Branch Migration Of Holliday Junctions Research Articles

Molecular mechanism of Holliday junction branch migration

Molecular mechanism of Holliday junction branch migration

Molecular mechanisms of Holliday junction branch migration catalyzed by an asymmetric RuvB hexamer

The Holliday junction (HJ) is a DNA intermediate of homologous recombination, involved in many fundamental physiological processes. RuvB, an ATPase motor protein, drives branch migration of the Holliday junction with a mechanism that had yet to be elucidated. Here we report two cryo-EM structures of RuvB, providing a comprehensive understanding of HJ branch migration. RuvB assembles into a spiral staircase, ring-like hexamer, encircling dsDNA. Four protomers of RuvB contact the DNA backbone with a translocation step size of 2 nucleotides. The variation of nucleotide-binding states in RuvB supports a sequential model for ATP hydrolysis and nucleotide recycling, which occur at separate, singular positions. RuvB’s asymmetric assembly also explains the 6:4 stoichiometry between the RuvB/RuvA complex, which coordinates HJ migration in bacteria. Taken together, we provide a mechanistic understanding of HJ branch migration facilitated by RuvB, which may be universally shared by prokaryotic and eukaryotic organisms.

Cryo-EM structure of the RuvAB-Holliday junction intermediate complex from Pseudomonas aeruginosa.

Holliday junction (HJ) is a four-way structured DNA intermediate in homologous recombination. In bacteria, the HJ-specific binding protein RuvA and the motor protein RuvB together form the RuvAB complex to catalyze HJ branch migration. Pseudomonas aeruginosa (P. aeruginosa, Pa) is a ubiquitous opportunistic bacterial pathogen that can cause serious infection in a variety of host species, including vertebrate animals, insects and plants. Here, we describe the cryo-Electron Microscopy (cryo-EM) structure of the RuvAB-HJ intermediate complex from P. aeruginosa. The structure shows that two RuvA tetramers sandwich HJ at the junction center and disrupt base pairs at the branch points of RuvB-free HJ arms. Eight RuvB subunits are recruited by the RuvA octameric core and form two open-rings to encircle two opposite HJ arms. Each RuvB subunit individually binds a RuvA domain III. The four RuvB subunits within the ring display distinct subdomain conformations, and two of them engage the central DNA duplex at both strands with their C-terminal β-hairpins. Together with the biochemical analyses, our structure implicates a potential mechanism of RuvB motor assembly onto HJ DNA.

Abstract 1230: Molecular Mechanisms of Holliday Junction Branch Migration Catalyzed by an Asymmetric RuvB Hexamer

Abstract 1230: Molecular Mechanisms of Holliday Junction Branch Migration Catalyzed by an Asymmetric RuvB Hexamer

Identification of small-molecule inhibitors of the DNA repair proteins RuvAB from Pseudomonas aeruginosa

The Holliday junction (HJ) branch migrator RuvAB complex plays a fundamental role during homologous recombination and DNA damage repair, and therefore, is an attractive target for the treatment of bacterial pathogens. Pseudomonas aeruginosa (P. aeruginosa, Pa) is one of the most common clinical opportunistic bacterial pathogens, which can cause a series of life-threatening acute or chronic infections. Here, we performed a high throughput small-molecule screening targeting PaRuvAB using the FRET-based HJ branch migration assay. We identified that corilagin, bardoxolone methyl (BM) and 10-(6ˊ-plastoquinonyl) decyltriphenylphosphonium (SKQ1) could efficiently inhibit the branch migration activity of PaRuvAB, with IC50 values of 0.40 ± 0.04 μM, 0.38 ± 0.05 μM and 4.64 ± 0.27 μM, respectively. Further biochemical and molecular docking analyses demonstrated that corilagin directly bound to PaRuvB at the ATPase domain, and thus prevented ATP hydrolysis. In contrast, BM and SKQ1 acted through blocking the interactions between PaRuvA and HJ DNA. Finally, these compounds were shown to increase the susceptibility of P. aeruginosa to UV-C irradiation. Our work, for the first time, reports the small-molecule inhibitors of RuvA and RuvB from any species, providing valuable chemical tools to dissect the functional role of each individual protein in vivo.

Single-Molecule Analysis of DNA Branch Migration under Biomimetic Environments.

Branch migration (BM) of DNA Holliday junctions (HJs) occurs through base-pair rearrangements between homologous DNA molecules during the repair of double-strand breaks (DSBs) by homologous recombination. Despite the fact that BM is conserved among organisms and is essential to stabilize recombination intermediates, which occurs by avoiding the reversal of strand exchange leading to the faithful repair of DSBs, molecular insights into the BM process including kinetics, the effects of microenvironments, and the role of HJ-binding proteins are poorly understood. In this article, using single-molecule fluorescence analysis of a synthetic, mobile HJ as a model system, we systematically investigated the effects of cell-mimic solvent composition and crowding on BM by varying the concentration of cosolutes, namely dimethyl sulfoxide (DMSO) and poly(ethylene glycol) (PEG). The single-molecule analyses revealed that BM is affected by cosolutes in a concentration-dependent manner. In addition, the kinetic analysis of the fluorescence resonance energy transfer (FRET) traces showed that the BM is significantly accelerated under the crowding environments. The finding that the mobility of the HJ is enhanced by cell-like environments not only provides a better insight into BM but also may open up new avenues for targeting HJs and the associated BM process for possible therapeutic interventions.

Mechanism of AAA+ ATPase-mediated RuvAB–Holliday junction branch migration

The Holliday junction is a key intermediate formed during DNA recombination across all kingdoms of life1. In bacteria, the Holliday junction is processed by two homo-hexameric AAA+ ATPase RuvB motors, which assemble together with the RuvA–Holliday junction complex to energize the strand-exchange reaction2. Despite its importance for chromosome maintenance, the structure and mechanism by which this complex facilitates branch migration are unknown. Here, using time-resolved cryo-electron microscopy, we obtained structures of the ATP-hydrolysing RuvAB complex in seven distinct conformational states, captured during assembly and processing of a Holliday junction. Five structures together resolve the complete nucleotide cycle and reveal the spatiotemporal relationship between ATP hydrolysis, nucleotide exchange and context-specific conformational changes in RuvB. Coordinated motions in a converter formed by DNA-disengaged RuvB subunits stimulate hydrolysis and nucleotide exchange. Immobilization of the converter enables RuvB to convert the ATP-contained energy into a lever motion, which generates the pulling force driving the branch migration. We show that RuvB motors rotate together with the DNA substrate, which, together with a progressing nucleotide cycle, forms the mechanistic basis for DNA recombination by continuous branch migration. Together, our data decipher the molecular principles of homologous recombination by the RuvAB complex, elucidate discrete and sequential transition-state intermediates for chemo-mechanical coupling of hexameric AAA+ motors and provide a blueprint for the design of state-specific compounds targeting AAA+ motors.

Uncovering an allosteric mode of action for a selective inhibitor of human Bloom syndrome protein.

BLM (Bloom syndrome protein) is a RECQ-family helicase involved in the dissolution of complex DNA structures and repair intermediates. Synthetic lethality analysis implicates BLM as a promising target in a range of cancers with defects in the DNA damage response; however, selective small molecule inhibitors of defined mechanism are currently lacking. Here, we identify and characterise a specific inhibitor of BLM's ATPase-coupled DNA helicase activity, by allosteric trapping of a DNA-bound translocation intermediate. Crystallographic structures of BLM-DNA-ADP-inhibitor complexes identify a hitherto unknown interdomain interface, whose opening and closing are integral to translocation of ssDNA, and which provides a highly selective pocket for drug discovery. Comparison with structures of other RECQ helicases provides a model for branch migration of Holliday junctions by BLM.

The ZGRF1 Helicase Promotes Recombinational Repair of Replication-Blocking DNA Damage in Human Cells

SUMMARYReplication-blocking DNA lesions are particularly toxic to proliferating cells because they can lead to chromosome mis-segregation if not repaired prior to mitosis. In this study, we report that ZGRF1 null cells accumulate chromosome aberrations following replication perturbation and show sensitivity to two potent replication-blocking anticancer drugs: mitomycin C and camptothecin. Moreover, ZGRF1 null cells are defective in catalyzing DNA damage-induced sister chromatid exchange despite accumulating excessive FANCD2, RAD51, and γ-H2AX foci upon induction of interstrand DNA crosslinks. Consistent with a direct role in promoting recombinational DNA repair, we show that ZGRF1 is a 5′-to-3′ helicase that catalyzes D-loop dissociation and Holliday junction branch migration. Moreover, ZGRF1 physically interacts with RAD51 and stimulates strand exchange catalyzed by RAD51-RAD54. On the basis of these data, we propose that ZGRF1 promotes repair of replication-blocking DNA lesions through stimulation of homologous recombination.

Freedom of movement.

Holliday junction resolvases lock dynamic DNA four-way junctions into specific structural conformations for symmetric DNA cleavage. Single-molecule studies now reveal that resolvases can relax their grip, enabling Holliday junction conformer transitions and branch migration in the enzyme-bound form.

Mechanistic View and Genetic Control of DNA Recombination during Meiosis

Meiotic recombination is essential for fertility and allelic shuffling. Canonical recombination models fail to capture the observed complexity of meiotic recombinants. Here, by combining genome-wide meiotic heteroduplex DNA patterns with meiotic DNA double-strand break (DSB) sites, we show that part of this complexity results from frequent template switching during synthesis-dependent strand annealing that yields noncrossovers and from branch migration of double Holliday junction (dHJ)-containing intermediates that mainly yield crossovers. This complexity also results from asymmetric positioning of crossover intermediates relative to the initiating DSB and Msh2-independent conversions promoted by the suspected dHJ resolvase Mlh1-3 as well as Exo1 and Sgs1. Finally, we show that dHJ resolution is biased toward cleavage of the pair of strands containing newly synthesized DNA near the junctions and that this bias can be decoupled from the crossover-biased dHJ resolution. These properties are likely conserved in eukaryotes containing ZMM proteins, which includes mammals.

RAD54 N-terminal domain is a DNA sensor that couples ATP hydrolysis with branch migration of Holliday junctions

In eukaryotes, RAD54 catalyzes branch migration (BM) of Holliday junctions, a basic process during DNA repair, replication, and recombination. RAD54 also stimulates RAD51 recombinase and has other activities. Here, we investigate the structural determinants for different RAD54 activities. We find that the RAD54 N-terminal domain (NTD) is responsible for initiation of BM through two coupled, but distinct steps; specific binding to Holliday junctions and RAD54 oligomerization. Furthermore, we find that the RAD54 oligomeric state can be controlled by NTD phosphorylation at S49, a CDK2 consensus site, which inhibits RAD54 oligomerization and, consequently, BM. Importantly, the effect of phosphorylation on RAD54 oligomerization is specific for BM, as it does not affect stimulation of RAD51 recombinase by RAD54. Thus, the transition of the oligomeric states provides an important control of the biological functions of RAD54 and, likely, other multifunctional proteins.

Mutator Phenotype and DNA Double-Strand Break Repair in BLM Helicase-Deficient Human Cells.

Bloom syndrome (BS), an autosomal recessive disorder of the BLM gene, predisposes sufferers to various cancers. To investigate the mutator phenotype and genetic consequences of DNA double-strand breaks (DSBs) in BS cells, we developed BLM helicase-deficient human cells by disrupting the BLM gene. Cells with a loss of heterozygosity (LOH) due to homologous recombination (HR) or nonhomologous end joining (NHEJ) can be restored with or without site-directed DSB induction. BLM cells exhibited a high frequency of spontaneous interallelic HR with crossover, but noncrossover events with long-tract gene conversions also occurred. Despite the highly interallelic HR events, BLM cells predominantly produced hemizygous LOH by spontaneous deletion. These phenotypes manifested during repair of DSBs. Both NHEJ and HR appropriately repaired DSBs in BLM cells, resulting in hemizygous and homozygous LOHs, respectively. However, the magnitude of the LOH was exacerbated in BLM cells, as evidenced by large deletions and long-tract gene conversions with crossover. BLM helicase suppresses the elongation of branch migration and crossover of double Holliday junctions (HJs) during HR repair, and a deficiency in this enzyme causes collapse, abnormal elongation, and/or preferable resolution to crossover of double HJs, resulting in a large-scale LOH. This mechanism underlies the predisposition for cancer in BS.

Active Control of Repetitive Structural Transitions between Replication Forks and Holliday Junctions by Werner Syndrome Helicase

The reactivation of stalled DNA replication via forkregression invokes Holliday junction formation, branch migration, and the recovery of the replication fork after DNA repair or error-free DNA synthesis. The coordination mechanism for these DNA structural transitions by molecular motors, however, remains unclear. Here we perform single-molecule fluorescence experiments with Werner syndrome protein (WRN) and model replication forks. The Holliday junction is readily formed once the lagging arm is unwound, and migrated unidirectionally with 3.2± 0.03 bases/s velocity. The recovery of the replication fork was controlled by branch migration reversal of WRN, resulting in repetitive fork regression. The Holliday junction formation, branch migration, and migration direction reversal are all ATP dependent, revealing that WRN uses the energy of ATP hydrolysis to actively coordinate the structural transitions of DNA.

Imaging of DNA and Protein-DNA Complexes with Atomic Force Microscopy.

This article reviews atomic force microscopy (AFM) studies of DNA structure and dynamics and protein-DNA complexes, including recent advances in the visualization of protein-DNA complexes with the use of cutting-edge, high-speed AFM. Special emphasis is given to direct nanoscale visualization of dynamics of protein-DNA complexes. In the area of DNA structure and dynamics, structural studies of local non-B conformations of DNA and the interplay of local and global DNA conformations are reviewed. The application of time-lapse AFM nanoscale imaging of DNA dynamics is illustrated by studies of Holliday junction branch migration. Structure and dynamics of protein-DNA interactions include problems related to site-specific DNA recombination, DNA replication, and DNA mismatch repair. Studies involving the structure and dynamics of chromatin are also described.

State transition analysis of spontaneous branch migration of the Holliday junction by photon-based single-molecule fluorescence resonance energy transfer

Branch migration of Holliday junction (HJ) DNA in solution is a spontaneous conformational change between multiple discrete states. We applied single-molecule fluorescence resonance energy transfer (smFRET) measurement to three-state branch migration. The photon-based variational Bayes-hidden Markov model (VB-HMM) method was applied to fluorescence signals to reproduce the state transition trajectories and evaluate the transition parameters, such as transition rate. The upper limit of time resolution suggested in simulation was nearly achieved for the state dynamics with relatively small FRET changes, and the distinctions in the populations of different states were successfully retrieved. We also discuss the suitability of the HJ as a standard sample for smFRET dynamics measurements and data analysis.

Human RECQ1 helicase-driven DNA unwinding, annealing, and branch migration: Insights from DNA complex structures

RecQ helicases are a widely conserved family of ATP-dependent motors with diverse roles in nearly every aspect of bacterial and eukaryotic genome maintenance. However, the physical mechanisms by which RecQ helicases recognize and process specific DNA replication and repair intermediates are largely unknown. Here, we solved crystal structures of the human RECQ1 helicase in complexes with tailed-duplex DNA and ssDNA. The structures map the interactions of the ssDNA tail and the branch point along the helicase and Zn-binding domains, which, together with reported structures of other helicases, define the catalytic stages of helicase action. We also identify a strand-separating pin, which (uniquely in RECQ1) is buttressed by the protein dimer interface. A duplex DNA-binding surface on the C-terminal domain is shown to play a role in DNA unwinding, strand annealing, and Holliday junction (HJ) branch migration. We have combined EM and analytical ultracentrifugation approaches to show that RECQ1 can form what appears to be a flat, homotetrameric complex and propose that RECQ1 tetramers are involved in HJ recognition. This tetrameric arrangement suggests a platform for coordinated activity at the advancing and receding duplexes of an HJ during branch migration.

The dissolution of double Holliday junctions.

Double Holliday junctions (dHJS) are important intermediates of homologous recombination. The separate junctions can each be cleaved by DNA structure-selective endonucleases known as Holliday junction resolvases. Alternatively, double Holliday junctions can be processed by a reaction known as "double Holliday junction dissolution." This reaction requires the cooperative action of a so-called "dissolvasome" comprising a Holliday junction branch migration enzyme (Sgs1/BLM RecQ helicase) and a type IA topoisomerase (Top3/TopoIIIα) in complex with its OB (oligonucleotide/oligosaccharide binding) fold containing accessory factor (Rmi1). This review details our current knowledge of the dissolution process and the players involved in catalyzing this mechanistically complex means of completing homologous recombination reactions.

Interaction of Branch Migration Translocases with the Holliday Junction-resolving Enzyme and Their Implications in Holliday Junction Resolution

Double-strand break repair involves the formation of Holliday junction (HJ) structures that need to be resolved to promote correct replication and chromosomal segregation. The molecular mechanisms of HJ branch migration and/or resolution are poorly characterized in Firmicutes. Genetic evidence suggested that the absence of the RuvAB branch migration translocase and the RecU HJ resolvase is synthetically lethal in Bacillus subtilis, whereas a recU recG mutant was viable. In vitro RecU, which is restricted to bacteria of the Firmicutes phylum, binds HJs with high affinity. In this work we found that RecU does not bind simultaneously with RecG to a HJ. RuvB by interacting with RecU bound to the central region of HJ DNA, loses its nonspecific association with DNA, and re-localizes with RecU to form a ternary complex. RecU cannot stimulate the ATPase or branch migration activity of RuvB. The presence of RuvB·ATPγS greatly stimulates RecU-mediated HJ resolution, but the addition of ATP or RuvA abolishes this stimulatory effect. A RecU·HJ·RuvAB complex might be formed. RecU does not increase the RuvAB activities but slightly inhibits them.

PRDM9 binding organizes hotspot nucleosomes and limits Holliday junction migration

In mammals, genetic recombination during meiosis is limited to a set of 1- to 2-kb regions termed hotspots. Their locations are predominantly determined by the zinc finger protein PRDM9, which binds to DNA in hotspots and subsequently uses its SET domain to locally trimethylate histone H3 at lysine 4 (H3K4me3). This sets the stage for double-strand break (DSB) formation and reciprocal exchange of DNA between chromatids, forming Holliday junctions. Here we report genome-wide analyses of PRDM9-dependent histone modifications using two inbred mouse strains differing only in their PRDM9 zinc finger domain. We show that PRDM9 binding actively reorganizes nucleosomes into a symmetrical pattern, creating an extended nucleosome-depleted region. These regions are centered by a consensus PRDM9 binding motif, whose location and identity was confirmed in vitro. We also show that DSBs are centered over the PRDM9 binding motif within the nucleosome-depleted region. Combining these results with data from genetic crosses, we find that crossing-over is restricted to the region marked by H3K4me3. We suggest that PRDM9-modified nucleosomes create a permissible environment that first directs the location of DSBs and then defines the boundaries of Holliday junction branch migration.