MOC1 cleaves Holliday junctions through a cooperative nick and counter-nick mechanism mediated by metal ions.

Holliday junction resolution is a crucial process in homologous recombination and DNA double-strand break repair. Complete Holliday junction resolution requires two stepwise incisions across the center of the junction, but the precise mechanism of metal ion-catalyzed Holliday junction cleavage remains elusive. Here, we perform a metal ion-triggered catalysis in crystals to investigate the mechanism of Holliday junction cleavage by MOC1. We capture the structures of MOC1 in complex with a nicked Holliday junction at various catalytic states, including the ground state, the one-metal ion binding state, and the two-metal ion binding state. Moreover, we also identify a third metal ion that may aid in the nucleophilic attack on the scissile phosphate. Further structural and biochemical analyses reveal a metal ion-mediated allosteric regulation between the two active sites, contributing to the enhancement of the second strand cleavage following the first strand cleavage, as well as the precise symmetric cleavage across the Holliday junction. Our work provides insights into the mechanism of metal ion-catalyzed Holliday junction resolution by MOC1, with implications for understanding how cells preserve genome integrity during the Holliday junction resolution phase.

Inhibition of the ATR-DNAPKcs-RB axis drives G1/S-phase transition and sensitizes triple-negative breast cancer (TNBC) to DNA holliday junctions.

Targeting the DNA damage response (DDR) is a promising strategy in oncotherapy, as most tumor cells are sensitive to excess damage due to their repair defects. Ataxia telangiectasia mutated and RAD3-related protein (ATR) is a damage response signal transduction sensor, and its therapeutic potential in tumor cells needs to be precisely investigated. Herein, we identified a new axis that could be targeted by ATR inhibitors to decrease the DNA-dependent protein kinase catalytic subunit (DNAPKcs), downregulate the expression of the retinoblastoma (RB), and drive G1/S-phase transition. Four-way DNA Holliday junctions (FJs) assembled in this process could trigger S-phase arrest and induce lethal chromosome damage in RB-positive triple-negative breast cancer (TNBC) cells. Furthermore, these unrepaired junctions also exerted toxic effects to RB-deficient TNBC cells when the homologous recombination repair (HRR) was inhibited. This study proposes a precise strategy for treating TNBC by targeting the DDR and extends our understanding of ATR and HJ in tumor treatment.

Molecular Insight into the Structural Dynamics of Holliday Junctions Modulated by Integration Host Factor.

The integration host factor (IHF) in Escherichia coli is a nucleoid-associated protein with multifaceted roles that encompass DNA packaging, viral DNA integration, and recombination. IHF binds to double-stranded DNA featuring a 13-base pair (bp) consensus sequence with high affinity, causing a substantial bend of approximately 160° upon binding. Although wild-type IHF (WtIHF) is principally involved in DNA bending to facilitate foreign DNA integration into the host genome, its engineered counterpart, single-chain IHF (ScIHF), was specifically designed for genetic engineering and biotechnological applications. Our study delves into the interactions of both IHF variants with Holliday junctions (HJs), pivotal intermediates in DNA repair, and homologous recombination. HJs are dynamic structures capable of adopting open or stacked conformations, with the open conformation facilitating processes such as branch migration and strand exchange. Using microscale thermophoresis, we quantitatively assessed the binding of IHF to four-way DNA junctions that harbor specific binding sequences H' and H1. Our findings demonstrate that both IHF variants exhibit a strong affinity for HJs, signifying a structure-based recognition mechanism. Circular dichroism (CD) experiments unveiled the impact of the protein on the junction's conformation. Furthermore, single-molecule Förster resonance energy transfer (smFRET) confirmed the influence of IHF on the junction's dynamicity. Intriguingly, our results revealed that WtIHF and ScIHF binding shifts the population toward the open conformation of the junction and stabilizes it in that state. In summary, our findings underscore the robust affinity of the IHF for HJs and its capacity to stabilize the open conformation of these junctions.

Heterozygosity alters Msh5 binding to meiotic chromosomes in the baker's yeast.

Meiotic crossovers are initiated from programmed DNA double-strand breaks. The Msh4-Msh5 heterodimer is an evolutionarily conserved mismatch repair-related protein complex that promotes meiotic crossovers by stabilizing strand invasion intermediates and joint molecule structures such as Holliday junctions. In vivo studies using homozygous strains of the baker's yeast Saccharomyces cerevisiae (SK1) show that the Msh4-Msh5 complex associates with double-strand break hotspots, chromosome axes, and centromeres. Many organisms have heterozygous genomes that can affect the stability of strand invasion intermediates through heteroduplex rejection of mismatch-containing sequences. To examine Msh4-Msh5 function in a heterozygous context, we performed chromatin immunoprecipitation and sequencing (ChIP-seq) analysis in a rapidly sporulating hybrid S. cerevisiae strain (S288c-sp/YJM789, containing sporulation-enhancing QTLs from SK1), using SNP information to distinguish reads from homologous chromosomes. Overall, Msh5 localization in this hybrid strain was similar to that determined in the homozygous strain (SK1). However, relative Msh5 levels were reduced in regions of high heterozygosity, suggesting that high mismatch densities reduce levels of recombination intermediates to which Msh4-Msh5 binds. Msh5 peaks were also wider in the hybrid background compared to the homozygous strain (SK1). We determined regions containing heteroduplex DNA by detecting chimeric sequence reads with SNPs from both parents. Msh5-bound double-strand break hotspots overlap with regions that have chimeric DNA, consistent with Msh5 binding to heteroduplex-containing recombination intermediates.

Method to generate Holliday junction recombination intermediates via RecA-mediated four-strand exchange.

DNA molecules that contain single Holliday junctions have served as model substrates to investigate the pathway in which homologous recombination intermediates are processed. However, the preparation of DNA containing Holliday junctions in high yield remains a challenge. In this work, we used a nicking endonuclease to generate gapped DNA, from which α-structured DNA or figure-8 DNA were created via RecA-mediated reactions. The resulting DNA molecules were found to serve as good substrates for Holliday junction resolvases. The simplified method negates the requirement for radioactive labelling of DNA, making the generation of Holliday junction DNA more accessible to non-experts.

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.

Attenuation of a DNA cruciform by a conserved regulator directs T3SS1 mediated virulence in Vibrio parahaemolyticus.

Pathogenic Vibrio species account for 3-5 million annual life-threatening human infections. Virulence is driven by bacterial hemolysin and toxin gene expression often positively regulated by the winged helix-turn-helix (wHTH) HlyU transcriptional regulator family and silenced by histone-like nucleoid structural protein (H-NS). In the case of Vibrio parahaemolyticus, HlyU is required for virulence gene expression associated with type 3 Secretion System-1 (T3SS1) although its mechanism of action is not understood. Here, we provide evidence for DNA cruciform attenuation mediated by HlyU binding to support concomitant virulence gene expression. Genetic and biochemical experiments revealed that upon HlyU mediated DNA cruciform attenuation, an intergenic cryptic promoter became accessible allowing for exsA mRNA expression and initiation of an ExsA autoactivation feedback loop at a separate ExsA-dependent promoter. Using a heterologous E. coli expression system, we reconstituted the dual promoter elements which revealed that HlyU binding and DNA cruciform attenuation were strictly required to initiate the ExsA autoactivation loop. The data indicate that HlyU acts to attenuate a transcriptional repressive DNA cruciform to support T3SS1 virulence gene expression and reveals a non-canonical extricating gene regulation mechanism in pathogenic Vibrio species.

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-(6'-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.

The Biochemical Mechanism of Fork Regression in Prokaryotes and Eukaryotes-A Single Molecule Comparison.

The rescue of stalled DNA replication forks is essential for cell viability. Impeded but still intact forks can be rescued by atypical DNA helicases in a reaction known as fork regression. This reaction has been studied at the single-molecule level using the Escherichia coli DNA helicase RecG and, separately, using the eukaryotic SMARCAL1 enzyme. Both nanomachines possess the necessary activities to regress forks: they simultaneously couple DNA unwinding to duplex rewinding and the displacement of bound proteins. Furthermore, they can regress a fork into a Holliday junction structure, the central intermediate of many fork regression models. However, there are key differences between these two enzymes. RecG is monomeric and unidirectional, catalyzing an efficient and processive fork regression reaction and, in the process, generating a significant amount of force that is used to displace the tightly-bound E. coli SSB protein. In contrast, the inefficient SMARCAL1 is not unidirectional, displays limited processivity, and likely uses fork rewinding to facilitate RPA displacement. Like many other eukaryotic enzymes, SMARCAL1 may require additional factors and/or post-translational modifications to enhance its catalytic activity, whereas RecG can drive fork regression on its own.

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.

Multifaceted regulation of the sumoylation of the Sgs1 DNA helicase.

Homologous recombination repairs DNA breaks and sequence gaps via the production of joint DNA intermediates such as Holliday junctions. Dissolving Holliday junctions into linear DNA repair products requires the activity of the Sgs1 helicase in yeast and of its homologs in other organisms. Recent studies suggest that the functions of these conserved helicases are regulated by sumoylation; however, the mechanisms that promote their sumoylation are not well understood. Here, we employed in vitro sumoylation systems and cellular assays to determine the roles of DNA and the scaffold protein Esc2 in Sgs1 sumoylation. We show that DNA binding enhances Sgs1 sumoylation in vitro. In addition, we demonstrate the Esc2's midregion (MR) with DNA-binding activity is required for Sgs1 sumoylation. Unexpectedly, we found that the sumoylation-promoting effect of Esc2-MR is DNA independent, suggesting a second function for this domain. In agreement with our biochemical data, we found the Esc2-MR domain, like its SUMO E2-binding C-terminal domain characterized in previous studies, is required for proficient sumoylation of Sgs1 and its cofactors, Top3 and Rmi1, in cells. Taken together, these findings provide evidence that while DNA binding enhances Sgs1 sumoylation, Esc2-based stimulation of this modification is mediated by two distinct domains.

The toposiomerase IIIalpha-RMI1-RMI2 complex orients human Bloom's syndrome helicase for efficient disruption of D-loops.

Homologous recombination (HR) is a ubiquitous and efficient process that serves the repair of severe forms of DNA damage and the generation of genetic diversity during meiosis. HR can proceed via multiple pathways with different outcomes that may aid or impair genome stability and faithful inheritance, underscoring the importance of HR quality control. Human Bloom's syndrome (BLM, RecQ family) helicase plays central roles in HR pathway selection and quality control via unexplored molecular mechanisms. Here we show that BLM's multi-domain structural architecture supports a balance between stabilization and disruption of displacement loops (D-loops), early HR intermediates that are key targets for HR regulation. We find that this balance is markedly shifted toward efficient D-loop disruption by the presence of BLM's interaction partners Topoisomerase IIIα-RMI1-RMI2, which have been shown to be involved in multiple steps of HR-based DNA repair. Our results point to a mechanism whereby BLM can differentially process D-loops and support HR control depending on cellular regulatory mechanisms.

Genetic Study of Four Candidate Holliday Junction Processing Proteins in the Thermophilic Crenarchaeon Sulfolobus acidocaldarius.

Homologous recombination (HR) is thought to be important for the repair of stalled replication forks in hyperthermophilic archaea. Previous biochemical studies identified two branch migration helicases (Hjm and PINA) and two Holliday junction (HJ) resolvases (Hjc and Hje) as HJ-processing proteins; however, due to the lack of genetic evidence, it is still unclear whether these proteins are actually involved in HR in vivo and how their functional relation is associated with the process. To address the above questions, we constructed hjc-, hje-, hjm-, and pina single-knockout strains and double-knockout strains of the thermophilic crenarchaeon Sulfolobus acidocaldarius and characterized the mutant phenotypes. Notably, we succeeded in isolating the hjm- and/or pina-deleted strains, suggesting that the functions of Hjm and PINA are not essential for cellular growth in this archaeon, as they were previously thought to be essential. Growth retardation in Δpina was observed at low temperatures (cold sensitivity). When deletion of the HJ resolvase genes was combined, Δpina Δhjc and Δpina Δhje exhibited severe cold sensitivity. Δhjm exhibited severe sensitivity to interstrand crosslinkers, suggesting that Hjm is involved in repairing stalled replication forks, as previously demonstrated in euryarchaea. Our findings suggest that the function of PINA and HJ resolvases is functionally related at lower temperatures to support robust cellular growth, and Hjm is important for the repair of stalled replication forks in vivo.

DisA Restrains the Processing and Cleavage of Reversed Replication Forks by the RuvAB-RecU Resolvasome.

DNA lesions that impede fork progression cause replisome stalling and threaten genome stability. Bacillus subtilis RecA, at a lesion-containing gap, interacts with and facilitates DisA pausing at these branched intermediates. Paused DisA suppresses its synthesis of the essential c-di-AMP messenger. The RuvAB-RecU resolvasome branch migrates and resolves formed Holliday junctions (HJ). We show that DisA prevents DNA degradation. DisA, which interacts with RuvB, binds branched structures, and reduces the RuvAB DNA-dependent ATPase activity. DisA pre-bound to HJ DNA limits RuvAB and RecU activities, but such inhibition does not occur if the RuvAB- or RecU-HJ DNA complexes are pre-formed. RuvAB or RecU pre-bound to HJ DNA strongly inhibits DisA-mediated synthesis of c-di-AMP, and indirectly blocks cell proliferation. We propose that DisA limits RuvAB-mediated fork remodeling and RecU-mediated HJ cleavage to provide time for damage removal and replication restart in order to preserve genome integrity.

Repeated strand invasion and extensive branch migration are hallmarks of meiotic recombination.

Currently favored models for meiotic recombination posit that both noncrossover and crossover recombination are initiated by DNA double-strand breaks but form by different mechanisms: noncrossovers by synthesis-dependent strand annealing and crossovers by formation and resolution of double Holliday junctions centered around the break. This dual mechanism hypothesis predicts different hybrid DNA patterns in noncrossover and crossover recombinants. We show that these predictions are not upheld, by mapping with unprecedented resolution parental strand contributions to recombinants at a model locus. Instead, break repair in both noncrossovers and crossovers involves synthesis-dependent strand annealing, often with multiple rounds of strand invasion. Crossover-specific double Holliday junction formation occurs via processes involving branch migration as an integral feature, one that can be separated from repair of the break itself. These findings reveal meiotic recombination to be a highly dynamic process and prompt a new view of the relationship between crossover and noncrossover recombination.

Distribution of Holliday junctions and repair forks during Escherichia coli DNA double-strand break repair.

Accurate repair of DNA double-strand breaks (DSBs) is crucial for cell survival and genome integrity. In Escherichia coli, DSBs are repaired by homologous recombination (HR), using an undamaged sister chromosome as template. The DNA intermediates of this pathway are expected to be branched molecules that may include 4-way structures termed Holliday junctions (HJs), and 3-way structures such as D-loops and repair forks. Using a tool creating a site-specific, repairable DSB on only one of a pair of replicating sister chromosomes, we have determined how these branched DNA intermediates are distributed across a DNA region that is undergoing DSB repair. In cells, where branch migration and cleavage of HJs are limited by inactivation of the RuvABC complex, HJs and repair forks are principally accumulated within a distance of 12 kb from sites of recombination initiation, known as Chi, on each side of the engineered DSB. These branched DNA structures can even be detected in the region of DNA between the Chi sites flanking the DSB, a DNA segment not expected to be engaged in recombination initiation, and potentially degraded by RecBCD nuclease action. This is observed even in the absence of the branch migration and helicase activities of RuvAB, RadA, RecG, RecQ and PriA. The detection of full-length DNA fragments containing HJs in this central region implies that DSB repair can restore the two intact chromosomes, into which HJs can relocate prior to their resolution. The distribution of recombination intermediates across the 12kb region beyond Chi is altered in xonA, recJ and recQ mutants suggesting that, in the RecBCD pathway of DSB repair, exonuclease I stimulates the formation of repair forks and that RecJQ promotes strand-invasion at a distance from the recombination initiation sites.

Genetic and Molecular Approaches to Study Chromosomal Breakage at Secondary Structure-Forming Repeats.

DNA repeats capable of adopting stable secondary structures are hotspots for double-strand break (DSB) formation and, hence, for homologous recombination and gross chromosomal rearrangements (GCR) in many prokaryotic and eukaryotic organisms, including humans. Here, we provide protocols for studying chromosomal instability triggered by hairpin- and cruciform-forming palindromic sequences in the budding yeast, Saccharomyces cerevisiae. First, we describe two sensitive genetic assays aimed to determine the recombinogenic potential of inverted repeats and their ability to induce GCRs. Then, we detail an approach to monitor chromosomal DSBs by Southern blot hybridization. Finally, we describe how to define the molecular structure of DSBs. We provide, as an example, the analysis of chromosomal fragility at a reporter system containing unstable Alu-inverted repeats. By using these approaches, any DNA sequence motif can be assessed for its breakage potential and ability to drive genome instability.

A novel protein-engineered dsDNA-binding protein (HU-Simulacrum) inspired by HU, a nucleoid-associated DNABII protein.

HU, a DNA-binding protein, has a helical N-terminal region (NTR) of ∼44 residues and a beta strand- and IDR-rich C-terminal region (CTR) of ∼46 residues. CTR binds to DNA through (i) a clasp (two arginine/lysine-rich, IDR-rich beta hairpins that bind to phosphate groups in the minor groove), (ii) a flat surface (comprising four antiparallel beta strands that abut the major groove), and (iii) a charge cluster (two lysine residues upon a short C-terminal helix). HU forms a dimer displaying extensive inter-subunit CTR-CTR contacts. A single-chain simulacrum of these contacts (HU-Simul) incorporating all DNA-binding elements was created by fusing together the CTRs of Escherichia coli HU-A and Thermus thermophilus HU. HU-Simul is monomeric, binds to dsDNA and cruciform DNA, but not to ssDNA.

Implications of Metastable Nicks and Nicked Holliday Junctions in Processing Joint Molecules in Mitosis and Meiosis.

Joint molecules (JMs) are intermediates of homologous recombination (HR). JMs rejoin sister or homolog chromosomes and must be removed timely to allow segregation in anaphase. Current models pinpoint Holliday junctions (HJs) as a central JM. The canonical HJ (cHJ) is a four-way DNA that needs specialized nucleases, a.k.a. resolvases, to resolve into two DNA molecules. Alternatively, a helicase-topoisomerase complex can deal with pairs of cHJs in the dissolution pathway. Aside from cHJs, HJs with a nick at the junction (nicked HJ; nHJ) can be found in vivo and are extremely good substrates for resolvases in vitro. Despite these findings, nHJs have been neglected as intermediates in HR models. Here, I present a conceptual study on the implications of nicks and nHJs in the final steps of HR. I address this from a biophysical, biochemical, topological, and genetic point of view. My conclusion is that they ease the elimination of JMs while giving genetic directionality to the final products. Additionally, I present an alternative view of the dissolution pathway since the nHJ that results from the second end capture predicts a cross-join isomerization. Finally, I propose that this isomerization nicely explains the strict crossover preference observed in synaptonemal-stabilized JMs in meiosis.

Essentiality of CENP-A Depends on Its Binding Mode to HJURP.

CENP-A incorporation is critical for centromere specification and is mediated by the chaperone HJURP. The CENP-A-targeting domain (CATD) of CENP-A specifically binds to HJURP, and this binding is conserved. However, the binding interface of CENP-A-HJURP is yet to be understood. Here, we identify the critical residues for chicken CENP-A or HJURP. The A59Q mutation in the α1-helix of chicken CENP-A causes CENP-A mis-incorporation and subsequent cell death, whereas the corresponding mutation in human CENP-A does not. We also find that W53 of HJURP, which is a contact site of A59 in CENP-A, is also essential in chicken cells. Our comprehensive analyses reveal that the affinities of HJURP to CATD differ between chickens and humans. However, the introduction of two arginine residues to the chicken HJURP αA-helix suppresses CENP-A mis-incorporation in chicken cells expressing CENP-AA59Q. Our data explain the mechanisms and evolution of CENP-A essentiality by the CENP-A-HJURP interaction.