Homology recognition without double-stranded DNA-strand separation in D-loop formation by RecA.

RecA protein and RecA/Rad51 orthologues are required for homologous recombination and DNA repair in all living creatures. RecA/Rad51 catalyzes formation of the D-loop, an obligatory recombination intermediate, through an ATP-dependent reaction consisting of two phases: homology recognition between double-stranded (ds)DNA and single-stranded (ss)DNA to form a hybrid-duplex core of 6-8 base pairs and subsequent hybrid-duplex/D-loop processing. How dsDNA recognizes homologous ssDNA is controversial. The aromatic residue at the tip of the ß-hairpin loop (L2) was shown to stabilize dsDNA-strand separation. We tested a model in which dsDNA strands were separated by the aromatic residue before homology recognition and found that the aromatic residue was not essential to homology recognition, but was required for D-loop processing. Contrary to the model, we found that the double helix was not unwound even a single turn during search for sequence homology, but rather was unwound only after the homologous sequence was recognized. These results suggest that dsDNA recognizes its homologous ssDNA before strand separation. The search for homologous sequence with homologous ssDNA without dsDNA-strand separation does not generate stress within the dsDNA; this would be an advantage for dsDNA to express homology-dependent functions in vivo and also in vitro.

CpG ODN enhances the efficacy of F protein vaccine against respiratory syncytial virus infection in the upper respiratory tract via CD4+ T cells.

Respiratory syncytial virus (RSV) is a leading cause of severe respiratory illness worldwide, particularly in infants and older adults. Vaccines targeting the fusion glycoprotein (F protein) -one of the surface antigens of RSV- are highly effective in preventing RSV-associated severe lower respiratory tract disease. However, the efficacy of these vaccines against upper respiratory tract challenge needs improvement. Here, we aimed to examine the efficacy of F protein vaccines with or without CpG oligodeoxynucleotide (CpG ODN) as an adjuvant in the upper and lower respiratory tracts in mice. F + CpG ODN induced higher levels of F-specific IgG than that induced by F alone; however, levels of neutralizing antibodies did not increase compared to those induced by F alone. F + CpG ODN induced T helper 1 (Th1) cells while F alone induced T helper 2 (Th2) cells. Moreover, F + CpG ODN improved the protection against RSV challenge in the upper respiratory tract compared to F alone, which was largely dependent on CD4+ T cells. Meanwhile, both F + CpG ODN and F alone protected the lower respiratory tract. In conclusion, we demonstrated that induction of F-specific Th1 cells is an effective strategy to prevent RSV challenge in the upper respiratory tract in F protein vaccines. These data support the development of novel F protein vaccines.

Nonfilament-forming RecA dimer catalyzes homologous joint formation.

Homologous recombination is essential to genome maintenance, and also to genome diversification. In virtually all organisms, homologous recombination depends on the RecA/Rad51-family recombinases, which catalyze ATP-dependent formation of homologous joints-critical intermediates in homologous recombination. RecA/Rad51 binds first to single-stranded (ss) DNA at a damaged site to form a spiral nucleoprotein filament, after which double-stranded (ds) DNA interacts with the filament to search for sequence homology and to form consecutive base pairs with ssDNA ('pairing'). How sequence homology is recognized and what exact role filament formation plays remain unknown. We addressed the question of whether filament formation is a prerequisite for homologous joint formation. To this end we constructed a nonpolymerizing (np) head-to-tail-fused RecA dimer (npRecA dimer) and an npRecA monomer. The npRecA dimer bound to ssDNA, but did not form continuous filaments upon binding to DNA; it formed beads-on-string structures exclusively. Although its efficiency was lower, the npRecA dimer catalyzed the formation of D-loops (a type of homologous joint), whereas the npRecA monomer was completely defective. Thus, filament formation contributes to efficiency, but is not essential to sequence-homology recognition and pairing, for which a head-to-tail dimer form of RecA protomer is required and sufficient.

Rad51 and RecA juxtapose dsDNA ends ready for DNA ligase-catalyzed end-joining under recombinase-suppressive conditions.

RecA-family recombinase-catalyzed ATP-dependent homologous joint formation is critical for homologous recombination, in which RecA or Rad51 binds first to single-stranded (ss)DNA and then interacts with double-stranded (ds)DNA. However, when RecA or Rad51 interacts with dsDNA before binding to ssDNA, the homologous joint-forming activity of RecA or Rad51 is quickly suppressed. We found that under these and adenosine diphosphate (ADP)-generating suppressive conditions for the recombinase activity, RecA or Rad51 at similar optimal concentrations enhances the DNA ligase-catalyzed dsDNA end-joining (DNA ligation) about 30- to 40-fold. The DNA ligation enhancement by RecA or Rad51 transforms most of the substrate DNA into multimers within 2-5 min, and for this enhancement, ADP is the common and best cofactor. Adenosine triphosphate (ATP) is effective for RecA, but not for Rad51. Rad51/RecA-enhanced DNA ligation depends on dsDNA-binding, as shown by a mutant, and is independent of physical interactions with the DNA ligase. These observations demonstrate the common and unique activities of RecA and Rad51 to juxtapose dsDNA-ends in preparation for covalent joining by a DNA ligase. This new in vitro function of Rad51 provides a simple explanation for our genetic observation that Rad51 plays a role in the fidelity of the end-joining of a reporter plasmid DNA, by yeast canonical non-homologous end-joining (NHEJ) in vivo.

A critical role of Gas6/Axl signal in allergic airway responses during RSV vaccine-enhanced disease.

Respiratory syncytial virus (RSV) is a common virus that causes lower respiratory infections across a wide range of ages. A licensed RSV vaccine is not available because vaccination with formalin-inactivated RSV (FI-RSV) and the subsequent RSV infection cause not only insufficient induction of neutralizing antibodies but also severe allergic airway responses, termed FI-RSV vaccine-enhanced disease (FI-RSV VED). However, the underlying mechanism has not been identified, although a Th2-biased immune response is known to be a hallmark of this disease. Our previous studies have shown that growth arrest-specific 6 (Gas6)/Axl signaling leads to Th2-biased immune responses during fungus-induced allergic airway inflammation. Here, we show that Gas6/Axl signaling also leads to FI-RSV VED and partially identify the mechanism in mice. Inhibiting Gas6/Axl signaling using Gas6-deficient mice, neutralizing antibodies, and a specific inhibitor of Axl attenuated allergic airway hyperresponsiveness, including airway inflammation, goblet cell hyperplasia, and Th2 cytokine production, in addition to increasing interferon-γ levels and the production of RSV-neutralizing IgG2a in FI-RSV VED. Gas6 was produced in lymph nodes during immunization with FI-RSV. Lymph node cells derived from immunized mice produced high levels of Gas6 and Th2 cytokines, but not IFN-γ, after restimulation with RSV. Finally, we found that dendritic cells stimulated with RSV-glycoprotein (G protein) produced Gas6 and that Axl signaling suppressed DC maturation and the induction of IL-12 production by the toll-like receptor 4 agonist RSV-fusion protein. Taken together, these results indicate that RSV-G protein-induced Gas6/Axl signaling causes allergic airway responses during FI-RSV VED.

Loop L1 governs the DNA-binding specificity and order for RecA-catalyzed reactions in homologous recombination and DNA repair.

In all organisms, RecA-family recombinases catalyze homologous joint formation in homologous genetic recombination, which is essential for genome stability and diversification. In homologous joint formation, ATP-bound RecA/Rad51-recombinases first bind single-stranded DNA at its primary site and then interact with double-stranded DNA at another site. The underlying reason and the regulatory mechanism for this conserved binding order remain unknown. A comparison of the loop L1 structures in a DNA-free RecA crystal that we originally determined and in the reported DNA-bound active RecA crystals suggested that the aspartate at position 161 in loop L1 in DNA-free RecA prevented double-stranded, but not single-stranded, DNA-binding to the primary site. This was confirmed by the effects of the Ala-replacement of Asp-161 (D161A), analyzed directly by gel-mobility shift assays and indirectly by DNA-dependent ATPase activity and SOS repressor cleavage. When RecA/Rad51-recombinases interact with double-stranded DNA before single-stranded DNA, homologous joint-formation is suppressed, likely by forming a dead-end product. We found that the D161A-replacement reduced this suppression, probably by allowing double-stranded DNA to bind preferentially and reversibly to the primary site. Thus, Asp-161 in the flexible loop L1 of wild-type RecA determines the preference for single-stranded DNA-binding to the primary site and regulates the DNA-binding order in RecA-catalyzed recombinase reactions.

Axl receptor blockade protects from invasive pulmonary aspergillosis in mice.

Aspergillus fumigatus is a sporulating fungus found ubiquitously in the environment, which is quickly contained in the immunocompetent host but can cause lethal invasive aspergillosis in the immunocompromised host. We have recently demonstrated that Axl (one member of the Tyro3, Axl, Mertk receptor family) is a key regulator of antiviral immune responses in the lung. In this study, we investigated the role of Axl in antifungal immunity in a model of invasive pulmonary aspergillosis (IPA). In this model, Aspergillus fumigatus conidia were administered into the lungs of neutrophil-depleted mice, and the mice were monitored for survival, lung inflammatory response, and fungal clearance. The lethal effect of IPA was significantly reduced in anti-Axl mAb-treated mice compared with IgG control-treated mice. Targeting Axl significantly inhibited pulmonary inflammation, including the expression of IL-1ß, IL-6, TNF-α, and chitinase-like proteins in whole lung. Further, anti-Axl mAb treatment significantly increased M1 macrophages that highly expressed inducible NO synthase and decreased M2 macrophages that expressed Arginase 1 and were found in inflammatory zone protein (Fizz1). More importantly, anti-Axl mAb treatment significantly increased the number of IFN-γ-producing T cells and NK cells compared with the IgG control group during IPA. Together, our results demonstrate that the Axl mAb treatment is protective during invasive aspergillosis in neutropenic mice. Collectively, these data suggest a potential deleterious role for Axl during primary immune responses directed against A. fumigatus and novel therapeutic strategy for IPA.

Axl receptor blockade ameliorates pulmonary pathology resulting from primary viral infection and viral exacerbation of asthma.

Viruses use Tyro3, Axl, and Mertk (TAM) receptor tyrosine kinases to infect and modulate the immune properties of various cell types, which led us to investigate whether TAM receptor activation affected primary viral infection and viral exacerbation of asthma in experimental models. In these lung-specific models, we observed that Axl was the most abundantly induced TAM receptor protein. During primary respiratory syncytial virus (RSV) infection, anti-Axl mAb treatment significantly increased the number of IFN-γ-producing T cells and NK cells and significantly suppressed RSV replication and whole lung levels of IL-4 and IL-13. Intrapulmonary H1N1 infection induced lethal pulmonary inflammation, but anti-Axl mAb treatment of infected mice significantly increased the number of IFN-ß-producing macrophages and dendritic cells and significantly suppressed neutrophil infiltration. Consequently, the lethal effect of H1N1 infection in this model was significantly reduced in the mAb-treated group compared with the IgG control-treated group. Targeting Axl also inhibited airway hyperresponsiveness, IL-4 and IL-13 production, and goblet cell metaplasia in an Aspergillus fumigatus-induced asthma model. Finally, infection of mice with RSV during fungal asthma significantly exacerbated airway inflammation, goblet cell metaplasia, and airway remodeling, but all of these features in this viral exacerbation model were ameliorated by anti-Axl mAb treatment. Taken together, these results demonstrate that Axl modulates the pulmonary immune response during viral and/or allergic pathology, and they also suggest that targeting this TAM receptor might provide a novel therapeutic approach in these infectious diseases.

Functional analyses of the C-terminal half of the Saccharomyces cerevisiae Rad52 protein.

The Saccharomyces cerevisiae Rad52 protein is essential for efficient homologous recombination (HR). An important role of Rad52 in HR is the loading of Rad51 onto replication protein A-coated single-stranded DNA (ssDNA), which is referred to as the recombination mediator activity. In vitro, Rad52 displays additional activities, including self-association, DNA binding and ssDNA annealing. Although Rad52 has been a subject of extensive genetic, biochemical and structural studies, the mechanisms by which these activities are coordinated in the various roles of Rad52 in HR remain largely unknown. In the present study, we found that an isolated C-terminal half of Rad52 disrupted the Rad51 oligomer and formed a heterodimeric complex with Rad51. The Rad52 fragment inhibited the binding of Rad51 to double-stranded DNA, but not to ssDNA. The phenylalanine-349 and tyrosine-409 residues present in the C-terminal half of Rad52 were critical for the interaction with Rad51, the disruption of Rad51 oligomers, the mediator activity of the full-length protein and for DNA repair in vivo in the presence of methyl methanesulfonate. Our studies suggested that phenylalanine-349 and tyrosine-409 are key residues in the C-terminal half of Rad52 and probably play an important role in the mediator activity.

Putative antirecombinase Srs2 DNA helicase promotes noncrossover homologous recombination avoiding loss of heterozygosity.

DNA damage alone or DNA replication fork arrest at damaged sites may induce DNA double-strand breaks and initiate homologous recombination. This event can result in a crossover with a homologous chromosome, causing loss of heterozygosity along the chromosome. It is known that Srs2 acts as an antirecombinase at the replication fork: it is recruited by the SUMO (a small ubiquitin-related modifier)-conjugated DNA-polymerase sliding clamp (PCNA) and interferes with Rad51/Rad52-mediated homologous recombination. Here, we report that Srs2 promotes another type of homologous recombination that produces noncrossover products only, in collaboration with PCNA and Rad51. Srs2 proteins lacking the Rad51-binding domain, PCNA-SUMO-binding motifs, or ATP hydrolysis-dependent DNA helicase activity reduce this noncrossover recombination. However, the removal of either the Rad51-binding domain or the PCNA-binding motif strongly increases crossovers. Srs2 gene mutations are epistatic to mutations in the PCNA modification-related genes encoding PCNA, Siz1 (a SUMO ligase) and Rad6 (a ubiquitin-conjugating protein). Knocking out RAD51 blocked this recombination but enhanced nonhomologous end-joining. We hypothesize that, during DNA double-strand break repair, Srs2 mediates collaboration between the Rad51 nucleofilament and PCNA-SUMO and directs the heteroduplex intermediate to DNA synthesis in a moving bubble. This Rad51/Rad52/Srs2/PCNA-mediated noncrossover pathway avoids both interchromosomal crossover and imprecise end-joining, two potential paths leading to loss of heterozygosity, and contributes to genome maintenance and human health.

Din7 and Mhr1 expression levels regulate double-strand-break-induced replication and recombination of mtDNA at ori5 in yeast.

The Ntg1 and Mhr1 proteins initiate rolling-circle mitochondrial (mt) DNA replication to achieve homoplasmy, and they also induce homologous recombination to maintain mitochondrial genome integrity. Although replication and recombination profoundly influence mitochondrial inheritance, the regulatory mechanisms that determine the choice between these pathways remain unknown. In Saccharomyces cerevisiae, double-strand breaks (DSBs) introduced by Ntg1 at the mitochondrial replication origin ori5 induce homologous DNA pairing by Mhr1, and reactive oxygen species (ROS) enhance production of DSBs. Here, we show that a mitochondrial nuclease encoded by the nuclear gene DIN7 (DNA damage inducible gene) has 5'-exodeoxyribonuclease activity. Using a small ρ(-) mtDNA bearing ori5 (hypersuppressive; HS) as a model mtDNA, we revealed that DIN7 is required for ROS-enhanced mtDNA replication and recombination that are both induced at ori5. Din7 overproduction enhanced Mhr1-dependent mtDNA replication and increased the number of residual DSBs at ori5 in HS-ρ(-) cells and increased deletion mutagenesis at the ori5 region in ρ(+) cells. However, simultaneous overproduction of Mhr1 suppressed all of these phenotypes and enhanced homologous recombination. Our results suggest that after homologous pairing, the relative activity levels of Din7 and Mhr1 modulate the preference for replication versus homologous recombination to repair DSBs at ori5.

Role of growth arrest-specific gene 6 in the development of fungal allergic airway disease in mice.

Growth arrest-specific gene (Gas)6 is a secreted vitamin K-dependent protein with pleiotropic effects via activation of receptor tyrosine kinase Tyro3, Axl, and Mertk receptors, but little is known about its role in allergic airway disease. We investigated the role of Gas6 in the development of fungal allergic airway disease in mice. The immune response was evaluated in Gas6-deficient (Gas6-/-) and wild-type (WT) mice and in recombinant Gas6-treated WT mice during Aspergillus fumigatus-induced allergic airway disease. Gas6 plasma levels were significantly elevated in adult clinical asthma of all severities compared with subjects without asthma. In a murine model of fungal allergic airway disease, increased protein expression of Axl and Mertk were observed in the lung. Airway hyperresponsiveness (AHR), whole lung Th2 cytokine levels, goblet cell metaplasia, and peribronchial fibrosis were ameliorated in Gas6-/- mice compared with WT mice with fungal allergic airway disease. Intranasal Gas6 administration into WT mice had a divergent effect on airway inflammation and AHR. Specifically, a total dose of 2 µg of exogenous Gas6 (i.e., low dose) significantly increased whole lung Th2 cytokine levels and subsequent AHR, whereas a total dose of 7 µg of exogenous Gas6 (i.e., high dose) significantly suppressed Th1 and Th2 cytokines and AHR compared with appropriate control groups. Mechanistically, Gas6 promoted Th2 activation via its highest affinity receptor Axl expressed by myeloid DCs. Intranasal administration of Gas6 consistently exacerbated airway remodeling compared with control WT groups. These results demonstrate that Gas6 enhances several features of fungal allergic airway disease.

Integrin-αvß3 regulates thrombopoietin-mediated maintenance of hematopoietic stem cells.

Throughout life, one's blood supply depends on sustained division of hematopoietic stem cells (HSCs) for self-renewal and differentiation. Within the bone marrow microenvironment, an adhesion-dependent or -independent niche system regulates HSC function. Here we show that a novel adhesion-dependent mechanism via integrin-ß3 signaling contributes to HSC maintenance. Specific ligation of ß3-integrin on HSCs using an antibody or extracellular matrix protein prevented loss of long-term repopulating (LTR) activity during ex vivo culture. The actions required activation of αvß3-integrin "inside-out" signaling, which is dependent on thrombopoietin (TPO), an essential cytokine for activation of dormant HSCs. Subsequent "outside-in" signaling via phosphorylation of Tyr747 in the ß3-subunit cytoplasmic domain was indispensable for TPO-dependent, but not stem cell factor-dependent, LTR activity in HSCs in vivo. This was accompanied with enhanced expression of Vps72, Mll1, and Runx1, 3 factors known to be critical for maintaining HSC activity. Thus, our findings demonstrate a mechanistic link between ß3-integrin and TPO in HSCs, which may contribute to maintenance of LTR activity in vivo as well as during ex vivo culture.

Stepwise chromatin remodelling by a cascade of transcription initiation of non-coding RNAs.

Recent transcriptome analyses using high-density tiling arrays and data from large-scale analyses of full-length complementary DNA libraries by the FANTOM3 consortium demonstrate that many transcripts are non-coding RNAs (ncRNAs). These transcriptome analyses indicate that many of the non-coding regions, previously thought to be functionally inert, are actually transcriptionally active regions with various features. Furthermore, most relatively large ( approximately several kilobases) polyadenylated messenger RNA transcripts are transcribed from regions harbouring little coding potential. However, the function of such ncRNAs is mostly unknown and has been a matter of debate. Here we show that RNA polymerase II (RNAPII) transcription of ncRNAs is required for chromatin remodelling at the fission yeast Schizosaccharomyces pombe fbp1(+) locus during transcriptional activation. The chromatin at fbp1(+) is progressively converted to an open configuration, as several species of ncRNAs are transcribed through fbp1(+). This is coupled with the translocation of RNAPII through the region upstream of the eventual fbp1(+) transcriptional start site. Insertion of a transcription terminator into this upstream region abolishes both the cascade of transcription of ncRNAs and the progressive chromatin alteration. Our results demonstrate that transcription through the promoter region is required to make DNA sequences accessible to transcriptional activators and to RNAPII.

Non-glycosylated G protein with CpG ODN provides robust protection against respiratory syncytial virus without inducing eosinophilia.

Introduction: Respiratory syncytial virus (RSV) vaccines targeting the fusion glycoprotein (F protein) are highly effective clinically in preventing RSV challenges. The attachment glycoprotein (G protein) is a potentially effective vaccine antigen candidate, as it is important for cell adhesion during infection. However, vaccine-associated enhanced diseases in mice, such as eosinophilic lung inflammation following RSV challenge, are a concern with G protein vaccines. This study aimed to design an effective G protein vaccine with enhanced safety and efficacy by evaluating the efficacy and adverse reactions of vaccines composed of different recombinant G proteins and adjuvants in mice. Methods: Mice were subcutaneously immunized with glycosylated G protein expressed in mammalian cells (mG), non-glycosylated G protein expressed in Escherichia coli (eG), or F protein with or without aluminum salts (alum), CpG oligodeoxynucleotide (CpG ODN), or AddaVax. After vaccination, the levels of G-specific antibody and T-cell responses were measured. The immunized mice were challenged with RSV and examined for the viral load in the lungs and nasal turbinates, lung-infiltrating cells, and lung pathology. Results: mG with any adjuvant was ineffective at inducing G-specific antibodies and had difficulty achieving both protection against RSV challenge and eosinophilia suppression. In particular, mG+CpG ODN induced G-specific T helper 1 (Th1) cells but only a few G-specific antibodies and did not protect against RSV challenge. However, eG+CpG ODN induced high levels of G-specific antibodies and Th1 cells and protected against RSV challenge without inducing pulmonary inflammation. Moreover, the combination vaccine of eG+F+CpG ODN showed greater protection against upper respiratory tract RSV challenge than using each single antigen vaccine alone. Discussion: These results indicate that the efficacy of recombinant G protein vaccines can be enhanced without inducing adverse reactions by using appropriate antigens and adjuvants, and their efficacy is further enhanced in the combination vaccine with F protein. These data provide valuable information for the clinical application of G protein vaccines.

Cationic-nanogel nasal vaccine containing the ectodomain of RSV-small hydrophobic protein induces protective immunity in rodents.

Respiratory syncytial virus (RSV) is a leading cause of upper and lower respiratory tract infection, especially in children and the elderly. Various vaccines containing the major transmembrane surface proteins of RSV (proteins F and G) have been tested; however, they have either afforded inadequate protection or are associated with the risk of vaccine-enhanced disease (VED). Recently, F protein-based maternal immunization and vaccines for elderly patients have shown promising results in phase III clinical trials, however, these vaccines have been administered by injection. Here, we examined the potential of using the ectodomain of small hydrophobic protein (SHe), also an RSV transmembrane surface protein, as a nasal vaccine antigen. A vaccine was formulated using our previously developed cationic cholesteryl-group-bearing pullulan nanogel as the delivery system, and SHe was linked in triplicate to pneumococcal surface protein A as a carrier protein. Nasal immunization of mice and cotton rats induced both SHe-specific serum IgG and mucosal IgA antibodies, preventing viral invasion in both the upper and lower respiratory tracts without inducing VED. Moreover, nasal immunization induced greater protective immunity against RSV in the upper respiratory tract than did systemic immunization, suggesting a critical role for mucosal RSV-specific IgA responses in viral elimination at the airway epithelium. Thus, our nasal vaccine induced effective protection against RSV infection in the airway mucosa and is therefore a promising vaccine candidate for further development.

A mechanism for single-stranded DNA-binding protein (SSB) displacement from single-stranded DNA upon SSB-RecO interaction.

Displacement of single-stranded DNA (ssDNA)-binding protein (SSB) from ssDNA is necessary for filament formation of RecA on ssDNA to initiate homologous recombination. The interaction between RecO and SSB is considered to be important for SSB displacement; however, the interaction has not been characterized at the atomic level. In this study, to clarify the mechanism underlying SSB displacement from ssDNA upon RecO binding, we examined the interaction between Thermus thermophilus RecO and cognate SSB by NMR analysis. We found that SSB interacts with the C-terminal positively charged region of RecO. Based on this result, we constructed some RecO mutants. The R127A mutant had considerably decreased binding affinity for SSB and could not anneal SSB-coated ssDNAs. Further, the mutant in the RecOR complex prevented the recovery of ssDNA-dependent ATPase activity of RecA from inhibition by SSB. These results indicated that the region surrounding Arg-127 is the binding site of SSB. We also performed NMR analysis using the C-terminal peptide of SSB and found that the acidic region of SSB is involved in the interaction with RecO, as seen in other protein-SSB interactions. Taken together with the findings of previous studies, we propose a model for SSB displacement from ssDNA where the acidic C-terminal region of SSB weakens the ssDNA binding affinity of SSB when the dynamics of the C-terminal region are suppressed by interactions with other proteins, including RecO.

Vital roles of the second DNA-binding site of Rad52 protein in yeast homologous recombination.

RecA/Rad51 proteins are essential in homologous DNA recombination and catalyze the ATP-dependent formation of D-loops from a single-stranded DNA and an internal homologous sequence in a double-stranded DNA. RecA and Rad51 require a "recombination mediator" to overcome the interference imposed by the prior binding of single-stranded binding protein/replication protein A to the single-stranded DNA. Rad52 is the prototype of recombination mediators, and the human Rad52 protein has two distinct DNA-binding sites: the first site binds to single-stranded DNA, and the second site binds to either double- or single-stranded DNA. We previously showed that yeast Rad52 extensively stimulates Rad51-catalyzed D-loop formation even in the absence of replication protein A, by forming a 2:1 stoichiometric complex with Rad51. However, the precise roles of Rad52 and Rad51 within the complex are unknown. In the present study, we constructed yeast Rad52 mutants in which the amino acid residues corresponding to the second DNA-binding site of the human Rad52 protein were replaced with either alanine or aspartic acid. We found that the second DNA-binding site is important for the yeast Rad52 function in vivo. Rad51-Rad52 complexes consisting of these Rad52 mutants were defective in promoting the formation of D-loops, and the ability of the complex to associate with double-stranded DNA was specifically impaired. Our studies suggest that Rad52 within the complex associates with double-stranded DNA to assist Rad51-mediated homologous pairing.

The double-stranded break-forming activity of plant SPO11s and a novel rice SPO11 revealed by a Drosophila bioassay.

BACKGROUND: SPO11 is a key protein for promoting meiotic recombination, by generating chromatin locus- and timing-specific DNA double-strand breaks (DSBs). The DSB activity of SPO11 was shown by genetic analyses, but whether SPO11 exerts DSB-forming activity by itself is still an unanswered question. DSB formation by SPO11 has not been detected by biochemical means, probably because of a lack of proper protein-folding, posttranslational modifications, and/or specific SPO11-interacting proteins required for this activity. In addition, plants have multiple SPO11-homologues. RESULTS: To determine whether SPO11 can cleave DNA by itself, and to identify which plant SPO11 homologue cleaves DNA, we developed a Drosophila bioassay system that detects the DSB signals generated by a plant SPO11 homologue expressed ectopically. We cytologically and genetically demonstrated the DSB activities of Arabidopsis AtSPO11-1 and AtSPO11-2, which are required for meiosis, in the absence of other plant proteins. Using this bioassay, we further found that a novel SPO11-homologue, OsSPO11D, which has no counterpart in Arabidopsis, displays prominent DSB-forming activity. Quantitative analyses of the rice SPO11 transcripts revealed the specific increase in OsSPO11D mRNA in the anthers containing meiotic pollen mother cells. CONCLUSIONS: The Drosophila bioassay system successfully demonstrated that some plant SPO11 orthologues have intrinsic DSB activities. Furthermore, we identified a novel SPO11 homologue, OsSPO11D, with robust DSB activity and a possible meiotic function.

Targeting ST2L potentiates CpG-mediated therapeutic effects in a chronic fungal asthma model.

IL-33 and its soluble receptor and cell-associated receptor (ST2L) are all increased in clinical and experimental asthma. The present study addressed the hypothesis that ST2L impairs the therapeutic effects of CpG in a fungal model of asthma. C57BL/6 mice were sensitized to Aspergillus fumigatus and challenged via i.t. instillation with live A. fumigatus conidia. Mice were treated with IgG alone, anti-ST2L monoclonal antibody (mAb) alone, CpG alone, IgG plus CpG, or anti-ST2L mAb plus CpG every other day from day 14 to day 28 and investigated on day 28 after conidia. Lung ST2L and toll-like receptor 9 protein expression levels concomitantly increased in a time-dependent manner during fungal asthma. Therapeutic blockade of ST2L with an mAb attenuated key pathological features of this model. At subtherapeutic doses, neither anti-ST2L mAb nor CpG alone affected fungal asthma severity. However, airway hyperresponsiveness, mucus cell metaplasia, peribronchial fibrosis, and fungus retention were markedly reduced in asthmatic mice treated with the combination of both. Whole lung CXCL9 levels were significantly elevated in the combination group but not in the controls. Furthermore, in asthmatic mice treated with the combination therapy, dendritic cells generated significantly greater IL-12p70 with CpG in vitro compared with control dendritic cells. The combination of anti-ST2L mAb with CpG significantly attenuated experimental asthma, suggesting that targeting ST2L might enhance the therapeutic efficacy of CpG during allergic inflammation.