Direct observation of RuvAB-catalyzed branch migration of single Holliday junctions

Holliday junctions form during DNA repair and homologous recombination processes. These processes entail branch migration, whereby the length of two arms of a cruciform increases at the expense of the two others. Branch migration is carried out in prokaryotic cells by the RuvAB motor complex. We study RuvAB-catalyzed branch migration by following the motion of a small paramagnetic bead tethered to a surface by two opposing arms of a single cruciform. The bead, pulled under the action of magnetic tweezers, exerts tension on the cruciform, which in turn transmits the force to a single RuvAB complex bound at the crossover point. This setup provides a unique means of measuring several kinetic parameters of interest such as the translocation rate, the processivity, and the force on the substrate against which the RuvAB complex cannot effect translocation. RuvAB-catalyzed branch migration proceeds with a small, discrete number of rates, supporting the view that the monomers comprising the RuvB hexameric rings are not functionally homogeneous and that dimers or trimers constitute the active subunits. The most frequently encountered rate, 98 +/- 3 bp/sec, is approximately five times faster than previously estimated. The apparent processivity of branch migration between pauses of inactivity is approximately 7,000 bp. Branch migration persists against opposing forces up to 23 pN.     

Structural transitions within human Rad51 nucleoprotein filaments

Rad51 is a core component of the eukaryotic homologous recombination machinery and is responsible for key mechanistic steps during strand invasion. Higher order oligomers of Rad51 display a remarkable degree of structural variation, forming rings, compressed filaments, and elongated filaments. It is unclear whether Rad51 can transition directly between these different oligomeric structures without disassembling first into monomers. We have used single-molecule microscopy to investigate the behavior of human Rad51 assembled on double-stranded DNA. Our results show that human Rad51 can form elongated nucleoprotein filaments on DNA, but ATP hydrolysis causes a decrease in their length without concomitant dissociation of protein. Compressed Rad51 filaments can re-elongate when presented with either ATP or the non-hydrolyzable analog AMP-PNP, and these cycles of elongation and compression are reversible. A Rad51 mutant deficient in ATP hydrolysis is locked into an extended conformation that is incapable of transitioning to a compressed filament. Similarly, wild-type Rad51 bound to DNA in the presence of AMP-PNP was trapped in the elongated state. Proteins incapable of transitioning to the compressed state were also highly resistant to dissociation from the DNA. Taken together, our results indicate that nucleotide hydrolysis by human Rad51 triggers a reversible structural transition leading to filaments with reduced helical pitch.     

Tension induces a base-paired overstretched DNA conformation

Mixed-sequence DNA molecules undergo mechanical overstretching by approximately 70% at 60–70 pN. Since its initial discovery 15 y ago, a debate has arisen as to whether the molecule adopts a new form [Cluzel P, et al. (1996) Science 271:792–794; Smith SB, Cui Y, Bustamante C (1996) Science 271:795–799], or simply denatures under tension [van Mameren J, et al. (2009) Proc Natl Acad Sci USA 106:18231–18236]. Here, we resolve this controversy by using optical tweezers to extend small 60–64 bp single DNA duplex molecules whose base content can be designed at will. We show that when AT content is high (70%), a force-induced denaturation of the DNA helix ensues at 62 pN that is accompanied by an extension of the molecule of approximately 70%. By contrast, GC-rich sequences (60% GC) are found to undergo a reversible overstretching transition into a distinct form that is characterized by a 51% extension and that remains base-paired. For the first time, results proving the existence of a stretched basepaired form of DNA can be presented. The extension observed in the reversible transition coincides with that produced on DNA by binding of bacterial RecA and human Rad51, pointing to its possible relevance in homologous recombination.     

Nutrition education: taking it one step at a time

Although it is important for most patients with diabetes to gain a broad background in nutrition information and exchange principles, for many these will be long-range goals. Don't be in such a hurry to teach so much that you lose the patient's interest and end up confusing the patient with too much knowledge and information. Keep the principles of adult learning in mind as you set your goals for initial, intermediate, and advanced learning objectives for each patient. And, finally, consider evaluating the DNIS as a useful addition to your patient education resources for initial level teaching tools.     

Treatment of chronic hepatitis C infection: one step at a time

Non-A, non-B hepatitis was recognised as an important cause of chronic liver disease long before the aetiological agent-hepatitis C virus-was identified in 1989. Recombinant interferons, initially developed as a treatment for malignancies, proved to be an effective treatment for this disease before the identification of the viral agent. Subsequent testing for hepatitis C virus RNA demonstrated that the virus appeared to be eradicated in a small proportion of treated patients. Treatment regimens have improved dramatically since 1989 with the addition of the oral nucleoside ribavirin and long-acting pegylated interferons to treatment regimens. Currently, more than half of treated patients can achieve durable viral clearance. This clearance is quite a remarkable feat; indeed, eradication is not possible in any other chronic viral infection. Considerable effort continues to be devoted to improving therapeutic regimens to make them more effective and tolerable. Drugs that directly act on the replicative machinery of the virus-protease and polymerase inhibitors-are under development and entering clinical trials in human beings.     

Direct single-molecule observation of a protein living in two opposed native structures

Biological activity in proteins requires them to share the energy landscape for folding and global conformational motions, 2 key determinants of function. Although most structural studies to date have focused on fluctuations around a single structural basin, we directly observe the coexistence of 2 symmetrically opposed conformations for a mutant of the Rop-homodimer (Repressor of Primer) in single-molecule fluorescence resonance energy transfer (smFRET) measurements. We find that mild denaturing conditions can affect the sensitive balance between the conformations, generating an equilibrium ensemble consisting of 2 equally occupied structural basins. Despite the need for large-scale conformational rearrangement, both native structures are dynamically and reversibly adopted for the same paired molecules without separation of the constituent monomers. Such an ability of some proteins or protein complexes to switch between conformations by thermal fluctuations and/or minor environmental changes could be central to their ability to control biological function.     

The DNA-gate of Bacillus subtilis gyrase is predominantly in the closed conformation during the DNA supercoiling reaction

Gyrase is the only type II topoisomerase that introduces negative supercoils into DNA. Supercoiling is catalyzed via a strand-passage mechanism, in which the gate DNA (gDNA) is transiently cleaved, and a second DNA segment, the transfer DNA (tDNA), is passed through the gap before the gDNA is religated. Strand passage requires an opening of the so-called DNA-gate by ≈2 nm. A single-molecule FRET study reported equal populations of open and closed DNA-gate in topoisomerase II. We present here single-molecule FRET experiments that monitor the conformation of DNA bound to the DNA-gate of Bacillus subtilis gyrase and the conformation of the DNA-gate itself. DNA bound to gyrase adopts two different conformations, one slightly, one severely distorted. DNA distortion requires cleavage, but neither ATP nor the presence of a tDNA. At the same time, the DNA-gate of gyrase is predominantly in the closed conformation. In agreement with the single molecule data and with the danger of dsDNA breaks for genome integrity, <5% of cleavage complexes are detected in equilibrium. Quinolone inhibitors favor DNA cleavage by B. subtilis gyrase, but disfavor DNA distortion, and the DNA-gate remains in the closed conformation. Our results demonstrate that DNA binding, distortion and cleavage, and gate-opening are mechanistically distinct events. During the relaxation and supercoiling reactions, gyrase with an open DNA-gate is not significantly populated, consistent with gate-opening as a very rare event that only occurs briefly to allow for strand passage.     

MutL traps MutS at a DNA mismatch

DNA mismatch repair (MMR) identifies and corrects errors made during replication. In all organisms except those expressing MutH, interactions between a DNA mismatch, MutS, MutL, and the replication processivity factor (β-clamp or PCNA) activate the latent MutL endonuclease to nick the error-containing daughter strand. This nick provides an entry point for downstream repair proteins. Despite the well-established significance of strand-specific nicking in MMR, the mechanism(s) by which MutS and MutL assemble on mismatch DNA to allow the subsequent activation of MutL’s endonuclease activity by β-clamp/PCNA remains elusive. In both prokaryotes and eukaryotes, MutS homologs undergo conformational changes to a mobile clamp state that can move away from the mismatch. However, the function of this MutS mobile clamp is unknown. Furthermore, whether the interaction with MutL leads to a mobile MutS–MutL complex or a mismatch-localized complex is hotly debated. We used single molecule FRET to determine that Thermus aquaticus MutL traps MutS at a DNA mismatch after recognition but before its conversion to a sliding clamp. Rather than a clamp, a conformationally dynamic protein assembly typically containing more MutL than MutS is formed at the mismatch. This complex provides a local marker where interaction with β-clamp/PCNA could distinguish parent/daughter strand identity. Our finding that MutL fundamentally changes MutS actions following mismatch detection reframes current thinking on MMR signaling processes critical for genomic stability.The DNA mismatch repair (MMR) system employs several proteins to locate and correct DNA replication errors that escape polymerase proofreading. Mutations in these proteins contribute to MMR dysfunction that is associated with carcinogenesis, such as Lynch syndrome and other diseases associated with high mutator phenotypes (1, 2). In all organisms, MMR is initiated by binding of MutS homologs to a base–base mismatch or an insertion/deletion loop (IDL), followed by ATP-dependent recruitment of MutL homologs to begin the process of repair (3, 4). Following MutL recruitment, a key event is the introduction of a nick that directs excision and resynthesis of the nascent DNA strand containing the error (5–7).In methyl-directed MMR, which occurs in Escherichia coli, the mismatch- and ATP-dependent MutS–MutL–DNA complex activates the protein MutH to nick transiently unmethylated d(GATC) sequences in the daughter strand. Notably, however, MutH is not widely conserved in prokaryotes and does not exist in eukaryotes. Recent in vitro studies of eukaryotic MMR indicate that in these MutH-free organisms, detection of a mismatch by MutS or MutSα [MutS(α)] licenses MutL(α) to interact with the processivity factor (β-clamp/PCNA), which in turn activates the latent endonuclease activity of MutL(α) to incise the daughter DNA strand on both the 3′ and 5′ sides of the error (8–11). The interaction between MutL and the β-clamp (or between MutLα and PCNA) provides the strand discrimination signal because the β-clamp (or PCNA) is loaded asymmetrically at the replication fork or at a nick in DNA (10, 12).The importance of the nicking activity of MutL homologs is highlighted by the observation that mutations that impair yeast MutLα endonuclease activity cause a significant mutator phenotype and genomic instability (11, 13, 14). Despite the well-established significance of strand-specific nicking in MMR, the mechanism(s) by which MutS and MutL assemble on mismatched DNA to allow subsequent activation of MutL endonuclease activity by β-clamp/PCNA remains elusive. There is general agreement that in both prokaryotes and eukaryotes, after binding a mismatch MutS or MutSα can undergo conformational changes to a mobile clamp state that can move away from the mismatch (6, 15). What happens after this step is mired in controversy. Several disparate models for MutS(α)–MutL(α) mismatch complex formation and the subsequent signaling of repair have been proposed (e.g., see refs. 6, 7, 15–21). One prominent model in the field has MutL(α) joining MutS(α) to form MutS(α)–MutL(α) sliding clamps that diffuse along the DNA to interact with the strand-discrimination signal (β-clamp/PCNA or MutH) (16). Other models include trapping of MutS(α) clamps near the mismatch by MutL(α) followed by DNA looping or, alternately, MutS(α)-induced polymerization of MutL(α) along the DNA to reach the strand-discrimination signal (6, 7, 15, 18, 22). Some degree of localization to the mismatch is suggested by in vitro studies of eukaryotic MMR proteins, indicating that although MutLα can introduce nicks across long stretches of DNA, they occur preferentially in the vicinity of the mismatch (9, 11, 12).In this study, we have used single molecule fluorescence to demonstrate that in the case of Thermus aquaticus (a MutH-free organism), MutL traps MutS at the mismatch after its ATP-induced activation but before its conversion into a sliding clamp. The resulting MutS–MutL mismatch complex typically contains more MutL than MutS, with one or two MutS dimers and up to four MutL dimers. MutS exists in a conformationally dynamic state within these complexes, which may be relevant for subsequent steps in MMR. In contrast to a mobile MutS–MutL complex, localization of MutS–MutL at the mismatch can restrict β-clamp/PCNA-activated MutL nicking to the vicinity of the mismatch, thereby enhancing MMR efficiency and limiting excessive excision and resynthesis that can destabilize the genome.     

Assembly,translocation, and activation of XerCD-dif recombination by FtsK translocase analyzed in real-time by FRET and two-color tethered fluorophore motion

The FtsK dsDNA translocase functions in bacterial chromosome unlinking by activating XerCD-dif recombination in the replication terminus region. To analyze FtsK assembly and translocation, and the subsequent activation of XerCD-dif recombination, we extended the tethered fluorophore motion technique, using two spectrally distinct fluorophores to monitor two effective lengths along the same tethered DNA molecule. We observed that FtsK assembled stepwise on DNA into a single hexamer, and began translocation rapidly (∼0.25 s). Without extruding DNA loops, single FtsK hexamers approached XerCD-dif and resided there for ∼0.5 s irrespective of whether XerCD-dif was synapsed or unsynapsed. FtsK then dissociated, rather than reversing. Infrequently, FtsK activated XerCD-dif recombination when it encountered a preformed synaptic complex, and dissociated before the completion of recombination, consistent with each FtsK–XerCD-dif encounter activating only one round of recombination.Understanding how molecular machines assemble and act requires a combination of biochemical, structural, and biophysical approaches. In recent years, single-molecule techniques have allowed the observation of biological reactions in real time, thereby avoiding the averaging of ensemble experiments. These single-molecule studies rely on information gained from existing biochemical characterization to set experimental parameters and to place their results in context. To follow complex multistep reactions involving multiple components, methods capable of tracking several observables simultaneously have been established (1–4). In this work, we push the boundaries of a recently developed technique, and apply it to further our understanding of the Escherichia coli XerCD-dif–FtsK molecular machine, which functions in chromosome segregation and coordinates it with cell division.FtsK is a 1,329-aa DNA translocase, which assembles at the division septum and functions in segregating sister chromosomes during the late stages of the cell cycle in a wide range of bacteria (5–8) by activating site-specific recombination by XerCD at dif (9). The two related Tyr recombinases, XerC and XerD, bind the 28-bp dif site located within the terminus region, ter, on the E. coli chromosome. They unlink catenated chromosomes and resolve chromosome dimers formed by homologous recombination (10–14). Independent of its role in activating XerCD-dif recombination, FtsK appears to play a direct role in the segregation of ter (8). FtsK consists of three domains: an essential 179-aa N-terminal domain that anchors it to the division septum, an ∼500-aa C-terminal motor domain, and an ∼650-aa linker domain (6, 7, 15, 16). The motor domain, FtsK_C, is composed of α-, β-, and γ-subdomains (17). The α- and β-subdomains form a dsDNA translocase, belonging to the RecA family of ATPases (17). The γ-subdomain plays a regulatory role in the recognition of the FtsK orientating polar sequence (KOPS) that guides FtsK translocation toward the dif site at ter (18–20), and in the activation of XerCD-dif recombination (9, 14, 21). Activation of recombination requires direct interaction between the γ-subdomain and XerD (22, 23).Our previous work, using a single tethered fluorophore motion (TFM) reporter, in combination with Förster resonance energy transfer (FRET), determined the conformational transitions in the XerCD-dif complex that occurred as XerD mediated an initial strand exchange to form a Holliday junction (HJ), which was resolved by a subsequent XerC-mediated strand exchange (24). In that work, we indirectly inferred the presence of FtsK_C, taking advantage of protein-induced fluorescence enhancement (PIFE) (25). Here, we have expanded TFM-FRET, using two spectrally distinct TFM reporters (one on FtsK_C and one on DNA), to directly observe FtsK_C as it assembles and translocates, and to correlate its behavior, upon arrival at XerCD, with the progress of the recombination reaction.Previous studies of FtsK_C assembly and translocation have used biochemical methods (26, 27) and single-molecule techniques, including magnetic and optical tweezers (18, 28–30); tethered particle motion (TPM) (18); and, more recently, DNA curtains (31, 32). Optical/magnetic tweezers and TPM experiments have relied on loop extrusion by FtsK_C to observe its action (looping by FtsK shortens the length between two DNA ends, hence displacing the bead used in TPM or optical/magnetic tweezers). Many of the single-molecule assays involved the attachment of FtsK_C to quantum dot (QD) labels or used derivatives that were known to aggregate, thereby potentially confounding the interpretation of data, because multiple motors could be present in the region of analysis. Experiments utilizing DNA curtains have revealed that FtsK_C can push, evict, and bypass proteins bound to DNA as it translocates (32). However, FtsK_C stops at least transiently and/or dissociates at XerCD bound to dif (27, 32). Reversals in translocation direction have been observed to occur spontaneously (18, 28, 30, 31) and in response to XerCD bound to dif (32). The use of a fluorophore label, along with singly tethered DNA, has allowed us to observe FtsK_C without requiring any loop extrusion, and to observe its interaction with synaptic complexes of XerCD, where previous single-molecule work has only dealt with unsynapsed XerCD-dif (32). Using this approach, we have determined that FtsK_C assembles on DNA as a single hexamer, and begins translocating rapidly (∼0.25 s), without extruding a loop of DNA. When it reached XerCD bound to dif, either in a synapsed or unsynapsed conformation, it resided briefly for ∼0.5 s and then dissociated without any evidence of reversal. FtsK_C activated recombination when it met synapsed XerCD-dif complexes, and then dissociated faster than the completion of recombination by XerCD.     

Single-molecule analysis of RAG-mediated V(D)J DNA cleavage

The recombination-activating gene products, RAG1 and RAG2, initiate V(D)J recombination during lymphocyte development by cleaving DNA adjacent to conserved recombination signal sequences (RSSs). The reaction involves DNA binding, synapsis, and cleavage at two RSSs located on the same DNA molecule and results in the assembly of antigen receptor genes. We have developed single-molecule assays to examine RSS binding by RAG1/2 and their cofactor high-mobility group-box protein 1 (HMGB1) as they proceed through the steps of this reaction. These assays allowed us to observe in real time the individual molecular events of RAG-mediated cleavage. As a result, we are able to measure the binding statistics (dwell times) and binding energies of the initial RAG binding events and characterize synapse formation at the single-molecule level, yielding insights into the distribution of dwell times in the paired complex and the propensity for cleavage on forming the synapse. Interestingly, we find that the synaptic complex has a mean lifetime of roughly 400 s and that its formation is readily reversible, with only ∼40% of observed synapses resulting in cleavage at consensus RSS binding sites.V(D)J recombination is responsible for assembling the variable regions of antigen receptor genes during B- and T-lymphocyte development. During V(D)J recombination, fragments of V, D, and J segments located together on particular chromosomes are rearranged into functional V(D)J or VJ alleles that are the specificity determinants for B-cell receptors or immunoglobulins (Igs) and T-cell receptors (TCR) (1). Adjacent to the V, D, and J gene segments are recombination signal sequences (RSSs) consisting of a conserved heptamer (consensus 5′-CACAGTG-3′) and nonamer (consensus 5′-ACAAAAACC-3′) separated by a spacer of 12 or 23 bp (referred to as 12RSS and 23RSS, respectively). Efficient recombination requires one 12RSS and one 23RSS, a constraint known as the 12/23 rule (1).The recombination-activating genes, RAG1 and RAG2, encode proteins that carry out V(D)J recombination by bringing a 12RSS and a 23RSS together into a paired (or synaptic) complex, nicking the RSS sites adjacent to the heptamer, and converting each nick into a double-strand break, leaving a hairpin at the coding end (Fig. 1). The hairpin is created by nucleophilic attack on the opposing strand by the 3′ hydroxyl group at the nick in a transesterification reaction. After RAG1/2 forms hairpins, it recruits the nonhomologous end joining machinery to repair the ends (1, 2). Since their discovery, the full-length RAG1 and RAG2 proteins have proven difficult to isolate and study in vitro (1). However, core domains (referred to as RAG1c and RAG2c) have been identified by removing a large region from the N terminus of RAG1 (which includes an E3 ubiquitin ligase domain) and a large region from the C terminus of RAG2 (which includes a plant homeodomain) (3, 4). These core proteins have been shown to tetramerize to form RAG1/2c, which retains RSS binding, nicking, and hairpin formation activities (5). High-mobility group-box protein 1 (HMGB1) acts as a cofactor and increases RAG1/2c affinity for the 23RSS (6). HMGB1 is required for paired complex formation and efficient conversion of RAG-mediated nicks into hairpins (7–10).Open in a separate windowFig. 1.Orchestration of V(D)J cleavage. (A) Schematic of key molecules in the V(D)J recombination process and nomenclature used throughout the paper. (B) During the cleavage phase of V(D)J recombination, RAG1/2c and HMGB1 bind and nick a 12RSS (magenta) and a 23RSS (yellow) spaced by a distance known as the intersignal distance. Then, the two RSSs are brought together, forming a looped paired complex. While in the paired complex state, hairpins are produced only in the presence of a 12RSS and a 23RSS, which is known as the 12/23 rule. *The stoichiometry of RAG1/2c and HMGB1 involved in forming the paired complex is unknown.Current in vitro assays that capture the paired complex with RAG1/2c generally place a 12RSS and a 23RSS on two different DNA molecules, typically short oligonucleotides. However, in vivo, antigen receptor loci are assembled using RSSs on the same DNA molecule (1, 11). One prior study captured RAG1/2c and HMGB1 bound to DNA with a 12RSS and a 23RSS on the same substrate using standard bulk assays (12). However, many key mechanistic questions remain unresolved concerning how a diverse immune repertoire is generated by the RAG recombinase. How long does an RAG complex spend bound to a 12RSS or a 23RSS? What is the lifetime of an RAG-mediated paired complex formed between a 12RSS and a 23RSS before DNA double-strand break (hairpin) formation? Is formation of this complex reversible, or does it inevitably go on to cleavage? These issues have implications for the mechanisms that determine Ig and TCR repertoires.To address these questions, we have explored the dynamics of various stages of the V(D)J recombination cleavage reaction on single DNA molecules in real time, allowing visualization and characterization of individual RAG–HMGB1–RSS complexes and paired complex formation using intersignal distances comparable with some of the shorter distances found in the assembly of V, D, and J gene segments in vivo (13). Using a tethered particle motion (TPM) assay (14), we observed RSS-dependent apparent shortening of DNA in the presence of RAG1/2c, probably a reflection of bending of the DNA, and were able to exploit this shortening as a signature of the RAG1/2c–RSS-bound state (Fig. 2A). This technique in conjunction with a statistical mechanical model of RAG binding have allowed us to determine single-molecule binding constants for RAG1/2c with or without HMGB1 (Fig. 2 A and B). These measurements are also used to determine the dwell time distributions for the 12RSS and 23RSS protein bound states. Furthermore, this same substrate length readout is used for direct detection of the paired complex before DNA cleavage (Fig. 2C), because bringing the two RSSs into close proximity effectively shortens the tether. We find that formation of the paired complex results in one of two outcomes: either the DNA is cleaved, causing bead release from its tether, or the complex dissociates, leaving the DNA template intact. Finally, we use bead loss on cleavage to quantify the time dependence of cleavage on the concentrations of RAG1/2c and HMGB1 (Fig. 2D). These experiments provide a quantitative picture of the stages leading up to DNA cleavage during V(D)J recombination and reveal the dynamic nature of the paired complex.Open in a separate windowFig. 2.Steps of V(D)J cleavage investigated by TPM in this study: RAG binding, RAG–HMGB1 binding, paired complex formation, and hairpin production. Schematic of constructs and experimental measurements to examine the V(D)J recombination cleavage reaction at various stages. (A) RAG1/2c binding to the 12RSS or the 23RSS site alone. (B) RAG1/2c binding to the 12RSS or the 23RSS site in the presence of HMGB1. (C) RAG1/2c binding to DNA substrates containing 12RSS and 23RSS for the purposes of observing formation of a looped paired complex. (D) Bead loss caused by DNA cleavage as a result of 12/23 rule-regulated hairpin production.     

Generation of double Holliday junction DNAs and their dissolution/resolution within a chromatin context

Four-way DNA intermediates, also known as Holliday junctions (HJs), are formed during homologous recombination and DNA repair, and their resolution is necessary for proper chromosome segregation. To facilitate the biochemical analysis of HJ processing, we developed a method involving DNAzyme self-cleavage to generate 1.8-kb DNA molecules containing either single (sHJ) or double Holliday junctions (dHJs). We show that dHJ DNAs (referred to as HoJo DNAs) are dissolved by the human BLM–TopIIIα–RMI1–RMI2 complex to form two noncrossover products. However, structure-selective endonucleases (human GEN1 and SMX complex) resolve DNA containing single or double HJs to yield a mixture of crossover and noncrossover products. Finally, we demonstrate that chromatin inhibits the resolution of the double HJ by GEN or SMX while allowing BTRR-mediated dissolution.

Homologous recombination (HR) provides an important mechanism for the repair of DNA double-stranded breaks and the restoration of broken replication forks (1). Recombination in mitotic cells generally occurs between sister chromatids and can lead to the formation of DNA intermediates in which the sisters are covalently linked by four-way DNA junctions, known as Holliday junctions (HJs) (2). Failure to process these DNA intermediates leads to improper chromosome segregation and cell death (3, 4). Recombination also plays an important role in meiotic cells, when interactions occur between homologous chromosomes, and is responsible for the generation of genetic diversity.In mitotic cells, HJs are primarily processed by “dissolution,” in which two adjacent HJs (double Holliday junctions [dHJs]) converge in an adenosine triphosphate (ATP)-hydrolysis–dependent reaction to form a hemicatenane that is subsequently decatenated by topoisomerase action. In human cells, this two-step process involves the BLM–topoisomerase IIIα–RMI1–RMI2 (BTRR) complex (5–7). In yeast, similar reactions are driven by the Sgs1–Top3–Rmi1 (STR) complex (8, 9). Dissolution yields exclusively noncrossover products, which help to maintain the heterozygous state, as a loss of heterozygosity can cause cancer development (10). Mutations in the BLM gene are linked to a human inherited disorder known as Bloom syndrome, which is characterized by short stature, sensitivity to sunlight, and a greatly increased risk of a broad range of cancers (11, 12). In the clinic, patients with Bloom syndrome are diagnosed by a cytogenetic test that detects elevated levels of sister chromatid exchanges. Pathogenic mutations in the TOP3A and RMI1 genes also cause a Bloom syndrome–like disorder, consistent with the fact that these genes participate in the same molecular pathway (12).Persistent HJs that escape processing by BTRR, and single Holliday junctions (sHJs), are resolved by structure-selective endonucleases (SSEs), which specifically recognize and cleave HJs by mediating a nucleolytic attack on two opposing strands at the junction point (2). In humans, these endonucleases include GEN1 and the SMX trinuclease, comprising SLX1–SLX4–MUS81–EME1–XPF–ERCC1 (3, 13–15). Unlike dissolution, resolution gives rise to both crossover and noncrossover products, thereby elevating the frequency of sister chromatid exchanges and increasing the potential for loss of heterozygosity.In contrast to BTRR-mediated dHJ dissolution, which is active throughout the cell cycle, the actions of SMX and GEN1 are tightly regulated. Firstly, SMX complex formation is restricted to prometaphase, as it is dependent upon the phosphorylation of EME1 by CDK1 and PLK1, which stimulates its association with the SLX4 scaffold (15, 16). Secondly, GEN1 is mainly sequestered from the cell nucleus and gains access to DNA after the breakdown of the nuclear envelope during cell division (17).Despite the importance of the BTRR complex in maintaining genetic stability, a detailed picture of dissolution is lacking. Mechanistic studies, using protein complexes from various organisms, led to a model in which two HJs are converged by the branch migration activity of the BLM helicase (18–20). Convergent migration generates positive supercoiling that is relaxed by topoisomerase IIIα and generates a hemicatenane that is processed by topoisomerase IIIα with the aid of RMI1–RMI2 (6–9, 21).Studies of dHJ dissolution have utilized two model systems: 1) a small dHJ prepared by annealing two synthetic oligos (5–7), and 2) a larger plasmid-sized molecule in which two HJs are separated by 165 bp (20, 22). However, the small size of the synthetic DNA substrate eliminates any possibility for branch migration as the two HJs are separated by only 14 bp, raising concerns as to whether these substrates recapitulate the physiological aspects of dissolution (19). The plasmid-sized substrate has been utilized for the dissolution of dHJs by Saccharomyces cerevisiae STR, Drosophila melanogaster BTR, and more recently human BTRR complex (8, 20, 23). However, there is the significant drawback that generation of this substrate is laborious (taking several weeks), requires purified Cre recombinase and reverse gyrase, and leads to low yields of product.To facilitate mechanistic analysis of dissolution and resolution, we developed a rapid and scalable methodology to prepare a 1.8-kb DNA containing single or double HJs. In the dHJ molecules, the two HJs are separated by a maximum of 746 bp of homologous sequence, allowing the two HJs to migrate within the region of homology. We demonstrate that these dHJ molecules are efficiently dissolved by the human BTRR complex to generate noncrossover products. We also show that GEN1 or SMX resolves the single or double HJs to yield the expected mixture of crossover and noncrossover products. Finally, we find that GEN1/SMX are unable to resolve HJs on chromatinized templates, whereas BTRR-mediated dissolution events are unaffected by nucleosome assembly, potentially indicative of an additional level of regulatory control that favors dissolution over resolution.     

Improving health care efficiency one click at a time

Computers are an integral component of modern hospitals. Mouse clicks are currently inherent to this use of computers. However, mouse clicks are not instantaneous. These clicks may be associated with significant costs. Estimated costs associated with 10 additional clicks per day for 20 000 staff exceed AU$500 000 annually. Workflow modifications that increase clicks should weigh the potential benefits of such changes against these costs. Future investigation of strategies to reduce low-value clicks may provide an avenue for health care savings.