Wuho/WDR4 deficiency inhibits cell proliferation and induces apoptosis via DNA damage in mouse embryonic fibroblasts.

Wuho known as WDR4 encodes a highly conserved WD40-repeat protein, which has known homologues of WDR4 in human and mouse. Wuho-FEN1 interaction may have a critical role in the growth and development, and in the maintenance of genome stability. However, how Wuho gene deletion contributes to cell growth inhibition and apoptosis is still unknown. We utilized CAGGCre-ER transgenic mice have a tamoxifen-inducible cre-mediated recombination cassette to prepare primary mouse embryonic fibroblasts (MEFs) with Wuho deficiency. We have demonstrated that Wuho deficiency would induces γH2AX protein level elevation, heterochromatin relaxation and DNA damage down-stream sequences, including p53 activation, caspase-mediated apoptotic pathway, and p21-mediated G2/M cell cycle arrest.

Synthesis of Hemicatenanes for the Study of Type IA Topoisomerases.

Hemicatenane is a structure that forms when two DNA duplexes are physically linked through a single-stranded crossover. It is proposed to be an intermediate resulting from double Holliday junction (dHJ) dissolution, repair of replication stalled forks and late stage replication. Our previous study has shown that hemicatenane can be synthesized and dissolved in vitro by hyperthermophilic type IA topoisomerases. Here we present the protocol of hemicatenane synthesis and its structure detection by 2D agarose gel electrophoresis. The generated product can be used as a substrate to study the biochemical mechanism of hemicatenane processing reactions.

Ubiquitination of tumor suppressor PML regulates prometastatic and immunosuppressive tumor microenvironment.

The tumor microenvironment plays an important role in tumor growth and metastasis. However, the mechanism by which tumor cells regulate the cell and non-cell constituents of surrounding stroma remains incompletely understood. Promyelocytic leukemia (PML) is a pleiotropic tumor suppressor, but its role in tumor microenvironment regulation is poorly characterized. PML is frequently downregulated in many cancer types, including lung cancer. Here, we identify a PML ubiquitination pathway that is mediated by WD repeat 4-containing cullin-RING ubiquitin ligase 4 (CRL4WDR4). Clinically, this PML degradation pathway is hyperactivated in lung cancer and correlates with poor prognosis. The WDR4/PML axis induces a set of cell-surface or secreted factors, including CD73, urokinase-type plasminogen activator receptor (uPAR), and serum amyloid A2 (SAA2), which elicit paracrine effects to stimulate migration, invasion, and metastasis in multiple lung cancer models. In xenograft and genetically engineered mouse models, the WDR4/PML axis elevates intratumoral Tregs and M2-like macrophages and reduces CD8+ T cells to promote lung tumor growth. These immunosuppressive effects were all reversed by CD73 blockade. Our study identifies WDR4 as an oncoprotein that negatively regulates PML via ubiquitination to promote lung cancer progression by fostering an immunosuppressive and prometastatic tumor microenvironment, suggesting the potential of immune-modulatory approaches for treating lung cancer with aberrant PML degradation.

Single-molecule Förster resonance energy transfer (FRET) analysis discloses the dynamics of the DNA-topoisomerase II (Top2) interaction in the presence of TOP2-targeting agents.

Topoisomerases play crucial roles in DNA replication, transcription, and recombination. For instance, topoisomerase II (Top2) is critically important for resolving DNA tangles during cell division, and as such, it is a broad anticancer drug target. Top2 regulates DNA topology by transiently breaking one double-stranded DNA molecule (cleavage), allowing a second double strand to pass through the opened DNA gate (opening), and then closing the gate by rejoining the broken ends. Drugs that modulate Top2 catalysis may therefore affect enzymatic activity at several different steps. Previous studies have focused on examining DNA cleavage and ligation; however, the dynamic opening and closing of the DNA gate has been less explored. Here, we used the single-molecule Förster resonance energy transfer (smFRET) method to observe the open and closed state of the DNA gate and to measure dwell times in each state. Our results show that Top2 binds and bends DNA to increase the energy transfer efficiency (EFRET), and ATP treatment further induces the fluctuation of EFRET, representing the gate opening and closing. Additionally, our results demonstrate that both types of Top2-targeting anticancer drugs, the catalytic inhibitor dexrazoxane (ICRF187) and mechanistic poison teniposide (VM26), can interfere with DNA gate dynamics and shorten the dwell time in the closed state. Moreover, Top2 bound to the nonhydrolyzable ATP analog 5'-adenylyl-ß,γ-imidodiphosphate exhibits altered DNA gate dynamics, but the DNA gate appears to open and close even after N-gate closure. In summary, we have utilized single-molecule detection to unravel Top2 DNA gate dynamics and reveal previously unknown effects of Top2 drugs on these dynamics.

Electron tomographic analysis reveals ultrastructural features of mitochondrial cristae architecture which reflect energetic state and aging.

Within mitochondria, the ability to produce energy relies upon the architectural hallmarks of double membranes and cristae invaginations. Herein, we describe novel features of mitochondrial cristae structure, which correspond to the energetic state of the organelle. In concordance with high-energy demand, mitochondria of Drosophila indirect flight muscle exhibited extensive intra-mitochondrial membrane switches between densely packed lamellar cristae that resulted in a spiral-like cristae network and allowed for bidirectional matrix confluency. This highly interconnected architecture is expected to allow rapid equilibration of membrane potential and biomolecules across integrated regions. In addition, mutant flies with mtDNA replication defect and an accelerated aging phenotype accumulated mitochondria that contained subsections of swirling membrane alongside normal cristae. The swirling membrane had impaired energy production capacity as measured by protein composition and function. Furthermore, mitochondrial fusion and fission dynamics were affected in the prematurely aged flies. Interestingly, the normal cristae that remained in the mitochondria with swirling membranes maintained acceptable function that camouflaged them from quality control elimination. Overall, structural features of mitochondrial cristae were described in three-dimension from serial section electron tomographic analysis which reflect energetic state and mtDNA-mediated aging.

Ribosomal Protein S3 Negatively Regulates Unwinding Activity of RecQ-like Helicase 4 through Their Physical Interaction.

Human RecQ-like helicase 4 (RECQL4) plays crucial roles in replication initiation and DNA repair; however, the contextual regulation of its unwinding activity is not fully described. Mutations in RECQL4 have been linked to three diseases including Rothmund-Thomson syndrome, which is characterized by osteoskeletal deformities, photosensitivity, and increased osteosarcoma susceptibility. Understanding regulation of RECQL4 helicase activity by interaction partners will allow deciphering its role as an enzyme and a signaling cofactor in different cellular contexts. We became interested in studying the interaction of RECQL4 with ribosomal protein S3 (RPS3) because previous studies have shown that RPS3 activity is sometimes associated with phenotypes mimicking those of mutated RECQL4. RPS3 is a small ribosomal protein that also has extraribosomal functions, including apurnic-apyrimidinic endonuclease-like activity suggested to be important during DNA repair. Here, we report a functional and physical interaction between RPS3 and RECQL4 and show that this interaction may be enhanced during cellular stress. We show that RPS3 inhibits ATPase, DNA binding, and helicase activities of RECQL4 through their direct interaction. Further domain analysis shows that N-terminal 1-320 amino acids of RECQL4 directly interact with the C-terminal 94-244 amino acids of RPS3 (C-RPS3). Biochemical analysis of C-RPS3 revealed that it comprises a standalone apurnic-apyrimidinic endonuclease-like domain. We used U2OS cells to show that oxidative stress and UV exposure could enhance the interaction between nuclear RPS3 and RECQL4. Regulation of RECQL4 biochemical activities by RPS3 along with nuclear interaction during UV and oxidative stress may serve to modulate active DNA repair.

Single-molecule analysis of ϕC31 integrase-mediated site-specific recombination by tethered particle motion.

Serine and tyrosine site-specific recombinases (SRs and YRs, respectively) provide templates for understanding the chemical mechanisms and conformational dynamics of strand cleavage/exchange between DNA partners. Current evidence suggests a rather intriguing mechanism for serine recombination, in which one half of the cleaved synaptic complex undergoes a 180° rotation relative to the other. The 'small' and 'large' SRs contain a compact amino-terminal catalytic domain, but differ conspicuously in their carboxyl-terminal domains. So far, only one serine recombinase has been analyzed using single substrate molecules. We now utilized single-molecule tethered particle motion (TPM) to follow step-by-step recombination catalyzed by a large SR, phage ϕC31 integrase. The integrase promotes unidirectional DNA exchange between attB and attP sites to integrate the phage genome into the host chromosome. The recombination directionality factor (RDF; ϕC31 gp3) activates the excision reaction (attL × attR). From integrase-induced changes in TPM in the presence or absence of gp3, we delineated the individual steps of recombination and their kinetic features. The gp3 protein appears to regulate recombination directionality by selectively promoting or excluding active conformations of the synapse formed by specific att site partners. Our results support a 'gated rotation' of the synaptic complex between DNA cleavage and joining.

DNA and RNA topoisomerase activities of Top3ß are promoted by mediator protein Tudor domain-containing protein 3.

Topoisomerase 3ß (Top3ß) can associate with the mediator protein Tudor domain-containing protein 3 (TDRD3) to participate in two gene expression processes of transcription and translation. Despite the apparent importance of TDRD3 in binding with Top3ß and directing it to cellular compartments critical for gene expression, the biochemical mechanism of how TDRD3 can affect the functions of Top3ß is not known. We report here sensitive biochemical assays for the activities of Top3ß on DNA and RNA substrates in resolving topological entanglements and for the analysis of TDRD3 functions. TDRD3 stimulates the relaxation activity of Top3ß on hypernegatively supercoiled DNA and changes the reaction from a distributive to a processive mode. Both supercoil retention assays and binding measurement by fluorescence anisotropy reveal a heretofore unknown preference for binding single-stranded nucleic acids over duplex. Whereas TDRD3 has a structure-specific binding preference, it does not discriminate between DNA and RNA. This unique property for binding with nucleic acids can have an important function in serving as a hub to form nucleoprotein complexes on DNA and RNA. To gain insight into the roles of Top3ß on RNA metabolism, we designed an assay by annealing two single-stranded RNA circles with complementary sequences. Top3ß is capable of converting two such single-stranded RNA circles into a double-stranded RNA circle, and this strand-annealing activity is enhanced by TDRD3. These results demonstrate that TDRD3 can enhance the biochemical activities of Top3ß on both DNA and RNA substrates, in addition to its function of targeting Top3ß to critical sites in subcellular compartments.

RNA topoisomerase is prevalent in all domains of life and associates with polyribosomes in animals.

DNA Topoisomerases are essential to resolve topological problems during DNA metabolism in all species. However, the prevalence and function of RNA topoisomerases remain uncertain. Here, we show that RNA topoisomerase activity is prevalent in Type IA topoisomerases from bacteria, archaea, and eukarya. Moreover, this activity always requires the conserved Type IA core domains and the same catalytic residue used in DNA topoisomerase reaction; however, it does not absolutely require the non-conserved carboxyl-terminal domain (CTD), which is necessary for relaxation reactions of supercoiled DNA. The RNA topoisomerase activity of human Top3ß differs from that of Escherichia coli topoisomerase I in that the former but not the latter requires the CTD, indicating that topoisomerases have developed distinct mechanisms during evolution to catalyze RNA topoisomerase reactions. Notably, Top3ß proteins from several animals associate with polyribosomes, which are units of mRNA translation, whereas the Top3 homologs from E. coli and yeast lack the association. The Top3ß-polyribosome association requires TDRD3, which directly interacts with Top3ß and is present in animals but not bacteria or yeast. We propose that RNA topoisomerases arose in the early RNA world, and that they are retained through all domains of DNA-based life, where they mediate mRNA translation as part of polyribosomes in animals.

Drosophila mitochondrial topoisomerase III alpha affects the aging process via maintenance of mitochondrial function and genome integrity.

BACKGROUND: Mitochondria play important roles in providing metabolic energy and key metabolites for synthesis of cellular building blocks. Mitochondria have additional functions in other cellular processes, including programmed cell death and aging. A previous study revealed Drosophila mitochondrial topoisomerase III alpha (Top3α) contributes to the maintenance of the mitochondrial genome and male germ-line stem cells. However, the involvement of mitochondrial Top3α in the mitochondrion-mediated aging process remains unclear. In this study, the M1L flies, in which Top3α protein lacks the mitochondrial import sequence and is thus present in cell nuclei but not in mitochondria, is used as a model system to examine the role of mitochondrial Top3α in the aging of fruit flies. RESULTS: Here, we reported that M1L flies exhibit mitochondrial defects which affect the aging process. First, we observed that M1L flies have a shorter life span, which was correlated with a significant reduction in the mitochondrial DNA copy number, the mitochondrial membrane potential, and ATP content compared with those of both wildtype and transgene-rescued flies of the same age. Second, we performed a mobility assay and electron microscopic analysis to demonstrate that the locomotion defect and mitophagy of M1L flies were enhanced with age, as compared with the controls. Finally, we showed that the correlation between the mtDNA deletion level and aging in M1L flies resembles what was reported in mammalian systems. CONCLUSIONS: The results reported here demonstrate that mitochondrial Top3α ablation results in mitochondrial genome instability and its dysfunction, thereby accelerating the aging process.

The C-terminal 20 Amino Acids of Drosophila Topoisomerase 2 Are Required for Binding to a BRCA1 C Terminus (BRCT) Domain-containing Protein, Mus101, and Fidelity of DNA Segregation.

Eukaryotic topoisomerase 2 (Top2) and one of its interacting partners, topoisomerase IIß binding protein 1 (TopBP1) are two proteins performing essential cellular functions. We mapped the interacting domains of these two proteins using co-immunoprecipitation and pulldown experiments with truncated or mutant Drosophila Top2 with various Ser-to-Ala substitutions. We discovered that the last 20 amino acids of Top2 represent the key region for binding with Mus101 (the Drosophila homolog of TopBP1) and that phosphorylation of Ser-1428 and Ser-1443 is important for Top2 to interact with the N terminus of Mus101, which contains the BRCT1/2 domains. The interaction between Mus101 and the Top2 C-terminal regulatory domain is phosphorylation-dependent because treatment with phosphatase abolishes their association in pulldown assays. The binding affinity of N-terminal Mus101 with a synthetic phosphorylated peptide spanning the last 25 amino acids of Top2 (with Ser(P)-1428 and Ser(P)-1443) was determined by surface plasmon resonance with a Kd of 0.57 µm In an in vitro decatenation assay, Mus101 can specifically reduce the decatenation activity of Top2, and dephosphorylation of Top2 attenuates this response. Next, we endeavored to establish a cellular system for testing the biological function of Top2-Mus101 interaction. Top2-silenced S2 cells rescued by Top2Δ20, Top2 with 20 amino acids truncated from the C terminus, developed abnormally high chromosome numbers, which implies that Top2-Mus101 interaction is important for maintaining the fidelity of chromosome segregation during mitosis.

Wuho Is a New Member in Maintaining Genome Stability through its Interaction with Flap Endonuclease 1.

Replication forks are vulnerable to wayward nuclease activities. We report here our discovery of a new member in guarding genome stability at replication forks. We previously isolated a Drosophila mutation, wuho (wh, no progeny), characterized by a severe fertility defect and affecting expression of a protein (WH) in a family of conserved proteins with multiple WD40 repeats. Knockdown of WH by siRNA in Drosophila, mouse, and human cultured cells results in DNA damage with strand breaks and apoptosis through ATM/Chk2/p53 signaling pathway. Mice with mWh knockout are early embryonic lethal and display DNA damage. We identify that the flap endonuclease 1 (FEN1) is one of the interacting proteins. Fluorescence microscopy showed the localization of WH at the site of nascent DNA synthesis along with other replication proteins, including FEN1 and PCNA. We show that WH is able to modulate FEN1's endonucleolytic activities depending on the substrate DNA structure. The stimulatory or inhibitory effects of WH on FEN1's flap versus gap endonuclease activities are consistent with the proposed WH's functions in protecting the integrity of replication fork. These results suggest that wh is a new member of the guardians of genome stability because it regulates FEN1's potential DNA cleavage threat near the site of replication.

Toward discovering new anti-cancer agents targeting topoisomerase IIα: a facile screening strategy adaptable to high throughput platform.

Topoisomerases are a family of vital enzymes capable of resolving topological problems in DNA during various genetic processes. Topoisomerase poisons, blocking reunion of cleaved DNA strands and stabilizing enzyme-mediated DNA cleavage complex, are clinically important antineoplastic and anti-microbial agents. However, the rapid rise of drug resistance that impedes the therapeutic efficacy of these life-saving drugs makes the discovering of new lead compounds ever more urgent. We report here a facile high throughput screening system for agents targeting human topoisomerase IIα (Top2α). The assay is based on the measurement of fluorescence anisotropy of a 29 bp fluorophore-labeled oligonucleotide duplex. Since drug-stabilized Top2α-bound DNA has a higher anisotropy compared with free DNA, this assay can work if one can use a dissociating agent to specifically disrupt the enzyme/DNA binary complexes but not the drug-stabilized ternary complexes. Here we demonstrate that NaClO4, a chaotropic agent, serves a critical role in our screening method to differentiate the drug-stabilized enzyme/DNA complexes from those that are not. With this strategy we screened a chemical library of 100,000 compounds and obtained 54 positive hits. We characterized three of them on this list and demonstrated their effects on the Top2α-mediated reactions. Our results suggest that this new screening strategy can be useful in discovering additional candidates of anti-cancer agents.

Top3α is required during the convergent migration step of double Holliday junction dissolution.

Although Blm and Top3α are known to form a minimal dissolvasome that can uniquely undo a double Holliday junction structure, the details of the mechanism remain unknown. It was originally suggested that Blm acts first to create a hemicatenane structure from branch migration of the junctions, followed by Top3α performing strand passage to decatenate the interlocking single strands. Recent evidence suggests that Top3α may also be important for assisting in the migration of the junctions. Using a mismatch-dHJ substrate (MM-DHJS) and eukaryotic Top1 (in place of Top3α), we show that the presence of a topoisomerase is required for Blm to substantially migrate a topologically constrained Holliday junction. When investigated by electron microscopy, these migrated structures did not resemble a hemicatenane. However, when Blm is together with Top3α, the dissolution reaction is processive with no pausing at a partially migrated structure. Potential mechanisms are discussed.

Synthesis and dissolution of hemicatenanes by type IA DNA topoisomerases.

Type IA DNA topoisomerases work with a unique mechanism of strand passage through an enzyme-bridged, ssDNA gate, thus enabling them to carry out diverse reactions in processing structures important for replication, recombination, and repair. Here we report a unique reaction mediated by an archaeal type IA topoisomerase, the synthesis and dissolution of hemicatenanes. We cloned, purified, and characterized an unusual type IA enzyme from a hyperthermophilic archaeum, Nanoarchaeum equitans, which is split into two pieces. The recombinant heterodimeric enzyme has the expected activities in its preference of relaxing negatively supercoiled DNA. Its amino acid sequence and cleavage site sequence analysis suggest that it is topoisomerase III, and therefore we named it "NeqTop3." At high enzyme concentrations, NeqTop3 can generate high-molecular-weight DNA networks. Biochemical and electron microscopic data indicate that the DNA networks are connected through hemicatenane linkages. The hemicatenane formation likely is mediated by the single-strand passage through denatured bubbles in the substrate DNA under high temperature. NeqTop3 at lower concentrations can reverse hemicatenanes. A complex of human topoisomerase 3α, Bloom helicase, and RecQ-mediated genome instability protein 1 and 2 can partially disentangle the hemicatenane network. Both the formation and dissolution of hemicatenanes by type IA topoisomerases demonstrate that these enzymes have an important role in regulating intermediates from replication, recombination, and repair.

New mechanistic and functional insights into DNA topoisomerases.

DNA topoisomerases are nature's tools for resolving the unique problems of DNA entanglement that occur owing to unwinding and rewinding of the DNA helix during replication, transcription, recombination, repair, and chromatin remodeling. These enzymes perform topological transformations by providing a transient DNA break, formed by a covalent adduct with the enzyme, through which strand passage can occur. The active site tyrosine is responsible for initiating two transesterifications to cleave and then religate the DNA backbone. The cleavage reaction intermediate is exploited by cytotoxic agents, which have important applications as antibiotics and anticancer drugs. The reactions mediated by these enzymes can also be regulated by their binding partners; one example is a DNA helicase capable of modulating the directionality of strand passage, enabling important functions like reannealing denatured DNA and resolving recombination intermediates. In this review, we cover recent advances in mechanistic insights into topoisomerases and their various cellular functions.

Induced pH-dependent shift by local surface plasmon resonance in functionalized gold nanorods.

Localized surface plasmon resonance (LSPR) spectroscopy of metallic nanoparticles is a powerful tool for chemical and biological sensing experiments. In this study, we observed LSPR shifts of 11-mercaptoundecanoic acid modified gold nanorods (GNR-MUA) for the pH range of 6.41 to 8.88. We proposed a mechanism involving changes of the dipole moment after protonation/deprotonation carboxylic groups of 11-mercaptoundecanoic acid (MUA) which plays an important role by modulating LSPR around the functionalized GNR. Such a stable and easily prepared GNR-MUA has potential to become one of the most efficient and promising pH nanosensors to study intra- or extra-cellular pH in a wide range of chemical or biological systems.

Improved methods for creating migratable Holliday junction substrates.

Previously, we published a method for creating a novel DNA substrate, the double Holliday junction substrate. This substrate contains two Holliday junctions that are mobile, topologically constrained and separated by a distance comparable with conversion tract lengths. Although useful for studying late stage homologous recombination in vitro, construction of the substrate requires significant effort. In particular, there are three bottlenecks: (i) production of large quantities of single-stranded DNA; (ii) the loss of a significant portion of the DNA following the recombination step; and (iii) the loss of DNA owing to inefficient gel extraction. To address these limitations, we have made the following changes to the protocol: (i) use of a helper plasmid, rather than exogenous helper phage, to produce single-stranded DNA; (ii) use of the unidirectional C31 integrase system in place of the bidirectional Cre recombinase reaction; and (iii) gel extraction by DNA diffusion. Here, we describe the changes made to the materials and methods and characterize the substrates that can be produced, including migratable single Holliday junctions, hemicatenanes and a quadruple Holliday junction substrate.

Restoration of topoisomerase 2 function by complementation of defective monomers in Drosophila.

Type II topoisomerases are essential ATP-dependent homodimeric enzymes required for transcription, replication, and chromosome segregation. These proteins alter DNA topology by generating transient enzyme-linked double-strand breaks for passage of one DNA strand through another. The central role of type II topoisomerases in DNA metabolism has made these enzymes targets for anticancer drugs. Here, we describe a genetic screen that generated novel alleles of Drosophila Topoisomerase 2 (Top2). Fifteen alleles were obtained, resulting from nonsense and missense mutations. Among these, 14 demonstrated recessive lethality, with one displaying temperature-sensitive lethality. Several newly generated missense alleles carry amino acid substitutions in conserved residues within the ATPase, Topoisomerase/Primase, and Winged helix domains, including four that encode proteins with alterations in residues associated with resistance to cancer chemotherapeutics. Animals lacking zygotic Top2 function can survive to pupation and display reduced cell division and altered polytene chromosome structure. Inter se crosses between six strains carrying Top2 missense alleles generated morphologically normal trans-heterozygous adults, which showed delayed development and were female sterile. Complementation occurred between alleles encoding Top2 proteins with amino acid substitutions in the same functional domain and between alleles encoding proteins with substitutions in different functional domains. Two complementing alleles encode proteins with amino acid substitutions associated with drug resistance. These observations suggest that dimerization of mutant Top2 monomers can restore enzymatic function. Our studies establish the first series of Top2 alleles in a multicellular organism. Future analyses of these alleles will enhance our knowledge about the contributions made by type II topoisomerases to development.

Probing conformational changes in human DNA topoisomerase IIα by pulsed alkylation mass spectrometry.

Type II topoisomerases are essential enzymes for solving DNA topological problems by passing one segment of DNA duplex through a transient double-strand break in a second segment. The reaction requires the enzyme to precisely control DNA cleavage and gate opening coupled with ATP hydrolysis. Using pulsed alkylation mass spectrometry, we were able to monitor the solvent accessibilities around 13 cysteines distributed throughout human topoisomerase IIα by measuring the thiol reactivities with monobromobimane. Most of the measured reactivities are in accordance with the predicted ones based on a homology structural model generated from available crystal structures. However, these results reveal new information for both the residues not covered in the structural model and potential differences between the modeled and solution holoenzyme structures. Furthermore, on the basis of the reactivity changes of several cysteines located at the N-gate and DNA gate, we could monitor the movement of topoisomerase II in the presence of cofactors and detect differences in the DNA gate between two closed clamp enzyme conformations locked by either 5'-adenylyl ß,γ-imidodiphosphate or the anticancer drug ICRF-193.