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Multivalency as an interaction principle is widely utilized in nature. It enables specific and strong binding by multiple weak interactions through enhanced avidity and is a core process in immune recognition and cellular signaling, which is also a current concept in drug design. Here, we use the high signals from plasmon-enhanced fluorescence of nanoparticles to extract binding kinetics and dynamics of multivalent interactions on the single-molecule level and in real time. We study mono-, bi-, and trivalent binding interactions using a DNA Holliday Junction as a model construct with programmable valency and introduce a step-binding model for binding kinetics relevant for structured macromolecules by including an experimentally extractable binding restriction term ω to quantify the effects from conformation, steric effects, and rigidity. We used this approach to explore how length and flexibility of the DNA ligands affect binding restriction and binding strength, where the overall binding strength decreased with spacer length. For trivalent systems, increasing spacer length additionally activated binding in the trivalent state, giving insight into the design of multivalent drug or targeting moieties. By systematically changing the receptor density, we explored the binding super selectivity of the multivalent HJ at the single-molecule level. We find a polynomial behavior of the trivalent binding strength that clearly shows receptor-density-dependent selective binding. Interestingly, we could exploit the rapidly decaying near fields of the plasmon that induce a strong dependence of the signal on the position of the dye to observe binding dynamics during single multivalent binding events.

DNA Holliday junctions (HJs) are crucial intermediates in genetic recombination and genome repair processes, characterized by a dynamic nature and transitioning among multiple conformations on the timescale ranging from sub-milliseconds to seconds. Although the influence of ions on HJ dynamics has been extensively studied, precise quantification of the thermodynamic feasibility of transitions and detailed kinetic cooperativity remain unexplored. Understanding the heterogeneity of stochastic gene recombination using ensemble-averaged experimental techniques is extremely difficult because of its lack of ability to differentiate dynamics and function in a high spatiotemporal resolution. Herein, we developed a new technique that combines single-molecule fluorescence resonance energy transfer (smFRET) experiments and molecular simulation to investigate the kinetic choreography and preferential stability of HJ conformations under ionic conditions that closely mimic the physiological environment relevant to cellular biology. Our findings predict the prevalence of three distinct conformational macrostates in HJ dynamics. At low ion concentrations, HJs transition rapidly among three thermodynamically stable conformational macrostates. However, in a physiological ionic environment, the open conformation becomes predominant. Using a kinetic network model based on the multi-order time correlation function (TCF), we delineated thermodynamic parameters that govern heterogeneous dynamics as a function of divalent ion concentration. Stabilization of conformations due to an ionic environment and activation barriers concertedly affect transition rates between open and closed conformations. Furthermore, we observed a significant enhancement of Mg 2+ condensation in the central region of HJs rather than branch ends, leading to a plausible conclusion that the differential stability of conformational states may be governed by the junction region of HJs rather than duplex branches. This study gives a new insight into the complex interplay between the ionic environment and HJ dynamics, offering a comprehensive understanding of their behavior under conditions relevant to cellular biology and roles in key biological processes for creating a heterogeneous nature of life.

In eubacteria, Holliday junction (HJ) resolvases (HJRs) are crucial for faithful segregation of newly replicated chromosomes, homologous recombination, and repair of stalled/collapsed DNA replication forks. However, compared with the Escherichia coli HJRs, little is known about their orthologs in mycobacterial species. A genome-wide analysis of Mycobacterium smegmatis identified two genes encoding putative HJRs, namely RuvC (MsRuvC) and RuvX (MsRuvX); but whether they play redundant, overlapping, or distinct roles remains unknown. Here, we reveal that MsRuvC exists as a homodimer while MsRuvX as a monomer in solution, and both showed high-binding affinity for branched DNAs compared with unbranched DNA species. Interestingly, the DNA cleavage specificities of MsRuvC and MsRuvX were found to be mutually exclusive: the former efficiently promotes HJ resolution, in a manner analogous to the Escherichia coli RuvC, but does not cleave other branched DNA species; whereas the latter is a versatile DNase capable of cleaving a variety of branched DNA structures, including 3' and 5' flap DNA, splayed-arm DNA and dsDNA with 3' and 5' overhangs but lacks the HJ resolution activity. Point mutations in the RNase H-like domains of MsRuvC and MsRuvX pinpointed critical residues required for their DNA cleavage activities and also demonstrated uncoupling between DNA-binding and DNA cleavage activities. Unexpectedly, we found robust evidence that MsRuvX possesses a double-strand/single-strand junction-specific endonuclease and ssDNA exonucleolytic activities. Combined, our findings highlight that the RuvC and RuvX DNases play distinct complementary, and not redundant, roles in the processing of branched DNA structures in M. smegmatis., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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., 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 Inc. All rights reserved.)

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., (© 2024. The Author(s).)

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.

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., Competing Interests: Conflicts of interest The authors declare no conflicts of interest., (© The Author(s) 2023. 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.)

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., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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., (© 2023. The Author(s).)

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 IC 50 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., 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 © 2022 Elsevier Ltd. All rights reserved.)

The Holliday junction is a key intermediate formed during DNA recombination across all kingdoms of life 1 . 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 reaction 2 . 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., (© 2022. The Author(s).)

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.

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., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (© 2022. The Author(s).)

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 Δ pina was observed at low temperatures (cold sensitivity). When deletion of the HJ resolvase genes was combined, Δ pina Δ hjc and Δ pina Δ hje exhibited severe cold sensitivity. Δ hjm 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.

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Inc.)

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.

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., Competing Interests: The authors have declared that no competing interests exist.

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., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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.

The SWI/SNF chromatin remodeling complex plays important roles in gene regulation and it is classified as the SWI/SNF complex in yeast and BAF complex in vertebrates. BAF57, one of the subunits that forms the chromatin remodeling complex core, is well conserved in the BAF complex of vertebrates, which is replaced by bap111 in the Drosophila BAP complex and does not have a counterpart in the yeast SWI/SNF complex. This suggests that BAF57 is a key component of the chromatin remodeling complex in higher eukaryotes. BAF57 contains a HMG domain, which is widely distributed among various proteins and functions as a DNA binding motif. Most proteins with HMG domain bind to four-way junction (4WJ) DNA. Here, we report the crystal structure of the HMG domain of BAF57 (BAF57 HMG ) at a resolution of 2.55 Å. The structure consists of three α-helices and adopts an L-shaped form. The overall structure is stabilized by a hydrophobic core, which is formed by hydrophobic residues. The binding affinity between BAF57 HMG and 4WJ DNA is determined as a 295.83 ± 1.05 nM using a fluorescence quenching assay, and the structure revealed 4WJ DNA binding site of BAF57 HMG . Our data will serve structural basis in understanding the roles of BAF57 during chromatin remodeling process., 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 © 2020. Published by Elsevier Inc.)

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.

The axial stiffness of DNA origami is determined as a function of key nanostructural characteristics. Different constructs of two-helix nanobeams with specified densities of nicks and Holliday junctions are synthesized and stretched by fluid flow. Implementing single particle tracking to extract force-displacement curves enables the measurement of DNA origami stiffness values at the enthalpic elasticity regime, i.e. for forces larger than 15 pN. Comparisons between ligated and nicked helices show that the latter exhibit nearly a two-fold decrease in axial stiffness. Numerical models that treat the DNA helices as elastic rods are used to evaluate the local loss of stiffness at the locations of nicks and Holliday junctions. It is shown that the models reproduce the experimental data accurately, indicating that both of these design characteristics yield a local stiffness two orders of magnitude smaller than the corresponding value of the intact double-helix. This local degradation in turn leads to a macroscopic loss of stiffness that is evaluated numerically for multi-helix DNA bundles., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The Saccharomyces cerevisiae Mus81-Mms4 complex is a highly conserved DNA structure-specific endonuclease that plays essential roles in the processing of recombination intermediates that arise during the repair of stalled replication forks and double-stranded breaks. To identify novel factors functioning conjointly with Mus81-Mms4, we performed a biochemical screen and found that Crp1, a cruciform DNA-recognizing protein that specifically binds to DNA four-way junction structures, could stimulate the Mus81-Mms4 endonuclease. The specific protein interaction between Mus81-Mms4 and Crp1 was responsible for the stimulation observed. Multicopy expression of Crp1 could partially rescue the sensitivity to DNA-damaging agents of the sgs1∆mus81∆21-24N mutant. Our results provide insight into the functional role and interaction of Crp1 with other proteins involved in DNA repair., (© 2020 Federation of European Biochemical Societies.)

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-A A59Q . Our data explain the mechanisms and evolution of CENP-A essentiality by the CENP-A-HJURP interaction., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Cruciform DNA is a causing factor of genome instability and chromosomal translocation, however, most studies about cruciform DNA in mammalian cells were based on palindromic sequences containing plasmids and reports about endogenous cruciform DNA are rare. In this study we observed the dynamics of endogenous cruciform DNA in mouse growing oocytes using immunofluorescence labeling method. We found cruciform DNA foci exist in transcription active growing oocytes but not in transcription inactive fully grown oocytes and colocalized with Parp1 but not with DNA damage marker γH2A.X. By analyzing the Genotype-Tissue Expression data, we found cruciform DNA-mediated chromosomal translocation in human spermatocytes is associated with the specific DNA transcription in testis. When inhibiting the transcription with α-amanitin in mouse oocytes, we found oocyte cruciform DNA foci decreased significantly. In summary, we observed the endogenous cruciform DNA in growing oocytes and our results showed that the cruciform DNA formation is transcription-dependent., Competing Interests: The authors have declared that no competing interests exist.

During prophase of the first meiotic division, cells deliberately break their DNA 1 . These DNA breaks are repaired by homologous recombination, which facilitates proper chromosome segregation and enables the reciprocal exchange of DNA segments between homologous chromosomes 2 . A pathway that depends on the MLH1-MLH3 (MutLγ) nuclease has been implicated in the biased processing of meiotic recombination intermediates into crossovers by an unknown mechanism 3-7 . Here we have biochemically reconstituted key elements of this pro-crossover pathway. We show that human MSH4-MSH5 (MutSγ), which supports crossing over 8 , binds branched recombination intermediates and associates with MutLγ, stabilizing the ensemble at joint molecule structures and adjacent double-stranded DNA. MutSγ directly stimulates DNA cleavage by the MutLγ endonuclease. MutLγ activity is further stimulated by EXO1, but only when MutSγ is present. Replication factor C (RFC) and the proliferating cell nuclear antigen (PCNA) are additional components of the nuclease ensemble, thereby triggering crossing-over. Saccharomyces cerevisiae strains in which MutLγ cannot interact with PCNA present defects in forming crossovers. Finally, the MutLγ-MutSγ-EXO1-RFC-PCNA nuclease ensemble preferentially cleaves DNA with Holliday junctions, but shows no canonical resolvase activity. Instead, it probably processes meiotic recombination intermediates by nicking double-stranded DNA adjacent to the junction points 9 . As DNA nicking by MutLγ depends on its co-factors, the asymmetric distribution of MutSγ and RFC-PCNA on meiotic recombination intermediates may drive biased DNA cleavage. This mode of MutLγ nuclease activation might explain crossover-specific processing of Holliday junctions or their precursors in meiotic chromosomes 4 .

During meiosis, crossover recombination connects homologous chromosomes to direct their accurate segregation 1 . Defective crossing over causes infertility, miscarriage and congenital disease. Each pair of chromosomes attains at least one crossover via the formation and biased resolution of recombination intermediates known as double Holliday junctions 2,3 . A central principle of crossover resolution is that the two Holliday junctions are resolved in opposite planes by targeting nuclease incisions to specific DNA strands 4 . The endonuclease activity of the MutLγ complex has been implicated in the resolution of crossovers 5-10 , but the mechanisms that activate and direct strand-specific cleavage remain unknown. Here we show that the sliding clamp PCNA is important for crossover-biased resolution. In vitro assays with human enzymes show that PCNA and its loader RFC are sufficient to activate the MutLγ endonuclease. MutLγ is further stimulated by a co-dependent activity of the pro-crossover factors EXO1 and MutSγ, the latter of which binds Holliday junctions 11 . MutLγ also binds various branched DNAs, including Holliday junctions, but does not show canonical resolvase activity, implying that the endonuclease incises adjacent to junction branch points to achieve resolution. In vivo, RFC facilitates MutLγ-dependent crossing over in budding yeast. Furthermore, PCNA localizes to prospective crossover sites along synapsed chromosomes. These data highlight similarities between crossover resolution and the initiation steps of DNA mismatch repair 12,13 and evoke a novel model for crossover-specific resolution of double Holliday junctions during meiosis.

Sequences of DNA typically adopt B-form duplexes in genomes, although noncanonical structures such as G-quadruplexes, i-motifs, Z-DNA, and cruciform structures can occur. A challenge is to determine the functions of these various structures in cellular processes. We and others have hypothesized that G-rich G-quadruplex-forming sequences in human genome promoters serve to sense oxidative damage generated during oxidative stress impacting gene regulation. Herein, chemical tools and a cell-based assay were used to study the oxidation of guanine to 8-oxo-7,8-dihydroguanine (OG) in the context of a cruciform-forming sequence in a gene promoter to determine the impact on transcription. We found that OG in the nontemplate strand in the loop of a cruciform-forming sequence could induce gene expression; conversely when OG was in the same sequence on the template strand, gene expression was inhibited. A model for the transcriptional changes observed is proposed in which OG focuses the DNA repair process on the promoter to impact expression. Our cellular and biophysical studies and literature sources support the idea that removal of OG from duplex DNA by OGG1 yields an abasic site (AP) that triggers a structural shift to the cruciform fold. The AP-bearing cruciform structure is presented to APE1, which functions as a conduit between DNA repair and gene regulation. The significance is enhanced by a bioinformatic study of all human gene promoters and transcription termination sites for inverted repeats (IRs). Comparison of the two regions showed that promoters have stable and G-rich IRs at a low frequency and termination sites have many AT-rich IRs with low stability.

In this study, we report experimental (Protein Data Bank (PDB) search) and theoretical (RI-MP2/def2-TZVP level of theory) evidence of the nature, stability, and directionality properties of intramolecular halogen bonding interactions (HaBs) between 5-bromo/5-iodoracil bases and backbone phosphate groups in nucleic acids (NAs). A PDB survey revealed relevant examples where intramolecular HaBs are undertaken and serve as a structural source of stability in RNA and DNA molecules. In order to develop suitable energy predictors, we started this investigation by calculating the interaction energy values and both the potential V ( r ) and kinetic G ( r ) energy densities (using Bader's "atoms in molecules" theory) of several halogen bond complexes involving 5-bromo/5-iodoracil molecules and biologically relevant electron donors. Once the energy predictors based on V ( r )/ G ( r ) energy densities were developed, we analyzed the HaBs observed in the biological examples retrieved from the PDB search in order to estimate the strength of the interaction. As far as our knowledge extends, intramolecular halogen bonds in NAs have not been previously quantified in the literature using this methodology and may be of great importance in understanding their structural properties as well as in the construction of molecular materials with DNA and other biological macromolecules.

Crossover recombination is critical for meiotic chromosome segregation, but how mammalian crossing over is accomplished is poorly understood. Here, we illuminate how strands exchange during meiotic recombination in male mice by analyzing patterns of heteroduplex DNA in recombinant molecules preserved by the mismatch correction deficiency of Msh2 -/- mutants. Surprisingly, MSH2-dependent recombination suppression was not evident. However, a substantial fraction of crossover products retained heteroduplex DNA, and some provided evidence of MSH2-independent correction. Biased crossover resolution was observed, consistent with asymmetry between DNA ends in earlier intermediates. Many crossover products yielded no heteroduplex DNA, suggesting dismantling by D-loop migration. Unlike the complexity of crossovers in yeast, these simple modifications of the original double-strand break repair model-asymmetry in recombination intermediates and D-loop migration-may be sufficient to explain most meiotic crossing over in mice while also addressing long-standing questions related to Holliday junction resolution., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The Holliday junction, a four-way DNA structure, is an important intermediate of homologous recombination. Proper Holliday junction resolution is critical to complete the recombination process. In most bacterial cells, the Holliday junction cleavage is mainly performed by a specific endonuclease RuvC. Here, we describe the biochemical properties and the crystal structure of RuvC from an opportunistic pathogen, Pseudomonas aeruginosa (PaRuvC). PaRuvC specifically binds to the Holliday junction DNA and preferentially cleaves it at the consensus 5'-TTC-3'. PaRuvC uses Mg 2+ as the preferred divalent metal cofactor for Holliday junction cleavage and its optimum pH is 8.0-9.0. Elevated temperatures (37-60 °C) boost the catalytic activity, but temperatures higher than 53 °C reduce the protein stability. The crystal structure of PaRuvC determined at 2.4 Å and mutagenesis analysis reveal key residues involved in the dimer formation, substrate binding and catalysis. Our results are expected to provide useful information to combat antibiotic resistance of Pseudomonas aeruginosa by targeting its homologous recombination system., Competing Interests: Declaration of competing interest The authors declare no conflict of interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Modified DNA bases functionally distinguish the taxonomic forms of life-5-methylcytosine separates prokaryotes from eukaryotes and 5-hydroxymethylcytosine (5hmC) invertebrates from vertebrates. We demonstrate here that mouse endonuclease G (mEndoG) shows specificity for both 5hmC and Holliday junctions. The enzyme has higher affinity (>50-fold) for junctions over duplex DNAs. A 5hmC-modification shifts the position of the cut site and increases the rate of DNA cleavage in modified versus unmodified junctions. The crystal structure of mEndoG shows that a cysteine (Cys69) is positioned to recognize 5hmC through a thiol-hydroxyl hydrogen bond. Although this Cys is conserved from worms to mammals, a two amino acid deletion in the vertebrate relative to the invertebrate sequence unwinds an α-helix, placing the thiol of Cys69 into the mEndoG active site. Mutations of Cys69 with alanine or serine show 5hmC-specificity that mirrors the hydrogen bonding potential of the side chain (C-H < S-H < O-H). A second orthogonal DNA binding site identified in the mEndoG structure accommodates a second arm of a junction. Thus, the specificity of mEndoG for 5hmC and junctions derives from structural adaptations that distinguish the vertebrate from the invertebrate enzyme, thereby thereby supporting a role for 5hmC in recombination processes., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Holliday junctions (HJs) are key DNA intermediates in genetic recombination and are eliminated by nuclease, termed resolvase, to ensure genome stability. HJ resolvases have been identified across all kingdoms of life, members of which exhibit sequence-dependent HJ resolution. However, the molecular basis of sequence selectivity remains largely unknown. Here, we present the chloroplast resolvase MOC1, which cleaves HJ in a cytosine-dependent manner. We determine the crystal structure of MOC1 with and without HJs. MOC1 exhibits an RNase H fold, belonging to the retroviral integrase family. MOC1 functions as a dimer, and the HJ is embedded into the basic cleft of the dimeric enzyme. We characterize a base recognition loop (BR loop) that protrudes into and opens the junction. Residues from the BR loop intercalate into the bases, disrupt the C-G base pairing at the crossover and recognize the cytosine, providing the molecular basis for sequence-dependent HJ resolution by a resolvase.

Fowlpox virus resolvase (Fpr) is an endonuclease that cleaves a broad range of branched DNA structures, including the Holliday junction (HJ), with little sequence-specificity. To better understand the mechanisms underlying its relaxed substrate specificity, we determined the crystal structures of Fpr and that in a novel complex with HJ at 3.1-Å resolution. In the Fpr-HJ complex, two Fpr dimers use several distinct regions to interact with different DNA structural motifs, showing versatility in DNA-binding. Biochemical and solution NMR data support the existence of non-canonical modes of HJ interaction in solution. The binding of Fpr to various DNA motifs are mediated by its flat DNA-binding surface, which is centered on a short loop spanning K61 to I72 and flanked by longer α-helices at the outer edges, and basic side grooves near the dimer interface. Replacing the Fpr loop K61~I72 with a longer loop from Thermus thermophilus RuvC (E71~A87) endows Fpr with an enhanced selectivity toward HJ cleavage but with a target sequence preference distinct from that of RuvC, highlighting a unique role of this loop region in Fpr-HJ interaction. Our work helps explain the broad substrate selectivity of Fpr and suggests a possible mode of its association with poxvirus hairpin telomeres.

Transcription challenges the integrity of replicating chromosomes by generating topological stress and conflicts with forks 1,2 . The DNA topoisomerases Top1 and Top2 and the HMGB family protein Hmo1 assist DNA replication and transcription 3-6 . Here we describe the topological architecture of genes in Saccharomyces cerevisiae during the G1 and S phases of the cell cycle. We found under-wound DNA at gene boundaries and over-wound DNA within coding regions. This arrangement does not depend on Pol II or S phase. Top2 and Hmo1 preserve negative supercoil at gene boundaries, while Top1 acts at coding regions. Transcription generates RNA-DNA hybrids within coding regions, independently of fork orientation. During S phase, Hmo1 protects under-wound DNA from Top2, while Top2 confines Pol II and Top1 at coding units, counteracting transcription leakage and aberrant hybrids at gene boundaries. Negative supercoil at gene boundaries prevents supercoil diffusion and nucleosome repositioning at coding regions. DNA looping occurs at Top2 clusters. We propose that Hmo1 locks gene boundaries in a cruciform conformation and, with Top2, modulates the architecture of genes that retain the memory of the topological arrangements even when transcription is repressed.

Extracellular DNA (eDNA) is a critical component of the extracellular matrix of bacterial biofilms that protects the resident bacteria from environmental hazards, which includes imparting significantly greater resistance to antibiotics and host immune effectors. eDNA is organized into a lattice-like structure, stabilized by the DNABII family of proteins, known to have high affinity and specificity for Holliday junctions (HJs). Accordingly, we demonstrated that the branched eDNA structures present within the biofilms formed by NTHI in the middle ear of the chinchilla in an experimental otitis media model, and in sputum samples recovered from cystic fibrosis patients that contain multiple mixed bacterial species, possess an HJ-like configuration. Next, we showed that the prototypic Escherichia coli HJ-specific DNA-binding protein RuvA could be functionally exchanged for DNABII proteins in the stabilization of biofilms formed by 3 diverse human pathogens, uropathogenic E. coli , nontypeable Haemophilus influenzae, and Staphylococcus epidermidis Importantly, while replacement of DNABII proteins within the NTHI biofilm matrix with RuvA was shown to retain similar mechanical properties when compared to the control NTHI biofilm structure, we also demonstrated that biofilm eDNA matrices stabilized by RuvA could be subsequently undermined upon addition of the HJ resolvase complex, RuvABC, which resulted in significant biofilm disruption. Collectively, our data suggested that nature has recapitulated a functional equivalent of the HJ recombination intermediate to maintain the structural integrity of bacterial biofilms., Competing Interests: The authors declare no competing interest.

Homologous recombination (HR) is a complex biological process and is central to meiosis and for repair of DNA double-strand breaks. Although the HR process has been the subject of intensive study for more than three decades, the complex protein-protein and protein-DNA interactions during HR present a significant challenge for determining the molecular mechanism(s) of the process. This knowledge gap is largely because of the dynamic interactions between HR proteins and DNA which is difficult to capture by routine biochemical or structural biology methods. In recent years, single-molecule fluorescence microscopy has been a popular method in the field of HR to visualize these complex and dynamic interactions at high spatiotemporal resolution, revealing mechanistic insights of the process. In this review, we describe recent efforts that employ single-molecule fluorescence microscopy to investigate protein-protein and protein-DNA interactions operating on three key DNA-substrates: single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), and four-way DNA called Holliday junction (HJ). We also outline the technological advances and several key insights revealed by these studies in terms of protein assembly on these DNA substrates and highlight the foreseeable promise of single-molecule fluorescence microscopy in advancing our understanding of homologous recombination.

Long Interspersed Elements (LINEs), also known as non-LTR retrotransposons, encode a multifunctional protein that reverse transcribes its mRNA into DNA at the site of insertion by target primed reverse transcription. The second half of the integration reaction remains very poorly understood. Second-strand DNA cleavage and second-strand DNA synthesis were investigated in vitro using purified components from a site-specific restriction-like endonuclease (RLE) bearing LINE. DNA structure was shown to be a critical component of second-strand DNA cleavage. A hitherto unknown and unexplored integration intermediate, an open '4-way' DNA junction, was recognized by the element protein and cleaved in a Holliday junction resolvase-like reaction. Cleavage of the 4-way junction resulted in a natural primer-template pairing used for second-strand DNA synthesis. A new model for RLE LINE integration is presented., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Bulk methods measure the ensemble behavior of molecules, in which individual reaction rates of the underlying steps are averaged throughout the population. Single-molecule Förster resonance energy transfer (smFRET) provides a recording of the conformational changes taking place by individual molecules in real-time. Therefore, smFRET is powerful in measuring structural changes in the enzyme or substrate during binding and catalysis. This work presents a protocol for single-molecule imaging of the interaction of a four-way Holliday junction (HJ) and gap endonuclease I (GEN1), a cytosolic homologous recombination enzyme. Also presented are single-color and two-color alternating excitation (ALEX) smFRET experimental protocols to follow the resolution of the HJ by GEN1 in real-time. The kinetics of GEN1 dimerization are determined at the HJ, which has been suggested to play a key role in the resolution of the HJ and has remained elusive until now. The techniques described here can be widely applied to obtain valuable mechanistic insights of many enzyme-DNA systems.

Holliday junction (HJ) resolution by resolving enzymes is essential for chromosome segregation and recombination-mediated DNA repair. HJs undergo two types of structural dynamics that determine the outcome of recombination: conformer exchange between two isoforms and branch migration. However, it is unknown how the preferred branch point and conformer are achieved between enzyme binding and HJ resolution given the extensive binding interactions seen in static crystal structures. Single-molecule fluorescence resonance energy transfer analysis of resolving enzymes from bacteriophages (T7 endonuclease I), bacteria (RuvC), fungi (GEN1) and humans (hMus81-Eme1) showed that both types of HJ dynamics still occur after enzyme binding. These dimeric enzymes use their multivalent interactions to achieve this, going through a partially dissociated intermediate in which the HJ undergoes nearly unencumbered dynamics. This evolutionarily conserved property of HJ resolving enzymes provides previously unappreciated insight on how junction resolution, conformer exchange and branch migration may be coordinated.

G-Quadruplexes formed in the 3' telomere overhang (∼200 nucleotides) have been shown to regulate biological functions of human telomeres. The mechanism governing the population pattern of multiple telomeric G-quadruplexes is yet to be elucidated inside the telomeric overhang in a time window shorter than thermodynamic equilibrium. Using a single-molecule force ramping assay, we quantified G-quadruplex populations in telomere overhangs over a full physiological range of 99-291 nucleotides. We found that G-quadruplexes randomly form in these overhangs within seconds, which leads to a population governed by a kinetic, rather than a thermodynamic, folding pattern. The kinetic folding gives rise to vacant G-tracts between G-quadruplexes. By targeting these vacant G-tracts using complementary DNA fragments, we demonstrated that binding to the telomeric G-quadruplexes becomes more efficient and specific for telomestatin derivatives.

GEN1 is a member of the FEN/EXO family of structure-selective nucleases that cleave 1 nt 3' to a variety of branchpoints. For each, the H2TH motif binds a monovalent ion and plays an important role in binding one helical arm of the substrates. We investigate here the importance of this metal ion on substrate specificity and GEN1 structure. In the presence of K+ ions the substrate specificity is wider than in Na+, yet four-way junctions remain the preferred substrate. In a combination of K+ and Mg2+ second strand cleavage is accelerated 17-fold, ensuring bilateral cleavage of the junction. We have solved crystal structures of Chaetomium thermophilum GEN1 with Cs+, K+ and Na+ bound. With bound Cs+ the loop of the H2TH motif extends toward the active site so that D199 coordinates a Mg2+, buttressed by an interaction of the adjacent Y200. With the lighter ions bound the H2TH loop changes conformation and retracts away from the active site. We hypothesize this conformational change might play a role in second strand cleavage acceleration.

PINA is a novel ATPase and DNA helicase highly conserved in Archaea, the third domain of life. The PINA from Sulfolobus islandicus (SisPINA) forms a hexameric ring in crystal and solution. The protein is able to promote Holliday junction (HJ) migration and physically and functionally interacts with Hjc, the HJ specific endonuclease. Here, we show that SisPINA has direct physical interaction with Hjm (Hel308a), a helicase presumably targeting replication forks. In vitro biochemical analysis revealed that Hjm, Hjc, and SisPINA are able to coordinate HJ migration and cleavage in a concerted way. Deletion of the carboxyl 13 amino acid residues impaired the interaction between SisPINA and Hjm. Crystal structure analysis showed that the carboxyl 70 amino acid residues fold into a type II KH domain which, in other proteins, functions in binding RNA or ssDNA. The KH domain not only mediates the interactions of PINA with Hjm and Hjc but also regulates the hexameric assembly of PINA. Our results collectively suggest that SisPINA, Hjm and Hjc work together to function in replication fork regression, HJ formation and HJ cleavage.

Interactions between DNA and motor proteins regulate nearly all biological functions of DNA such as gene expression, DNA replication and repair, and transcription. During the late stages of homologous recombination (HR), the Escherichia coli recombination machinery, RuvABC, resolves the four-way DNA motifs called Holliday junctions (HJs) that are formed during exchange of nucleotide sequences between two homologous duplex DNA. Although the formation of the RuvA-HJ complex is known to be the first critical step in the RuvABC pathway, the mechanism for the binding interaction between RuvA and HJ has remained elusive. Here, using single-molecule fluorescence resonance energy transfer (smFRET) and ensemble analyses, we show that RuvA stably binds to the HJ, halting its conformational dynamics. Our FRET experiments in different ionic environments created by Mg 2+ and Na + ions suggest that RuvA binds to the HJ via electrostatic interaction. Further, while recent studies have indicated that the HR process can be modulated for therapeutic applications by selective targeting of the HJ by chemotherapeutic drugs, we investigated the effect of drug-modified HJ on binding. Using cisplatin as a proof-of-concept drug, we show that RuvA binds to the cisplatin-modified HJ as efficiently as to the unmodified HJ, demonstrating that RuvA accommodates for the cisplatin-introduced charges and/or topological changes on the HJ.

Replication fork reversal is one of the major pathways for reactivating stalled DNA replication. Many enzymes with replication fork reversal activity have DNA-unwinding activity as well, but none of the fork reversal enzymes in the SWI/SNF family shows a separate DNA-unwinding activity, raising the question of how they initiate the remodeling process. Here, we found ATP binding to Rad5 induces the unwinding of the leading arm of the replication fork and proximally positions the leading and lagging arms. This facilitates the spontaneous remodeling of the replication fork into a four-way junction. Once the four-way junction is formed, Rad5 migrates the four-way junction at a speed of 7.1 ± 0.14 nt/s. The 3' end anchoring of the leading arm by Rad5's HIRAN domain is critical for both branch migration and the recovery of the three-way junction, but not for the structural transition to the four-way junction., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

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.

Archaeal DNA recombination mechanism and the related proteins are similar to those in eukaryotes. However, no functional homolog of eukaryotic MutSγ, which recognizes Holliday junction to promote homologous recombination, has been identified in archaea. Hence, the whole molecular mechanism of archaeal homologous recombination has not yet been revealed. In this study, to identify the archaeal functional homolog of MutSγ, we focused on a functionally uncharacterized MutS homolog, MutS5, from a hyperthermophilic archaeon Pyrococcus horikoshii (phMutS5). Archaeal MutS5 has a Walker ATPase motif-containing amino acid sequence that shows similarity to the ATPase domain of MutSγ. It is known that the ATPase domain of MutS homologs is also a dimerization domain. Chemical cross-linking revealed that purified phMutS5 has an ability to dimerize in solution. phMutS5 bound to Holliday junction with a higher affinity than to other branched and linear DNAs, which resembles the DNA-binding specificities of MutSγ and bacterial MutS2, a Holliday junction-resolving MutS homolog. However, phMutS5 has no nuclease activity against branched DNA unlike MutS2. The ATPase activity of phMutS5 was significantly stimulated by the presence of Holliday junction similarly to MutSγ. Furthermore, site-directed mutagenesis revealed that the ATPase activity is dependent on the Walker ATPase motif of the protein. These results suggest that archaeal MutS5 should stabilize the Holliday junction and play a role in homologous recombination, which is analogous to the function of eukaryotic MutSγ., (© 2017 Federation of European Biochemical Societies.)

Elucidation of the function of synaptonemal complex (SC) in Saccharomyces cerevisiae has mainly focused on in vivo analysis of recombination-defective meiotic mutants. Consequently, significant gaps remain in the mechanistic understanding of the activities of various SC proteins and the functional relationships among them. S. cerevisiae Hop1 and Red1 are essential structural components of the SC axial/lateral elements. Previous studies have demonstrated that Hop1 is a structure-selective DNA-binding protein exhibiting high affinity for the Holliday junction and promoting DNA bridging, condensation, and pairing between double-stranded DNA molecules. However, the exact mode of action of Red1 remains unclear, although it is known to interact with Hop1 and to suppress the spore viability defects of hop1 mutant alleles. Here, we report the purification and functional characterization of the full-length Red1 protein. Our results revealed that Red1 forms a stable complex with Hop1 in vitro and provided quantitative insights into their physical interactions. Mechanistically, Red1 preferentially associated with the Holliday junction and 3-way junction rather than with single- or double-stranded DNA with overhangs. Although Hop1 and Red1 exhibited similar binding affinities toward several DNA substrates, the two proteins displayed some significant differences. Notably, Red1, by itself, lacked DNA-pairing ability; however, it potentiated Hop1-promoted intermolecular pairing between double-stranded DNA molecules. Moreover, Red1 exhibited nonhomologous DNA end-joining activity, thus revealing an unexpected role for Red1 in recombination-based DNA repair. Collectively, this study presents the first direct insights into Red1's mode of action and into the mechanism underlying its role in chromosome synapsis and recombination., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)