new high-throughput technologies for the characterization of protein–DNA binding, and how these technologies are uncovering new complexities in protein–DNA recognition. Finally, we review the concept of multi-protein recognition codes in which new DNA-binding specificities are achieved by the assembly of multi-protein complexes.     

Evidence for direct contact between the RPA3 subunit of the human replication protein A and single-stranded DNA

Replication Protein A is a single-stranded (ss) DNA-binding protein that is highly conserved in eukaryotes and plays essential roles in many aspects of nucleic acid metabolism, including replication, recombination, DNA repair and telomere maintenance. It is a heterotrimeric complex consisting of three subunits: RPA1, RPA2 and RPA3. It possesses four DNA-binding domains (DBD), DBD-A, DBD-B and DBD-C in RPA1 and DBD-D in RPA2, and it binds ssDNA via a multistep pathway. Unlike the RPA1 and RPA2 subunits, no ssDNA-RPA3 interaction has as yet been observed although RPA3 contains a structural motif found in the other DBDs. We show here using 4-thiothymine residues as photoaffinity probe that RPA3 interacts directly with ssDNA on the 3′-side on a 31 nt ssDNA.The replication protein A (RPA) is a single-stranded (ss) DNA-binding protein that is highly conserved in eukaryotes (1–3). RPA is one of the key players in various essential processes of DNA metabolism including replication, recombination, DNA repair and telomere maintenance (1,2,4–9). The functions of this protein are based on its DNA-binding activity and specific protein–protein interactions. Its ssDNA binding properties depend on DNA length and nucleotide sequence (6,10–13). RPA is a heterotrimeric protein, composed of 70-, 32- and 14-kDa subunits, commonly referred to as RPA1, RPA2 and RPA3, respectively. There are four DNA-binding domains (DBD) located in RPA1 (DBD A, DBD B, DBD C and DBD F), one located in RPA2 (DBD D) and one belongs to RPA3 (DBD E). RPA interacts with ssDNA via four DBD: DBD A, DBD B, DBD C and DBD D (14).It is now accepted (11) that RPA binds to ssDNA in a sequential pathway with a defined polarity (15–17). RPA binds ssDNA with three different binding modes. First, binding initially involves an unstable recognition site of 8–10 nt with the high-affinity DBD A and DBD B domains on the 5′-side of the occluded ssDNA; it is designated ‘compact conformation’ or 8–10 nt binding mode. Second, this step is followed by the weaker binding of DBD C, on the 3′-side, leading to an intermediate or ‘elongated contracted’ (13–22 nt) binding mode (18–19). Finally binding of DBD D on the 3′-side forms a stable ‘elongated extended’ complex characterized by a 30 nt long occluded binding site (30 nt binding mode). Although RPA3 contains an Oligonucleotide-Binding (OB)-fold motif found in the other DBDs, there is presently no biochemical evidence that this subunit directly contacts DNA. Thus positioning of the RPA3 subunit relative to the other domains is still speculative (11,20). It has been clearly demonstrated that RPA3 is crucial for RPA function (1,2): RPA3 is involved in heterotrimer formation and is responsible for the polarity of binding to DNA (11,21,22). The scope of the data indicates that either RPA3 participates only in protein–protein interactions or that putative interaction of RPA3 with ssDNA is unstable and too transient to be detected by standard biochemical experiments. This latter possibility is likely if such interaction is provided by the 3′-side of the ssDNA, since it has been suggested that this region might be transiently accessible to the RPA DBD domains (23,24).In the past few years, thionucleobases have been extensively used as intrinsic photolabels to probe the structure in solution of folded molecules and to identify transient contacts within nucleic acids and/or between nucleic acids and proteins, in nucleoprotein assemblies (25). Thio residues such as 4-thiothymine and 6-thioguanine absorb light at wavelengths longer than 320 nm, and thus can be selectively photo-activated. Owing to the high photo-reactivity of their triplet state, they exhibit high photo-cross-linking ability towards nucleic acid bases as well as towards amino acid residues. Here we used a combination of approaches including gel retardation assays, chemical cross-linking and cross-linking with photoreactive ssDNA probes containing 4-thiothymine, introduced at a defined site in the sequence of the ssDNA, to study interactions present in human RPA (hRPA): ssDNA complexes. These studies coupled with the identification of cross-linked targets using specific antibodies revealed that in the elongated extended hRPA:ssDNA complex RPA3 closely contacts the 3′-end positioned nucleotide and yields a covalent adduct with zero-length photolabel.     

Rotations of the 2B sub-domain of E. coli UvrD helicase/translocase coupled to nucleotide and DNA binding

Escherichia coli UvrD is a superfamily 1 DNA helicase and single-stranded DNA (ssDNA) translocase that functions in DNA repair and plasmid replication and as an anti-recombinase by removing RecA protein from ssDNA. UvrD couples ATP binding and hydrolysis to unwind double-stranded DNA and translocate along ssDNA with 3′-to-5′ directionality. Although a UvrD monomer is able to translocate along ssDNA rapidly and processively, DNA helicase activity in vitro requires a minimum of a UvrD dimer. Previous crystal structures of UvrD bound to a ssDNA/duplex DNA junction show that its 2B sub-domain exists in a “closed” state and interacts with the duplex DNA. Here, we report a crystal structure of an apo form of UvrD in which the 2B sub-domain is in an “open” state that differs by an ∼ 160° rotation of the 2B sub-domain. To study the rotational conformational states of the 2B sub-domain in various ligation states, we constructed a series of double-cysteine UvrD mutants and labeled them with fluorophores such that rotation of the 2B sub-domain results in changes in fluorescence resonance energy transfer. These studies show that the open and closed forms can interconvert in solution, with low salt favoring the closed conformation and high salt favoring the open conformation in the absence of DNA. Binding of UvrD to DNA and ATP binding and hydrolysis also affect the rotational conformational state of the 2B sub-domain, suggesting that 2B sub-domain rotation is coupled to the function of this nucleic acid motor enzyme.     

DNA polymerase III chi subunit ties single-stranded DNA binding protein to the bacterial replication machinery

Single-stranded DNA binding (SSB) protein binds to single-stranded DNA (ssDNA) at the lagging strand of the replication fork in Escherichia coli cells. This protein is essential for the survival of the E.coli cell, presumably because it shields the ssDNA and holds it in a suitable conformation for replication by DNA polymerase III. In this study we undertook a biophysical analysis of the interaction between the SSB protein of E.coli and the χ subunit of DNA polymerase III. Using analytical ultracentrifugation we show that at low salt concentrations there is an increase in the stability in the physical interaction between χ and an EcoSSB/ssDNA complex when compared to that of χ to EcoSSB alone. This increase in stability disappeared in high salt conditions. The sedimentation of an EcoSSB protein lacking its C-terminal 26 amino acids remains unchanged in the presence of χ, showing that χ interacts specifically with the C-terminus of EcoSSB. In DNA melting experiments we demonstrate that χ specifically enhances the ssDNA stabilization by EcoSSB. Thus, the binding of EcoSSB to χ at the replication fork prevents premature dissociation of EcoSSB from the lagging strand and thereby enhances the processivity of DNA polymerase III.     

Single-molecule visualization of human RECQ5 interactions with single-stranded DNA recombination intermediates

RECQ5 is one of five RecQ helicases found in humans and is thought to participate in homologous DNA recombination by acting as a negative regulator of the recombinase protein RAD51. Here, we use kinetic and single molecule imaging methods to monitor RECQ5 behavior on various nucleoprotein complexes. Our data demonstrate that RECQ5 can act as an ATP-dependent single-stranded DNA (ssDNA) motor protein and can translocate on ssDNA that is bound by replication protein A (RPA). RECQ5 can also translocate on RAD51-coated ssDNA and readily dismantles RAD51–ssDNA filaments. RECQ5 interacts with RAD51 through protein–protein contacts, and disruption of this interface through a RECQ5–F666A mutation reduces translocation velocity by ∼50%. However, RECQ5 readily removes the ATP hydrolysis-deficient mutant RAD51–K133R from ssDNA, suggesting that filament disruption is not coupled to the RAD51 ATP hydrolysis cycle. RECQ5 also readily removes RAD51–I287T, a RAD51 mutant with enhanced ssDNA-binding activity, from ssDNA. Surprisingly, RECQ5 can bind to double-stranded DNA (dsDNA), but it is unable to translocate. Similarly, RECQ5 cannot dismantle RAD51-bound heteroduplex joint molecules. Our results suggest that the roles of RECQ5 in genome maintenance may be regulated in part at the level of substrate specificity.