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Repair of broken DNA is essential for life; the reactions involved can also promote genetic recombination to aid evolution. In Escherichia coli, RecBCD enzyme is required for the major pathway of these events. RecBCD is a complex ATP-dependent DNA helicase with nuclease activity controlled by Chi recombination hotspots (5'-GCTGGTGG-3'). During rapid DNA unwinding, when Chi is in a RecC tunnel, RecB nuclease nicks DNA at Chi. Here, we test our signal transduction model - upon binding Chi (step 1), RecC signals RecD helicase to stop unwinding (step 2); RecD then signals RecB (step 3) to nick at Chi (step 4) and to begin loading RecA DNA strand-exchange protein (step 5). We discovered that ATP-γ-S, like the small molecule RecBCD inhibitor NSAC1003, causes RecBCD to nick DNA, independent of Chi, at novel positions determined by the DNA substrate length. Two RecB ATPase-site mutants nick at novel positions determined by their RecB:RecD helicase rate ratios. In each case, we find that nicking at the novel position requires steps 3 and 4 but not step 1 or 2, as shown by mutants altered at the intersubunit contacts specific for each step; nicking also requires RecD helicase and RecB nuclease activities. Thus, altering the RecB ATPase site, by small molecules or mutation, sensitizes RecD to signal RecB to nick DNA (steps 4 and 3, respecitvely) without the signal from RecC or Chi (steps 1 and 2). These new, enzymatic results strongly support the signal transduction model and provide a paradigm for studying other complex enzymes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Much is still unknown about the mechanisms by which helicases unwind duplex DNA. Whereas structure-based models describe DNA unwinding as occurring by the ATPase motors mechanically pulling the DNA duplex across a wedge domain in the helicase, biochemical data show that processive DNA unwinding by E. coli RecBCD helicase can occur in the absence of ssDNA translocation by the canonical RecB and RecD motors. Here we show that DNA unwinding is not a simple consequence of ssDNA translocation by the motors. Using stopped-flow fluorescence approaches, we show that a RecB nuclease domain deletion variant (RecB ΔNuc CD) unwinds dsDNA at significantly slower rates than RecBCD, while the ssDNA translocation rate is unaffected. This effect is primarily due to the absence of the nuclease domain since a nuclease-dead mutant (RecB D1080A CD), which retains the nuclease domain, showed no change in ssDNA translocation or dsDNA unwinding rates relative to RecBCD on short DNA substrates (≤60 base pairs). Hence, ssDNA translocation is not rate-limiting for DNA unwinding. RecB ΔNuc CD also initiates unwinding much slower than RecBCD from a blunt-ended DNA. RecB ΔNuc CD also unwinds DNA ∼two-fold slower than RecBCD on long DNA (∼20 kilo base pair) in single molecule optical tweezer experiments, although the rates for RecB D1080A CD unwinding are intermediate between RecBCD and RecB ΔNuc CD. Surprisingly, significant pauses in DNA unwinding occur even in the absence of chi (crossover hotspot instigator) sites. We hypothesize that the nuclease domain influences the rate of DNA base pair melting, possibly allosterically and that RecB ΔNuc CD may mimic a post-chi state of RecBCD., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Escherichia coli RecBCD helicase-nuclease promotes vital homologous recombination-based repair of DNA double-strand breaks. The RecB nuclease domain (Nuc) is connected to the RecB helicase domain by a 19-amino-acid tether. When DNA binds to RecBCD, published evidence suggests that Nuc moves ∼50 Å from the exit of a RecC tunnel, from which the 3'-ended strand emerges during unwinding, to a distant position on RecC's surface. During subsequent ATP-dependent unwinding of DNA, Nuc nicks the 3'-ended strand near 5'-GCTGGTGG-3' (Chi recombination hotspot). Here, we test our model of Nuc swinging on the tether from the RecC tunnel exit to the RecC distant surface and back to the RecC tunnel exit to cut at Chi. We identify positions in a flexible surface loop on RecC and on RecB Nuc with complementary charges, mutation of which strongly reduces but does not eliminate Chi hotspot activity in cells. The recC loop mutation interacts with recB mutations hypothesized to be in the Chi-activated intramolecular signal transduction pathway; the double mutants, but not the single mutants, eliminate Chi hotspot activity. A RecC amino acid near the flexible loop is also essential for full Chi activity; its alteration likewise synergizes with a signal transduction mutation to eliminate Chi activity. We infer that altering the RecC surface loop reduces coordination among the subunits, which is critical for Chi hotspot activity. We discuss other RecBCD mutants with related properties., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Genetics Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

E. coli RecBCD, a helicase/nuclease involved in double stranded (ds) DNA break repair, binds to a dsDNA end and melts out several DNA base pairs (bp) using only its binding free energy. We examined RecBCD-DNA initiation complexes using thermodynamic and structural approaches. Measurements of enthalpy changes for RecBCD binding to DNA ends possessing pre-melted ssDNA tails of increasing length suggest that RecBCD interacts with ssDNA as long as 17-18 nucleotides and can melt at least 10-11 bp upon binding a blunt DNA end. Cryo-EM structures of RecBCD alone and in complex with a blunt-ended dsDNA show significant conformational heterogeneities associated with the RecB nuclease domain (RecB Nuc ) and the RecD subunit. In the absence of DNA, 56% of RecBCD molecules show no density for the RecB nuclease domain, RecB Nuc , and all RecBCD molecules show only partial density for RecD. DNA binding reduces these conformational heterogeneities, with 63% of the molecules showing density for both RecD and RecB Nuc . This suggests that the RecB Nuc domain is dynamic and influenced by DNA binding. The major RecBCD-DNA structural class in which RecB Nuc is docked onto RecC shows melting of at least 11 bp from a blunt DNA end, much larger than previously observed. A second structural class in which RecB Nuc is not docked shows only four bp melted suggesting that RecBCD complexes transition between states with different extents of DNA melting and that the extent of melting regulates initiation of helicase activity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Coordinating multiple activities of complex enzymes is critical for life, including transcribing, replicating and repairing DNA. Bacterial RecBCD helicase-nuclease must coordinate DNA unwinding and cutting to repair broken DNA. Starting at a DNA end, RecBCD unwinds DNA with its fast RecD helicase on the 5'-ended strand and its slower RecB helicase on the 3'-ended strand. At Chi hotspots (5' GCTGGTGG 3'), RecB's nuclease cuts the 3'-ended strand and loads RecA strand-exchange protein onto it. We report that a small molecule NSAC1003, a sulfanyltriazolobenzimidazole, mimics Chi sites by sensitizing RecBCD to cut DNA at a Chi-independent position a certain percent of the DNA substrate's length. This percent decreases with increasing NSAC1003 concentration. Our data indicate that NSAC1003 slows RecB relative to RecD and sensitizes it to cut DNA when the leading helicase RecD stops at the DNA end. Two previously described RecBCD mutants altered in the RecB ATP-binding site also have this property, but uninhibited wild-type RecBCD lacks it. ATP and NSAC1003 are competitive; computation docks NSAC1003 into RecB's ATP-binding site, suggesting NSAC1003 acts directly on RecB. NSAC1003 will help elucidate molecular mechanisms of RecBCD-Chi regulation and DNA repair. Similar studies could help elucidate other DNA enzymes with activities coordinated at chromosomal sites., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The RecBCD complex plays key roles in phage DNA degradation, CRISPR array acquisition (adaptation) and host DNA repair. The switch between these roles is regulated by a DNA sequence called Chi. We report cryo-EM structures of the Escherichia coli RecBCD complex bound to several different DNA forks containing a Chi sequence, including one in which Chi is recognized and others in which it is not. The Chi-recognized structure shows conformational changes in regions of the protein that contact Chi and reveals a tortuous path taken by the DNA. Sequence specificity arises from interactions with both the RecC subunit and the sequence itself. These structures provide molecular details for how Chi is recognized and insights into the changes that occur in response to Chi binding that switch RecBCD from bacteriophage destruction and CRISPR spacer acquisition to constructive host DNA repair.

The presence of adducts on the DNA double-helix can have major consequences for the efficient functioning of DNA repair enzymes. E. coli RecBCD (exonuclease V) is involved in recombinational repair of double-strand breaks that are caused by defective DNA replication, DNA damaging agents and other factors. The holoenzyme possesses a bipolar helicase activity which helps unwind DNA from both 3'- and 5'-directions and is coupled with a potent exonuclease activity that is also capable of digesting DNA from both 3'- and 5'-ends. In this study, DNA sequences were damaged with cisplatin or UV followed by RecBCD treatment. DNA damaging agents such as cisplatin and UV induce the formation of intrastrand adducts in the DNA template. It was demonstrated that RecBCD degradation was inhibited by either cisplatin-damaged or UV-damaged DNA sequences. This is the first occasion that RecBCD has been demonstrated to be inhibited by DNA adducts induced by cisplatin or UV. In addition, we quantified the amounts of DNA remaining after RecBCD treatment and observed that the level of inhibition was concentration and dose dependent. A DNA-targeted 9-aminoacridinecarboxamide cisplatin analogue was also found to inhibit RecBCD activity., (Crown Copyright © 2017. Published by Elsevier Inc. All rights reserved.)

In bacterial cells, processing of double-stranded DNA breaks for repair by homologous recombination is catalysed by AddAB, AdnAB or RecBCD-type helicase-nucleases. These enzyme complexes are highly processive, duplex unwinding and degrading machines that require tight regulation. Here, we report the structure of E.coli RecBCD, determined by cryoEM at 3.8 Å resolution, with a DNA substrate that reveals how the nuclease activity of the complex is activated once unwinding progresses. Extension of the 5'-tail of the unwound duplex induces a large conformational change in the RecD subunit, that is transferred through the RecC subunit to activate the nuclease domain of the RecB subunit. The process involves a SH3 domain that binds to a region of the RecB subunit in a binding mode that is distinct from others observed previously in SH3 domains and, to our knowledge, this is the first example of peptide-binding of an SH3 domain in a bacterial system., Competing Interests: The authors declare that no competing interests exist.

RecBCD enzyme is a complex, three-subunit protein machine essential for the major pathway of DNA double-strand break repair and homologous recombination in Escherichia coli Upon encountering a Chi recombination-hotspot during DNA unwinding, RecBCD nicks DNA to produce a single-stranded DNA end onto which it loads RecA protein. Conformational changes that regulate RecBCD's helicase and nuclease activities are induced upon its interaction with Chi, defined historically as 5' GCTGGTGG 3'. Chi is thought to be recognized as single-stranded DNA passing through a tunnel in RecC. To define the Chi recognition-domain in RecC and thus the mechanism of the RecBCD-Chi interaction, we altered by random mutagenesis eight RecC amino acids lining the tunnel. We screened for loss of Chi activity with Chi at one site in bacteriophage λ. The 25 recC mutants analyzed thoroughly had undetectable or strongly reduced Chi-hotspot activity with previously reported Chi sites. Remarkably, most of these mutants had readily detectable, and some nearly wild-type, activity with Chi at newly generated Chi sites. Like wild-type RecBCD, these mutants had Chi activity that responded dramatically (up to fivefold, equivalent to Chi's hotspot activity) to nucleotide changes flanking 5' GCTGGTGG 3'. Thus, these and previously published RecC mutants thought to be Chi-recognition mutants are actually Chi context-dependence mutants. Our results fundamentally alter the view that Chi is a simple 8-bp sequence recognized by the RecC tunnel. We propose that Chi hotspots have dual nucleotide sequence interactions, with both the RecC tunnel and the RecB nuclease domain., (Copyright © 2016 by the Genetics Society of America.)

RecBCD is a multifunctional enzyme that possesses both helicase and nuclease activities. To gain insight into the mechanism of its helicase function, RecBCD unwinding at low adenosine triphosphate (ATP) (2-4 μM) was measured using an optical-trapping assay featuring 1 base-pair (bp) precision. Instead of uniformly sized steps, we observed forward motion convolved with rapid, large-scale (∼4 bp) variations in DNA length. We interpret this motion as conformational dynamics of the RecBCD-DNA complex in an unwinding-competent state, arising, in part, by an enzyme-induced, back-and-forth motion relative to the dsDNA that opens and closes the duplex. Five observations support this interpretation. First, these dynamics were present in the absence of ATP. Second, the onset of the dynamics was coupled to RecBCD entering into an unwinding-competent state that required a sufficiently long 5' strand to engage the RecD helicase. Third, the dynamics were modulated by the GC-content of the dsDNA. Fourth, the dynamics were suppressed by an engineered interstrand cross-link in the dsDNA that prevented unwinding. Finally, these dynamics were suppressed by binding of a specific non-hydrolyzable ATP analog. Collectively, these observations show that during unwinding, RecBCD binds to DNA in a dynamic mode that is modulated by the nucleotide state of the ATP-binding pocket., (Published by Oxford University Press on behalf of Nucleic Acids Research 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

Type I restriction-modification enzymes are multifunctional heteromeric complexes with DNA cleavage and ATP-dependent DNA translocation activities located on motor subunit HsdR. Functional coupling of DNA cleavage and translocation is a hallmark of the Type I restriction systems that is consistent with their proposed role in horizontal gene transfer. DNA cleavage occurs at nonspecific sites distant from the cognate recognition sequence, apparently triggered by stalled translocation. The X-ray crystal structure of the complete HsdR subunit from E. coli plasmid R124 suggested that the triggering mechanism involves interdomain contacts mediated by ATP. In the present work, in vivo and in vitro activity assays and crystal structures of three mutants of EcoR124I HsdR designed to probe this mechanism are reported. The results indicate that interdomain engagement via ATP is indeed responsible for signal transmission between the endonuclease and helicase domains of the motor subunit. A previously identified sequence motif that is shared by the RecB nucleases and some Type I endonucleases is implicated in signaling.

A fundamental feature of many nucleic-acid binding proteins is their ability to move along DNA either by diffusion-based mechanisms or by ATP-hydrolysis driven translocation. For example, most site-specific DNA-binding proteins must diffuse to some extent along DNA to either find their target sites, or to otherwise fulfill their biological roles. Similarly, nucleic-acid translocases such as helicases and polymerases must move along DNA to fulfill their functions. In both instances, the proteins must also be capable of moving in crowded environments while navigating through DNA-bound obstacles. These types of behaviors can be challenging to analyze by bulk biochemical methods because of the transient nature of the interactions, and/or heterogeneity of the reaction intermediates. The advent of single-molecule methodologies has overcome some of these problems, and has led to many new insights into the mechanisms that contribute to protein motion along DNA. We have developed DNA curtains as a tool to facilitate single molecule observations of protein-nucleic acid interactions, and we have applied these new research tools to systems involving both diffusive-based motion as well as ATP directed translocation. Here we highlight these studies by first discussing how diffusion contributes to target searches by proteins involved in post-replicative mismatch repair. We then discuss DNA curtain assays of two different DNA translocases, RecBCD and FtsK, which participate in homologous DNA recombination and site-specific DNA recombination, respectively., (Copyright © 2014 Elsevier B.V. All rights reserved.)

AddAB and RecBCD-type helicase-nuclease complexes control the first stage of bacterial homologous recombination (HR) - the resection of double strand DNA breaks. A switch in the activities of the complexes to initiate repair by HR is regulated by a short, species-specific DNA sequence known as a Crossover Hotspot Instigator (Chi) site. It has been shown that, upon encountering Chi, AddAB and RecBCD pause translocation before resuming at a reduced rate. Recently, the structure of B.subtilis AddAB in complex with its regulatory Chi sequence revealed the nature of Chi binding and the paused translocation state. Here the structural features associated with Chi binding are described in greater detail and discussed in relation to the related E.coli RecBCD system.

The heterotrimeric Escherichia coli RecBCD enzyme comprises two helicase motors with different polarities: RecB (3'-to-5') and RecD (5'-to-3'). This superfamily I helicase is responsible for initiating DNA double-strand-break (DSB) repair in the homologous recombination pathway. We used single-molecule tethered particle motion (TPM) experiments to visualize the RecBCD helicase translocation over long single-stranded (ss) DNA (>200 nt) with no apparent secondary structure. The bead-labeled RecBCD helicases were found to bind to the surface-immobilized blunt-end DNA, and translocate along the DNA substrates containing an ssDNA gap, resulting in a gradual decrease in the bead Brownian motion. Successful observation of RecBCD translocation over a long gap in either 3'-to-5' or 5'-to-3' ssDNA direction indicates that RecBCD helicase possesses ssDNA translocase activities in both polarities. Most RecBCD active tethers showed full translocation across the ssDNA to the dsDNA region, with about 19% of enzymes dissociated from the ss/dsDNA junction after translocating across the ssDNA region. In addition, we prepared DNA substrates containing two opposite polarities (5'-to-3' and 3'-to-5') of ssDNA regions intermitted by duplex DNA. RecBCD was able to translocate across both ssDNA regions in either ssDNA orientation orders, with less than 40% of tethers dissociating when entering into the second ssDNA region. These results suggest a model that RecBCD is able to switch between ssDNA translocases and rethread the other strand of ssDNA.

The repair of DNA double-strand breaks (DSBs) is essential for cell viability and important for homologous genetic recombination. In enteric bacteria such as Escherichia coli, the major pathway of DSB repair requires the RecBCD enzyme, a complex helicase-nuclease regulated by a simple unique DNA sequence called Chi. How Chi regulates RecBCD has been extensively studied by both genetics and biochemistry, and two contrasting mechanisms to generate a recombinogenic single-stranded DNA tail have been proposed: the nicking of one DNA strand at Chi versus the switching of degradation from one strand to the other at Chi. Which of these reactions occurs in cells has remained unproven because of the inability to detect intracellular DNA intermediates in bacterial recombination and DNA break repair. Here, I discuss evidence from a combination of genetics and biochemistry indicating that nicking at Chi is the intracellular (in vivo) reaction. This example illustrates the need for both types of analysis (i.e., molecular biology) to uncover the mechanism and control of complex processes in living cells.

In bacterial cells, processing of double-stranded DNA breaks for repair by homologous recombination is dependent upon the recombination hotspot sequence Chi and is catalysed by either an AddAB- or RecBCD-type helicase-nuclease. Here, we report the crystal structure of AddAB bound to DNA. The structure allows identification of a putative Chi-recognition site in an inactivated helicase domain of the AddB subunit. By generating mutant protein complexes that do not respond to Chi, we show that residues responsible for Chi recognition are located in positions equivalent to the signature motifs of a conventional helicase. Comparison with the related RecBCD complex, which recognizes a different Chi sequence, provides further insight into the structural basis for sequence-specific ssDNA recognition. The structure suggests a simple mechanism for DNA break processing, explains how AddAB and RecBCD can accomplish the same overall reaction with different sets of functional modules and reveals details of the role of an Fe-S cluster in protein stability and DNA binding.

In this issue, Wu et al. show that the RecBC helicase, which is involved in repairing double-strand DNA breaks,uses one ATPase motor to drive two translocases along opposite strands of DNA—much as an all-wheel drive engine controls movement of both front and back wheels. This mechanism may allow RecBC to load onto blunt-end DNA more efficiently and to move through obstacles such as gaps and DNA damage.

E. coli RecBCD is a DNA helicase with two ATPase motors (RecB, a 3'→5' translocase, and RecD, a 5'→3' translocase) that function in repair of double-stranded DNA breaks. The RecBC heterodimer, with only the RecB motor, remains a processive helicase. Here we examined RecBC translocation along single-stranded DNA (ssDNA). Notably, we found RecBC to have two translocase activities: the primary translocase moves 3'→5', whereas the secondary translocase moves RecBC along the opposite strand of a forked DNA at a similar rate. The secondary translocase is insensitive to the ssDNA backbone polarity, and we propose that it may fuel RecBCD translocation along double-stranded DNA ahead of the unwinding fork and ensure that the unwound single strands move through RecBCD at the same rate after interaction with a crossover hot-spot indicator (Chi) sequence.

RecD2 from Deinococcus radiodurans is a superfamily 1 DNA helicase that is homologous to the Escherichia coli RecD protein but functions outside the context of RecBCD enzyme. We report here on the kinetics of DNA unwinding by RecD2 under single and multiple turnover conditions. There is little unwinding of 20-bp substrates by preformed RecD2-dsDNA complexes when excess ssDNA is present to trap enzyme molecules not bound to the substrate. A shorter 12-bp substrate is unwound rapidly under single turnover conditions. The 12-bp unwinding reaction could be simulated with a mechanism in which the DNA is unwound in two kinetic steps with rate constant of k(unw) = 5.5 s(-1) and a dissociation step from partially unwound DNA of k(off) = 1.9 s(-1). These results indicate a kinetic step size of about 3-4 bp, unwinding rate of about 15-20 bp/s, and low processivity (p = 0.74). The reaction time courses with 20-bp substrates, determined under multiple turnover conditions, could be simulated with a four-step mechanism and rate constant values very similar to those for the 12-bp substrate. The results indicate that the faster unwinding of a DNA substrate with a forked end versus only a 5'-terminal single-stranded extension can be accounted for by a difference in the rate of enzyme binding to the DNA substrates. Analysis of reactions done with different RecD2 concentrations indicates that the enzyme forms an inactive dimer or other oligomer at high enzyme concentrations. RecD2 oligomers can be detected by glutaraldehyde cross-linking but not by size exclusion chromatography.

In traditional biochemical experiments, the behavior of individual proteins is obscured by ensemble averaging. To better understand the behavior of proteins that bind to and/or translocate on DNA, we have developed instrumentation that uses optical trapping, microfluidic solution delivery, and fluorescent microscopy to visualize either individual proteins or assemblies of proteins acting on single molecules of DNA. The general experimental design involves attaching a single DNA molecule to a polystyrene microsphere that is then used as a microscopic handle to manipulate individual DNA molecules with a laser trap. Visualization is achieved by fluorescently labeling either the DNA or the protein of interest, followed by direct imaging using high-sensitivity fluorescence microscopy. We describe the sample preparation and instrumentation used to visualize the interaction of individual proteins with single molecules of DNA. As examples, we describe the application of these methods to the study of proteins involved in recombination-mediated DNA repair, a process essential for the maintenance of genomic integrity., (Copyright 2010 Elsevier Inc. All rights reserved.)

RecBCD is a processive molecular motor composed of two independent helicase domains and a nuclease domain. Understanding the molecular mechanism of its motor activity involves, in part, determining RecBCD's translocation properties (e.g., velocity, propensity to pause, pause duration). Single-molecule techniques, in general, and optical trapping, specifically, provide for measuring the translocation of individual molecules along DNA. We developed a high-spatial resolution optical-trapping assay for RecBCD. The RecBCD is anchored to the surface via a genetically engineered biotin. This RecBCD-bio exhibited native activity, as measured by biochemical assays. Motion is continuous down to a detection limit of 2 nm, implying a unitary step size below 6 base pairs. Unexpectedly, the catalytic rate changes abruptly and persists at different values for tens of seconds. This technically demanding, high-resolution optical-trapping assay is complemented by a simpler single-molecule assay-the tethered particle motion assay.

The E. coli RecBCD enzyme facilitates the loading of RecA onto single-stranded DNA produced by the combined helicase/nuclease activity of RecBCD. The nuclease domain of RecB protein, RecB(nuc), has been previously shown to bind RecA. Surprisingly, RecB(nuc) also binds to phage and eukaryotic homologs of RecA, leading to the suggestion that RecB(nuc) interacts with the polymerization motif that is present in all three proteins. This mode of interaction could only be with monomeric RecA, as this motif would be buried in filaments. We show that RecB(nuc) binds extensively to the outside of RecA-DNA filaments. Three-dimensional reconstructions suggest that RecB(nuc) binds to the ATP-binding core of RecA, with a displacement of the C-terminal domain of RecA. Solution experiments confirm that the interaction of RecB(nuc) is only with the RecA core. Since the RecA C-terminal domain has been shown to be regulatory, the interaction observed may be part of the loading mechanism where RecB displaces the RecA C-terminal domain and activates a RecA monomer for polymerization.

Escherichia coli RecBCD helicase unwinds blunt-end duplex DNA to repair damaged DNA molecules in the homologous recombination pathway. Previous single-molecule experiments showed that RecBCD recognizes an 8 nt DNA sequence, chi, and lowers its unwinding rate afterward under saturating ATP condition. We have developed a single-molecule force-tethered particle motion (FTPM) method, which is modified from the conventional TPM method, and applied it to study RecBCD motion in detail. In the FTPM experiment, a stretching force is applied to the DNA-bead complex that suppresses the bead's Brownian motion, resulting in an improved spatial resolution at long DNA substrates. Based on the equipartition theorem, the mean-square displacement of the bead's Brownian motion measured by FTPM correlates linearly to DNA extension length with a predicted slope, circumventing the difficulties of conventional TPM experiments, such as nonlinearity and low resolution of long DNA substrates. The FTPM method offers the best resolution in the presence of only a small stretching force (0.20 pN). We used the FTPM method to investigate RecBCD helicase motion along 4.1 kb long chi-containing duplex DNA molecules, and observed that the translocation rate of RecBCD changes after the chi sequence under limited ATP concentrations. This suggests that chi recognition by RecBCD does not require saturating ATP conditions, contrary to what was previously reported.

The molecular mechanism of superfamily 1Balpha helicases remains unclear. We present here the crystal structure of the RecD2 helicase from Deinococcus radiodurans at 2.2-A resolution. The structure reveals the folds of the 1B and 2B domains of RecD that were poorly ordered in the structure of the Escherichia coli RecBCD enzyme complex reported previously. The 2B domain adopts an SH3 fold which, although common in eukaryotes, is extremely rare in bacterial systems. In addition, the D. radiodurans RecD2 structure has aided us in deciphering lower resolution (3.6 A) electron density maps for the E. coli RecBCD enzyme in complex with a long DNA substrate that interacts with the RecD subunit. Taken together, these structures indicated an important role for the 1B domain of RecD, a beta-hairpin that extends from the surface of the 1A domain and interacts with the DNA substrate. On the basis of these structural data, we designed a mutant RecD2 helicase that lacks this pin. The 'pin-less' mutant protein is a fully active ssDNA-dependent ATPase but totally lacks helicase activity.

The Type I restriction-modification enzyme EcoR124I is an ATP-dependent endonuclease that uses dsDNA translocation to locate and cleave distant non-specific DNA sites. Bioinformatic analysis of the HsdR subunits of EcoR124I and related Type I enzymes showed that in addition to the principal PD-(E/D)xK Motifs, I, II and III, a QxxxY motif is also present that is characteristic of RecB-family nucleases. The QxxxY motif resides immediately C-terminal to Motif III within a region of predicted alpha-helix. Using mutagenesis, we examined the role of the Q and Y residues in DNA binding, translocation and cleavage. Roles for the QxxxY motif in coordinating the catalytic residues or in stabilizing the nuclease domain on the DNA are discussed.

RecD is essential for growth at low temperature in the Antarctic psychrotrophic bacterium Pseudomonas syringae Lz4W. To examine the essential nature of its activity, we analyzed wild-type and mutant RecD proteins with substitutions of important residues in each of the seven conserved helicase motifs. The wild-type RecD displayed DNA-dependent ATPase and helicase activity in vitro, with the ability to unwind short DNA duplexes containing only 5' overhangs or forked ends. Five of the mutant proteins, K229Q (in motif I), D323N and E324Q (in motif II), Q354E (in motif III) and R660A (in motif VI) completely lost both ATPase and helicase activities. Three other mutants, T259A in motif Ia, R419A in motif IV and E633Q in motif V exhibited various degrees of reduction in ATPase activity, but had no helicase activity. While all RecD proteins had DNA-binding activity, the mutants of motifs IV and V displayed reduced binding, and the motif II mutant showed a higher degree of binding to ssDNA. Significantly, only RecD variants with in vitro ATPase activity could complement the cold-sensitive growth of a recD-inactivated strain of P. syringae at 4 degrees C. These results suggest that the requirement for RecD at lower temperatures lies in its ATP-hydrolyzing activity.

RecBCD is a DNA helicase comprising two motor subunits, RecB and RecD. Recognition of the recombination hotspot, chi, causes RecBCD to pause and reduce translocation speed. To understand this control of translocation, we used single-molecule visualization to compare RecBCD to the RecBCD(K177Q) mutant with a defective RecD motor. RecBCD(K177Q) paused at chi but did not change its translocation velocity. RecBCD(K177Q) translocated at the same rate as the wild-type post-chi enzyme, implicating RecB as the lead motor after chi. P1 nuclease treatment eliminated the wild-type enzyme's velocity changes, revealing a chi-containing ssDNA loop preceding chi recognition and showing that RecD is the faster motor before chi. We conclude that before chi, RecD is the lead motor but after chi, the slower RecB motor leads, implying a switch in motors at chi. We suggest that degradation of foreign DNA needs fast translocation, whereas DNA repair uses slower translocation to coordinate RecA loading onto ssDNA.

The DNA helicase RecBCD pauses when it reaches recombination hotspots known as Chi sites and then proceeds at a slower speed of translocation than before Chi recognition. Reporting in this issue, Spies et al. (2007) now show that this reduction in translocation velocity occurs when RecBCD changes which of its two motor subunits is in the lead.

The role of the RecBCD recombination pathway in PilE antigenic variation in Neisseria gonorrhoeae is contentious and appears to be strain dependent. In this study, N. gonorrhoeae strain MS11 recB mutants were assessed for recombination/repair. MS11 recB mutants were found to be highly susceptible to DNA treatments that caused double-chain breaks and were severely impaired for growth; recB growth suppressor mutants arose at high frequencies. When the recombination/repair capacity of strain MS11 was compared to that of strains FA1090 and P9, innate differences were observed between the strains, with FA1090 and P9 rec(+) bacteria presenting pronounced recombination/repair defects. Consequently, MS11 recB mutants present a more robust phenotype than the other strains that were tested. In addition, MS11 recB mutants are also shown to be defective for pilE/pilS recombination. Moreover, pilE/pilS recombination is shown to proceed with gonococci that carry inverted pilE loci. Consequently, a novel RecBCD-mediated double-chain-break repair model for PilE antigenic variation is proposed.

In Escherichia coli, RecBCD processes double-stranded DNA breaks during the initial stages of homologous recombination. RecBCD contains helicase and nuclease activities, and unwinds and digests the blunt-ended DNA until a specific eight-nucleotide sequence, Chi, is encountered. Chi modulates the nuclease activity of RecBCD and results in a resected DNA end, which is a substrate for RecA during subsequent steps in recombination. RecBCD also acts as a defence mechanism against bacteriophage infection by digesting linear viral DNA present during virus replication or resulting from the action of restriction endonucleases. To avoid this fate, bacteriophage lambda encodes the gene Gam whose product is an inhibitor of RecBCD. Gam has been shown to bind to RecBCD and inhibit its helicase and nuclease activities. We show that Gam inhibits RecBCD by preventing it from binding DNA. We have solved the crystal structure of Gam from two different crystal forms. Using the published crystal structure of RecBCD in complex with DNA we suggest models for the molecular mechanism of Gam-mediated inhibition of RecBCD. We also propose that Gam could be a mimetic of single-stranded, and perhaps also double-stranded, DNA.

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.

The F plasmid TraI protein (DNA helicase I) plays an essential role in conjugative DNA transfer as both a transesterase and a helicase. Previous work has shown that the 192-kDa TraI protein is a highly processive helicase, catalytically separating >850 bp under steady-state conditions. In this report, we examine the kinetic mechanism describing DNA unwinding of TraI. The kinetic step size of TraI was measured under both single turnover and pre-steady-state conditions. The resulting kinetic step-size estimate was approximately 6-8 bp step(-1). TraI can separate double-stranded DNA at a rate of approximately 1100 bp s(-1), similar to the measured unwinding rate of the RecBCD helicase, and appears to dissociate very slowly from the 3' terminus following translocation and strand-separation events. Analyses of pre-steady-state burst amplitudes indicate that TraI can function as a monomer, similar to the bacteriophage T4 helicase, Dda. However, unlike Dda, TraI is a highly processive monomeric helicase, making it unique among the DNA helicases characterized thus far.

The equilibrium binding of Escherichia coli RecBC and RecBCD helicases to duplex DNA ends containing varying lengths of polyethylene glycol (PEG) spacers within pre-formed 3'-single-stranded (ss) DNA ((dT)n) tails was studied. These studies were designed to test a previous proposal that the 3'-(dT)n tail can be looped out upon binding RecBC and RecBCD for 3'-ssDNA tails with n>or=6 nucleotides. Equilibrium binding of protein to unlabeled DNA substrates with ends containing PEG-substituted 3'-ssDNA tails was examined by competition with a Cy3-labeled reference DNA which undergoes a Cy3 fluorescence enhancement upon protein binding. We find that the binding affinities of both RecBC and RecBCD for a DNA end are unaffected upon substituting PEG for the ssDNA between the sixth and the final two nucleotides of the 3'-(dT)n tail. However, placing PEG at the end of the 3'-(dT)n tail increases the binding affinities to their maximum values (i.e. the same as binding constants for RecBC or RecBCD to a DNA end with only a 3'-(dT)6 tail). Equilibrium binding studies of a RecBC mutant containing a nuclease domain deletion, RecB(Deltanuc)C, suggest that looping of the 3'-tail (when n>or=6 nucleotides) occurs even in the absence of the RecB nuclease domain, although the nuclease domain stabilizes such loop formation. Computer modeling of the RecBCD-DNA complexes suggests that the loop in the 3'-ssDNA tail may form at the RecB/RecC interface. Based on these results we suggest a model for how a loop in the 3'-ssDNA tail might form upon encounter of a "Chi" recognition sequence during unwinding of DNA by the RecBCD helicase.

Telomeres, the protein-DNA complexes at the ends of eukaryotic chromosomes, are essential for chromosome stability, and their maintenance is achieved by the specialized reverse transcriptase activity of telomerase or the homologous recombination pathway in most eukaryotes. Here, we identified a human helicase, hPif1 that inhibits telomerase activity. The primary sequence and biochemical analysis suggest that hPif1 is a potential homologue of Escherichia coli RecD, an ATP-dependent 5' to 3' DNA helicase. Ectopic expression of wild-type, but not the ATPase/helicase-deficient hPif1, causes telomere shortening in HT1080 cells. hPif1 reduces telomerase processivity and unwinds DNA/RNA duplex in vitro. hPif1 preferentially binds telomeric DNA in vitro and in vivo. We propose that the mechanism of hPif1's inhibition on telomerase involves unwinding of the DNA/RNA duplex formed by telomerase RNA and telomeric DNA, and RecD homologues in eukaryotes may have evolved gaining additional functions.

RecBCD enzyme facilitates loading of RecA protein onto ssDNA produced by its helicase/nuclease activity. This process is essential for RecBCD-mediated homologous recombination. Here, we establish that the C-terminal nuclease domain of the RecB subunit (RecBnuc) forms stable complexes with RecA. Interestingly, RecBnuc also interacts with and loads noncognate DNA strand exchange proteins. Interaction is with a conserved element of the RecA-fold, but because the binding to noncognate proteins decreases in a phylogenetically consistent way, species-specific interactions are also present. RecBnuc does not impede activities of RecA that are important to DNA strand exchange, consistent with its role in targeting of RecA. Modeling predicts the interaction interface for the RecA-RecBCD complex. Because a similar interface is involved in the binding of human Rad51 to the conserved BRC repeat of BRCA2 protein, the RecB-domain may be one of several structural domains that interact with and recruit DNA strand exchange proteins to DNA.

RecBCD enzyme is a heterotrimeric helicase/nuclease that initiates homologous recombination at double-stranded DNA breaks. The enzyme is driven by two motor subunits, RecB and RecD, translocating on opposite single-strands of the DNA duplex. Here we provide evidence that, although both motor subunits can support the translocation activity for the enzyme, the activity of the RecB subunit is necessary for proper function of the enzyme both in vivo and in vitro. We demonstrate that the RecBCD(K177Q) enzyme, in which RecD helicase is disabled by mutation of the ATPase active site, complements recBCD deletion in vivo and displays all of the enzymatic activities that are characteristic of the wild-type enzyme in vitro. These include helicase and nuclease activities and the abilities to recognize the recombination hotspot chi and to coordinate the loading of RecA protein onto the ssDNA it produces. In contrast, the RecB(K29Q)CD enzyme, carrying a mutation in the ATPase site of RecB helicase, fails to complement recBCD deletion in vivo. We further show that even though RecB(K29Q)CD enzyme displays helicase and nuclease activities, its inability to translocate along the 3'-terminated strand results in the failure to recognize chi and to load RecA protein. Our findings argue that translocation by the RecB motor is required to deliver RecC subunit to chi, whereas the RecD subunit has a dispensable motor activity but an indispensable regulatory function.

Background: The PD-(D/E)xK superfamily, containing a wide variety of other exo- and endonucleases, is a notable example of general function conservation in the face of extreme sequence and structural variation. Almost all members employ a small number of shared conserved residues to bind catalytically essential metal ions and thereby effect DNA cleavage. The crystal structure of the RecBCD prokaryotic DNA repair machinery shows that RecB contains such a nuclease domain at its C-terminus. The RecC C-terminal region was reported as having a novel fold., Results: The RecC C-terminal region can be divided into an alpha/beta domain and a smaller alpha-helical bundle domain. Here we show that the alpha/beta domain is homologous to the RecB nuclease domain but lacks the features necessary for catalysis. Instead, the domain has a novel function within the nuclease superfamily--providing a hoop through which single-stranded DNA passes. Comparison with other structures of nuclease domains bound to DNA reveals strikingly different modes of ligand binding. The alpha-helical bundle domain contributes the pin which splits the DNA duplex., Conclusion: The demonstrated homology of RecB and RecC shows how evolution acted to produce the present RecBCD complex through aggregation of new domains as well as functional divergence and structural redeployment of existing domains. Distantly homologous nuclease(-like) domains bind DNA in highly diverse manners.

Restriction endonucleases and other nucleic acid cleaving enzymes form a large and extremely diverse superfamily that display little sequence similarity despite retaining a common core fold responsible for cleavage. The lack of significant sequence similarity between protein families makes homology inference a challenging task and hinders new family identification with traditional sequence-based approaches. Using the consensus fold recognition method Meta-BASIC that combines sequence profiles with predicted protein secondary structure, we identify nine new restriction endonuclease-like fold families among previously uncharacterized proteins and predict these proteins to cleave nucleic acid substrates. Application of transitive searches combined with gene neighborhood analysis allow us to confidently link these unknown families to a number of known restriction endonuclease-like structures and thus assign folds to the uncharacterized proteins. Finally, our method identifies a novel restriction endonuclease-like domain in the C-terminus of RecC that is not detected with structure-based searches of the existing PDB database.

We study the effect of a coupling between two motor domains in highly processive motor protein complexes. A simple stochastic discrete model, in which the two parts of the protein molecule interact through some energy potential, is presented. The exact analytical solutions for the dynamic properties of the combined motor species, such as the velocity and dispersion, are derived in terms of the properties of free individual motor domains and the interaction potential. It is shown that the coupling between the motor domains can create a more efficient motor protein that can move faster than individual particles. The results are applied to analyze the motion of RecBCD helicase molecules.

Catalytic DNA cleavage reactions by an ATP-dependent deoxyribonuclease (DNase) from Micrococcus luteus were monitored directly with a DNA-immobilized 27-MHz quartz-crystal microbalance (QCM). The 27-MHz QCM is a very sensitive mass-measuring device in aqueous solution, as the frequency decreases linearly with increasing mass on the electrode at a nanogram level. Three steps in ATP-dependent DNA hydrolysis reactions, including (1) binding of DNase to the end of double-stranded DNA (dsDNA) on the QCM electrode (mass increase), (2) degradation of one strand of dsDNA in the 3' --> 5' direction depending on ATP (mass decrease), and (3) release of the enzyme from the nonhydrolyzed 5'-free-ssDNA (mass decrease), could be monitored stepwise from the time dependencies of QCM frequency changes. Kinetic parameters for each step were obtained as follows. The binding constant (K(a)) of DNase to the dsDNA was determined as (28 +/- 2) x 10(6) M(-)(1) (k(on) = (8.0 +/- 0.3) x 10(3) M (-)(1) s(-)(1) and k(off) = (0.29 +/-0.01) x 10(-)(3) s(-)(1)), and it decreased to (0.79 +/- 0.16) x 10(6) M(-)(1) (k'(on) = (2.3 +/- 0.2) x 10(3) M (-)(1) s(-)(1) and k'(off) = (2.9 +/- 0.1) x 10(-)(3) s(-)(1)) for the completely nonhydrolyzed 5'-free ssDNA. This is the reason the DNase bound to the dsDNA substrate can easily release from the nonhydrolyzed 5'-free-ssDNA after the complete hydrolysis of the 3' --> 5' direction of the complementary ssDNA. K(a) values depended on the DNA structures on the QCM, and the order of these values was as follows: the dsDNA having a 4-base-mismatched base-pair end (3) > the dsDNA having a 5' 15-base overhanging end (2) > the dsDNA having a blunt end (1) > the ssDNA having a 3'-free end (4) >> the ssDNA having a 5'-free end (5). Thus, DNase hardly recognized the free 5' end of ssDNA. Michaelis-Menten parameters (K(m) for ATP and k(cat)) of the hydrolysis process also could be obtained, and the order of k(cat)/K(m) was as follows: the dsDNA having a blunt end (1) approximately the dsDNA having a 4-base-mismatched base-pair end (3) > the ssDNA having a free 3' end (4) >> the ssDNA having a free 5' end (5). Thus, DNase could not recognize and not hydrolyze the free 5' end of ssDNA. The DNA hydrolysis reaction could be driven by dATP and GTP (purine base) as well as ATP, whereas the cleavage efficiency was very low driven with UTP, CTP (pyrimidine base), ADP, and AMP.

The bacterium Deinococcus radiodurans is extremely resistant to high levels of DNA-damaging agents, including gamma rays and ultraviolet light that can lead to double-stranded DNA breaks. Surprisingly, the organism does not appear to have a RecBCD enzyme, an enzyme that is critical for double-strand break repair in many other bacteria. The D. radiodurans genome does encode a protein whose closest characterized homologues are RecD subunits of RecBCD enzymes in other bacteria. We have purified this novel D. radiodurans RecD protein and characterized its biochemical activities. The D. radiodurans RecD protein is a DNA helicase that unwinds short (20 base pairs) DNA duplexes with either a 5'-single-stranded tail or a forked end, but not blunt-ended or 3'-tailed duplexes. Duplexes with 10-12 nucleotide (nt) 5'-tails are good unwinding substrates and are bound tightly, while DNA with shorter tails (4-8 nt) are poor unwinding substrates and are bound much less tightly. The RecD protein is much less efficient at unwinding slightly longer substrates (52 or 76 base pairs, with 12 nt 5'-tails). Unwinding of the longer substrates is stimulated somewhat (4-5-fold) by the single-stranded DNA-binding protein from D. radiodurans. These results show that the D. radiodurans RecD protein is a DNA helicase with 5'-3' polarity and low processivity.

RecBCD is a multi-functional enzyme complex that processes DNA ends resulting from a double-strand break. RecBCD is a bipolar helicase that splits the duplex into its component strands and digests them until encountering a recombinational hotspot (Chi site). The nuclease activity is then attenuated and RecBCD loads RecA onto the 3' tail of the DNA. Here we present the crystal structure of RecBCD bound to a DNA substrate. In this initiation complex, the DNA duplex has been split across the RecC subunit to create a fork with the separated strands each heading towards different helicase motor subunits. The strands pass along tunnels within the complex, both emerging adjacent to the nuclease domain of RecB. Passage of the 3' tail through one of these tunnels provides a mechanism for the recognition of a Chi sequence by RecC within the context of double-stranded DNA. Gating of this tunnel suggests how nuclease activity might be regulated.

RecBCD is a processive, DNA-based motor enzyme with both helicase and nuclease activities. We used high-resolution optical trapping to study individual RecBCD molecules moving against applied forces up to 8 pN. Fine-scale motion was smooth down to a detection limit of 2 nm, implying a unitary step size below six basepairs (bp). Episodes of constant-velocity motion over hundreds to thousands of basepairs were punctuated by abrupt switches to a different speed or by spontaneous pauses of mean length 3 s. RecBCD occasionally reversed direction, sliding backward along DNA. Backsliding could be halted by reducing the force, after which forward motion sometimes resumed, often after a delay. Elasticity measurements showed that the DNA substrate was partially denatured during backsliding events, but reannealed concomitant with the resumption of forward movement. Our observations show that RecBCD-DNA complexes can exist in multiple, functionally distinct states that persist for many catalytic turnovers: such states may help tune enzyme activity for various biological functions.

DNA polymerases delta and epsilon (pol delta and epsilon) are the two major replicative polymerases in the budding yeast Saccharomyces cerevisiae. The fidelity of pol delta is influenced by its 3'-5' proofreading exonuclease activity, which corrects misinsertion errors, and by enzyme cofactors. PCNA is a pol delta cofactor, called the sliding clamp, which increases the processivity of pol delta holoenzyme. This study measures the fidelity of 3'-5' exonuclease-proficient and -deficient pol delta holoenzyme using a synthetic 30mer primer/100mer template in the presence and absence of PCNA. Although PCNA increases pol delta processivity, the presence of PCNA decreased pol delta fidelity 2-7-fold. In particular, wild-type pol delta demonstrated the following nucleotide substitution efficiencies for mismatches in the absence of PCNA: G.G, 0.728 x 10(-4); T.G, 1.82 x 10(-4); A.G, <0.01 x 10(-4). In the presence of PCNA these values increased as follows: G.G, 1.30 x 10(-4); T.G, 2.62 x 10(-4); A.G, 0.074 x 10(-4). A similar but smaller effect was observed for exonuclease-deficient pol delta (i.e., 2-4-fold increase in nucleotide substitution efficiencies in the presence of PCNA). Thus, the fidelity of wild-type pol delta in the presence of PCNA is more than 2 orders of magnitude lower than the fidelity of wild-type pol epsilon holoenzyme and is comparable to the fidelity of exonuclease-deficient pol epsilon holoenzyme.