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Homologous recombination (HR) is a universal phenomenon conserved from viruses to humans. The mechanisms of HR are essentially the same in humans and simple unicellular eukaryotes like yeast. Two highly diverged yeast species, Saccharomyces cerevisiae and Schizosaccharomyces pombe, have proven exceptionally useful in understanding the fundamental mechanisms of eukaryotic HR by serving as a source for unique biological insights and also complementing each other. Here, we will review the features of S. pombe HR mechanisms in comparison to S. cerevisiae and other model organisms. Particular emphasis will be put on the biochemical characterization of HR mechanisms uncovered using S. pombe proteins.

During homologous recombination, Rad51 forms a nucleoprotein filament on single-stranded DNA to promote DNA strand exchange. This filament binds to double-stranded DNA (dsDNA), searches for homology, and promotes transfer of the complementary strand, producing a new heteroduplex. Strand exchange proceeds via two distinct three-strand intermediates, C1 and C2. C1 contains the intact donor dsDNA whereas C2 contains newly formed heteroduplex DNA. Here, we show that the conserved DNA binding motifs, loop 1 (L1) and loop 2 (L2) in site I of Rad51, play distinct roles in this process. L1 is involved in formation of the C1 complex whereas L2 mediates the C1–C2 transition, producing the heteroduplex. Another DNA binding motif, site II, serves as the DNA entry position for initial Rad51 filament formation, as well as for donor dsDNA incorporation. Our study provides a comprehensive molecular model for the catalytic process of strand exchange mediated by eukaryotic RecA-family recombinases., Rad51 drives DNA strand exchange, the central reaction in recombinational DNA repair. Two sites of Rad51 are responsible for DNA binding, but the function of these sites has proven elusive. Here, the authors employ real-time assays to reveal catalytic roles for the two DNA binding sites of Rad51.

In this real-world clinical study, in which we determined eligibility for allogenic hematopoietic stem cell transplantation by prognostic factors and minimal residual disease status, we retrospectively evaluated cytogenetic, genetic, and clinical features in 96 patients with core-binding factor acute myeloid leukemia (CBF-AML) including 62 patients with RUNX1/RUNX1T1 and 34 patients with CBFβ/MYH11. Multivariate analyses for 5-year overall survival (OS) in CBF-AML patients revealed that age of 50 years or older (HR: 3.46, 95% CI 1.47-8.11, P = 0.004) and receiving 2 or more induction cycles (HR: 3.55, 95% CI 1.57-8.05, P = 0.002) were independently associated with worse OS and that loss of sex chromosome (LOS) was independently associated with better OS (HR: 0.09, 95% CI 0.01-0.71, P = 0.022). At the time of complete remission, all 21 karyotyped patients with LOS had a normal karyotype. Furthermore, in all 9 patients with LOS who had a mosaic of metaphase cells with and without t(8;21) or inv(16), the metaphase cells without t(8;21)/inv(16) showed a normal karyotype. These results proved that LOS was not age-related and physiological, but rather a neoplastic chromosomal abnormality.

Rad51 is the key protein in homologous recombination that plays important roles during DNA replication and repair. Auxiliary factors regulate Rad51 activity to facilitate productive recombination, and prevent inappropriate, untimely or excessive events, which could lead to genome instability. Previous genetic analyses identified a function for Rrp1 (a member of the Rad5/16-like group of SWI2/SNF2 translocases) in modulating Rad51 function, shared with the Rad51 mediator Swi5-Sfr1 and the Srs2 anti-recombinase. Here, we show that Rrp1 overproduction alleviates the toxicity associated with excessive Rad51 levels in a manner dependent on Rrp1 ATPase domain. Purified Rrp1 binds to DNA and has a DNA-dependent ATPase activity. Importantly, Rrp1 directly interacts with Rad51 and removes it from double-stranded DNA, confirming that Rrp1 is a translocase capable of modulating Rad51 function. Rrp1 affects Rad51 binding at centromeres. Additionally, we demonstrate in vivo and in vitro that Rrp1 possesses E3 ubiquitin ligase activity with Rad51 as a substrate, suggesting that Rrp1 regulates Rad51 in a multi-tiered fashion., Graphical Abstract Graphical AbstractRrp1 translocase removes Rad51 from double stranded DNA and is an E3 ubiquitin ligase with Rad51 as a substrate.

DNA is the molecule that stores the chemical instructions necessary for life and its stability is therefore of the utmost importance. Despite this, DNA is damaged by both exogenous and endogenous factors at an alarming frequency. The most severe type of DNA damage is a double-strand break (DSB), in which a scission occurs in both strands of the double helix, effectively dividing a single normal chromosome into two pathological chromosomes. Homologous recombination (HR) is a universal DSB repair mechanism that solves this problem by identifying another region of the genome that shares high sequence similarity with the DSB site and using it as a template for repair. Rad51 possess the enzymatic activity that is essential for this repair but several auxiliary factors are required for Rad51 to fulfil its function. It is becoming increasingly clear that many HR factors are subjected to post-translational modification. Here, we review what is known about how these modifications affect HR. We first focus on cases where there is experimental evidence to support a function for the modification, then discuss speculative cases where a function can be inferred. Finally, we contemplate why such modifications might be necessary.

Mating-type switching (MTS) in fission yeast Schizosaccharomyces pombe is a highly regulated gene conversion event. In the process, heterochromatic donors of genetic information are selected based on the P or M cell type and on the use of two recombination enhancers, SRE2 promoting use of mat2-P and SRE3 promoting use of mat3-M. Recently, we found that the histone H3K4 methyltransferase complex Set1C participates in donor selection, raising the question of how a complex best known for its effects in euchromatin controls recombination in heterochromatin. Here, we report that the histone H2BK119 ubiquitin ligase complex HULC functions with Set1C in MTS, as mutants in the shf1, brl1, brl2 and rad6 genes showed defects similar to Set1C mutants and belonged to the same epistasis group as set1Δ. Moreover, using H3K4R and H2BK119R histone mutants and a Set1-Y897A catalytic mutant indicated that ubiquitylation of histone H2BK119 by HULC and methylation of histone H3K4 by Set1C are functionally coupled in MTS. Cell-type biases in mutants further showed that the regulation might be by inhibiting use of the SRE3 enhancer in M cells, in favor of SRE2. Consistently, imbalanced switching in the mutants was traced to compromised association of the directionality factor Swi6 with the recombination enhancers in M cells. Based on their known effects at other chromosomal locations, we speculate that HULC and Set1C might control nucleosome mobility and strand invasion near the SRE elements. In addition, we uncovered distinct effects of HULC and Set1C on histone H3K9 methylation and gene silencing, consistent with additional functions in the heterochromatic domain. Author Summary Mating-type switching in the fission yeast Schizosaccharomyces pombe occurs by gene conversion using a donor, mat2 or mat3 located in a heterochromatin region. Multiple studies have shown that donor selection is critically affected by heterochromatic factors. Here, we document the role of euchromatic factors, the histone H2BK119 ubiquitin ligase complex HULC and the H3K4 methyltransferase complex Set1C, in donor selection. Mutational analysis indicated that HULC and Set1C inhibit donor choice at the mat3 cis-acting recombination enhancer SRE3 in M cells, through concerted histone modifications by the two complexes. Linking this effect with heterochromatin, mutants in each complex, shf1Δ and set1Δ strains, exhibited decreased association of the HP1 heterochromatic factor Swi6 with the SRE2 and SRE3 recombination enhancers at the mat2 and mat3 silent loci in M cells. These joint effects by the two complexes on MTS were observed even though other aspects, the methylation state of histone H3K9 and effects on gene silencing differed between the shf1Δ and set1Δ strains. The results provide insight into the regulation of recombination by chromatin structure.

The Mre11-Rad50-Nbs1 complex (MRN) is important for repairing DNA double-strand breaks (DSBs) by homologous recombination (HR). The endonuclease activity of MRN is critical for resecting 5′-ended DNA strands at DSB ends, producing 3′-ended single-strand DNA, a prerequisite for HR. This endonuclease activity is stimulated by Ctp1, the Schizosaccharomyces pombe homolog of human CtIP. Here, with purified proteins, we show that Ctp1 phosphorylation stimulates MRN endonuclease activity by inducing the association of Ctp1 with Nbs1. The highly conserved extreme C terminus of Ctp1 is indispensable for MRN activation. Importantly, a polypeptide composed of the conserved 15 amino acids at the C terminus of Ctp1 (CT15) is sufficient to stimulate Mre11 endonuclease activity. Furthermore, the CT15 equivalent from CtIP can stimulate human MRE11 endonuclease activity, arguing for the generality of this stimulatory mechanism. Thus, we propose that Nbs1-mediated recruitment of CT15 plays a pivotal role in the activation of the Mre11 endonuclease by Ctp1/CtIP.

Rad51 is the key protein in homologous recombination DNA repair and has important roles during DNA replication. Auxiliary factors regulate Rad51 activity to facilitate productive, and prevent inappropriate, recombination that could lead to genome instability. Previous genetic analyses identified a function for Rrp1 (a member of the Rad5/16-like group of SWI2/SNF2 translocases) in counteracting Rad51 activity, shared with the Rad51 mediator Swi5-Sfr1 and the Srs2 anti-recombinase. Here, we show that Rrp1 overproduction alleviates the toxicity associated with excessive Rad51 activity in a manner dependent on Rrp1 ATPase domain. Purified Rrp1 binds to DNA and has a DNA-dependent ATPase activity. Importantly, Rrp1 directly interacts with Rad51 and removes it from double-stranded DNA, confirming that Rrp1 is a translocase capable of modulating Rad51 activity. Additionally, we demonstrate that Rrp1 possesses E3 ubiquitin ligase activity with Rad51 as a substrate, suggesting that Rrp1 regulates Rad51 in a multi-tiered fashion.

TP0446131, developed as an antidepressant agent, was found to cause lenticular opacity in a 13-week repeated-dose study in dogs. Histopathologically, the lenticular opacity was observed as a degeneration of the lens fibers, characterized by irregularity in the ordered arrangement of the fibers which is necessary to maintain the transparency of the lens, and was considered to manifest clinically as cataract. To evaluate the development mechanism of the lenticular opacity, the chemical constituents of the lens, which is known to be associated with the development of cataract, were examined. The results of liquid chromatography-tandem mass spectrometry analysis revealed an increase in the amplitudes of 3 unknown peaks in a dose- and time-dependent manner in the lens, with no remarkable changes in the other chemical components tested. In addition, the content of cholesterol, alterations of which have been reported to be associated with cataract, remained unchanged. The mass spectral data and chromatographic behavior of the 3 peaks indicated that these peaks corresponded to sterol-related substances, and that one of them was 7-dehydrocholesterol, a precursor of cholesterol biosynthesis. This finding suggested that TP0446131 exerts some effects on the cholesterol biosynthesis pathway, which could be involved in the development of the cataracts. Furthermore, increases in the levels of these sterol-related substances were also detected in the serum, and were, in fact, noted prior to the onset of the cataract, suggesting the possibility that these substances in the serum could be used as potential safety biomarkers for predicting the onset of cataract induced by TP0446131.

Although Rad51 is the key protein in homologous recombination (HR), a major DNA double-strand break repair pathway, several auxiliary factors interact with Rad51 to promote productive HR. We present an interdisciplinary characterization of the interaction between Rad51 and Swi5-Sfr1, a conserved auxiliary factor. Two distinct sites within the intrinsically disordered N-terminus of Sfr1 (Sfr1N) were found to cooperatively bind Rad51. Deletion of this domain impaired Rad51 stimulation in vitro and rendered cells sensitive to DNA damage. By contrast, amino acid-substitution mutants, which had comparable biochemical defects, could promote DNA repair, suggesting that Sfr1N has another role in addition to Rad51 binding. Unexpectedly, the DNA repair observed in these mutants was dependent on Rad55-Rad57, another auxiliary factor complex hitherto thought to function independently of Swi5-Sfr1. When combined with the finding that they form a higher-order complex, our results imply that Swi5-Sfr1 and Rad55-Rad57 can collaboratively stimulate Rad51 in Schizosaccharomyces pombe., eLife digest The DNA within cells contains the instructions necessary for life and it must be carefully maintained. DNA is constantly being damaged by radiation and other factors so cells have evolved an arsenal of mechanisms that repair this damage. An enzyme called Rad51 drives one such DNA repair process known as homologous recombination. A pair of regulatory proteins known as the Swi5-Sfr1 complex binds to Rad51 and activates it. The complex can be thought of as containing two modules with distinct roles: one comprising the first half of the Sfr1 protein and that is capable of binding to Rad51, and a second consisting of the rest of Sfr1 bound to Swi5, which is responsible for activating Rad51. Here, Argunhan, Sakakura et al. used genetic and biochemical approaches to study how this first module, known as “Sfr1N”, interacts with Rad51 in a microbe known as fission yeast. The experiments showed that both modules of Swi5-Sfr1 were important for Rad51 to drive homologous recombination. Swi5-Sfr1 complexes carrying mutations in the region of Sfr1N that binds to Rad51 were unable to activate Rad51 in a test tube. However, fission yeast cells containing the same mutations were able to repair their DNA without problems. This was due to the presence of another pair of proteins known as the Rad55-Rad57 complex that also bound to Swi5-Sfr1. The findings of Argunhan, Sakakura et al. suggest that the Swi5-Sfr1 and Rad55-Rad57 complexes work together to activate Rad51. Many genetically inherited diseases and cancers have been linked to mutations in DNA repair proteins. The fundamental mechanisms of DNA repair are very similar from yeast to humans and other animals, therefore, understanding the details of DNA repair in yeast may ultimately benefit human health in the future.

During homologous recombination, Rad51 forms a nucleoprotein filament with single-stranded DNA (ssDNA) that undergoes strand exchange with homologous double-stranded DNA (dsDNA). Here, we use real-time analysis to show that strand exchange by fission yeast Rad51 proceeds via two distinct three-strand intermediates, C1 and C2. Both intermediates contain Rad51, but whereas the donor duplex remains intact in C1, the ssDNA strand is intertwined with the complementary strand of the donor duplex in C2. Swi5–Sfr1, an evolutionarily conserved recombination activator, facilitates the C1–C2 transition and subsequent ssDNA release from C2 to complete strand exchange in an ATP-hydrolysis-dependent manner. In contrast, Ca2+, which activates the Rad51 filament by curbing ATP hydrolysis, facilitates the C1–C2 transition but does not promote strand exchange. These results reveal that Swi5–Sfr1 and Ca2+ have different activation modes in the late synaptic phase, despite their common function in stabilizing the presynaptic filament. Real-time FRET analyses and biochemical assays reveal that Rad51 recombinase promotes DNA strand exchange via two distinct three-strand intermediate states.

In the present study, the free-energy landscape of a hydrated alanine dipeptide has been studied by the 3D-RISM theory. We calculate the hydration free energy as a function of dihedral angles and using both the 3D-RISM theory and the umbrella sampling method. The 3D-RISM results are found to be in good agreement with those by the umbrella sampling calculations, demonstrating the reliability of the integral equation theory applied to bimolecular systems.

Resolution achieved Chloroplasts possess multiple copies of their own chloroplast DNA that are packaged into DNA-protein complexes known as nucleoids. The shape, number, and distribution of chloroplast nucleoids change markedly depending on the cell cycle, developmental stage, and nutritional environment. Kobayashi et al. identified Holliday junction resolvase as a key factor in the dynamism of chloroplast nucleoids in the unicellular green algae Chlamydomonas reinhardtii . The gene encoding the resolvase is ubiquitously conserved among green plants. Disruption or down-regulation of this gene also disturbed chloroplast nucleoid organization and segregation in the land plant Arabidopsis thaliana . Science , this issue p. 631

Homologous recombination (HR) is the process whereby two DNA molecules that share high sequence similarity are able to recombine to generate hybrid DNA molecules. Throughout evolution, the ability of HR to identify highly similar DNA sequences has been adopted for numerous biological phenomena including DNA repair, meiosis, telomere maintenance, ribosomal DNA amplification and immunological diversity. Although Rad51 and Dmc1 are the key proteins that promote HR in mitotic and meiotic cells, respectively, accessory proteins that allow Rad51 and Dmc1 to effectively fulfil their functions have been identified in all examined model systems. In this Review, we discuss the roles of the highly conserved Swi5‐Sfr1 accessory complex in yeast, mice and humans, and explore similarities and differences between these species.

During homologous recombination, Rad51 forms a nucleoprotein filament on single-stranded DNA to promote DNA strand exchange. This filament binds to double-stranded DNA (dsDNA), searches for homology, and promotes transfer of the complementary strand, producing a new heteroduplex. Strand exchange proceeds via two distinct three-strand intermediates, C1 and C2. C1 contains the intact donor dsDNA whereas C2 contains newly formed heteroduplex DNA. Here, we show that conserved DNA binding motifs, loop 1 (L1) and loop 2 (L2) in site I of Rad51, play distinct roles in this process. L1 is involved in formation of the C1 complex whereas L2 mediates the C1-C2 transition, producing the heteroduplex. Another DNA binding motif, site II, serves as the DNA entry position for initial Rad51 filament formation, as well as for second donor dsDNA incorporation. Our study provides a comprehensive molecular model for the catalytic process of strand exchange mediated by eukaryotic RecA family recombinases.

Homologous recombination between repetitive sequences can lead to gross chromosomal rearrangements (GCRs). At fission yeast centromeres, Rad51-dependent conservative recombination predominantly occurs between inverted repeats, thereby suppressing formation of isochromosomes whose arms are mirror images. However, it is unclear how GCRs occur in the absence of Rad51 and how GCRs are prevented at centromeres. Here, we show that homology-mediated GCRs occur through Rad52-dependent single-strand annealing (SSA). The rad52-R45K mutation, which impairs SSA activity of Rad52 protein, dramatically reduces isochromosome formation in rad51 deletion cells. A ring-like complex Msh2–Msh3 and a structure-specific endonuclease Mus81 function in the Rad52-dependent GCR pathway. Remarkably, mutations in replication fork components, including DNA polymerase α and Swi1/Tof1/Timeless, change the balance between Rad51-dependent recombination and Rad52-dependent SSA at centromeres, increasing Rad52-dependent SSA that forms isochromosomes. Our results uncover a role of DNA replication machinery in the recombination pathway choice that prevents Rad52-dependent GCRs at centromeres., Onaka et al. show in fission yeast that Rad52-dependent single-strand annealing (SSA) causes gross chromosomal rearrangements in cells defective in Rad51-dependent recombination. Furthermore, mutations in replication fork proteins increase Rad52-dependent SSA at centromeres, demonstrating that the DNA replication machinery plays an important role in recombination pathway choice at centromeres.

Although Rad51 is the key protein in homologous recombination (HR), a major DNA double-strand break repair pathway, several auxiliary factors interact with Rad51 to promote productive HR. Here, we present an interdisciplinary characterization of the interaction between Rad51 and Swi5-Sfr1, a widely conserved auxiliary factor. NMR and site-specific crosslinking experiments revealed two distinct sites within the intrinsically disordered N-terminus of Sfr1 that cooperatively bind to Rad51. Although disruption of this binding severely impaired Rad51 stimulation in vitro, interaction mutants did not show any defects in DNA repair. Unexpectedly, in the absence of the Rad51 paralogs Rad55-Rad57, which constitute another auxiliary factor complex, these interaction mutants were unable to promote DNA repair. Our findings provide molecular insights into Rad51 stimulation by Swi5-Sfr1 and suggest that, rather than functioning in an independent subpathway of HR as was previously proposed, Rad55-Rad57 facilitates the recruitment of Swi5-Sfr1 to Rad51.

The DNA strand exchange reaction mediated by Rad51 is a critical step of homologous recombination. In this reaction, Rad51 forms a nucleoprotein filament on single-stranded DNA (ssDNA) and captures double-stranded DNA (dsDNA) non-specifically to interrogate it for a homologous sequence. After encountering homology, Rad51 catalyzes DNA strand exchange to mediate pairing of the ssDNA with the complementary strand of the dsDNA. This reaction is highly regulated by numerous accessary proteins in vivo. Although conventional biochemical assays have been successfully employed to examine the role of such accessory protein in vitro, kinetic analysis of intermediate formation and its progression into a final product has proven challenging due to the unstable and transient nature of the reaction intermediates. To observe these reaction steps directly in solution, fluorescence resonance energy transfer (FRET)-based real-time observation systems of this reaction were established. Kinetic analysis of real-time observations shows that the DNA strand exchange reaction mediated by Rad51 obeys a three-step reaction model involving the formation of a three-strand DNA intermediate, maturation of this intermediate, and the release of ssDNA from the mature intermediate. The Swi5-Sfr1 complex, an accessary protein conserved in eukaryotes, strongly enhances the second and third steps of this reaction. The FRET-based assays presented here enable us to uncover the molecular mechanisms through which recombination accessary proteins stimulate the DNA strand exchange activity of Rad51. The primary goal of this protocol is to enhance the repertoire of techniques available to researchers in the field of homologous recombination, particularly those working with proteins from species other than Schizosaccharomyces pombe, so that the evolutionary conservation of the findings presented herein can be determined.

Multiple myeloma (MM) is a hematological malignancy that grows in multiple sites of the axial skeleton and causes debilitating osteolytic disease. Interleukin-34 (IL-34) is a newly discovered cytokine that acts as a ligand of colony-stimulating factor-1 (CSF-1) receptor and can replace CSF-1 for osteoclast differentiation. In this study, we identify IL-34 as an osteoclastogenic cytokine that accelerates osteolytic disease in MM. IL-34 was found to be expressed in the murine MM cell line MOPC315.BM, and the expression of IL-34 was enhanced by stimulation with proinflammatory cytokines or by bone marrow (BM) stromal cells. MM-cell–derived IL-34 promoted osteoclast formation from mouse BM cells in vitro. Targeting Il34 by specific small interfering RNA impaired osteoclast formation in vitro and attenuated osteolytic disease in vivo. In BM aspirates from MM patients, the expression levels of IL-34 in CD138+ populations vary among patients from high to weak to absent. MM cell–derived IL-34 promoted osteoclast formation from human CD14+ monocytes, which was reduced by a neutralizing antibody against IL-34. Taken together, this study describes for the first time the expression of IL-34 in MM cells, indicating that it may enhance osteolysis and suggesting IL-34 as a potential therapeutic target to control pathological osteoclastogenesis in MM patients.

Eukaryotic Rad51 protein is essential for homologous-recombination repair of DNA double-strand breaks. Rad51 recombinases first assemble onto single-stranded DNA to form a nucleoprotein filament, required for function in homology pairing and strand exchange. This filament assembly is the first regulation step in homologous recombination. Rad51 nucleation is kinetically slow, and several accessory factors have been identified to regulate this step. Swi5–Sfr1 (S5S1) stimulates Rad51-mediated homologous recombination by stabilizing Rad51 nucleoprotein filaments, but the mechanism of stabilization is unclear. We used single-molecule tethered particle motion experiments to show that mouse S5S1 (mS5S1) efficiently stimulates mouse RAD51 (mRAD51) nucleus formation and inhibits mRAD51 dissociation from filaments. We also used single-molecule fluorescence resonance energy transfer experiments to show that mS5S1 promotes stable nucleus formation by specifically preventing mRAD51 dissociation. This leads to a reduction of nucleation size from three mRAD51 to two mRAD51 molecules in the presence of mS5S1. Compared with mRAD51, fission yeast Rad51 (SpRad51) exhibits fast nucleation but quickly dissociates from the filament. SpS5S1 specifically reduces SpRad51 disassembly to maintain a stable filament. These results clearly demonstrate the conserved function of S5S1 by primarily stabilizing Rad51 on DNA, allowing both the formation of the stable nucleus and the maintenance of filament length.

Summary The ring-shaped structural maintenance of chromosome (SMC) complexes are multi-subunit ATPases that topologically encircle DNA. SMC rings make vital contributions to numerous chromosomal functions, including mitotic chromosome condensation, sister chromatid cohesion, DNA repair, and transcriptional regulation. They are thought to do so by establishing interactions between more than one DNA. Here, we demonstrate DNA-DNA tethering by the purified fission yeast cohesin complex. DNA-bound cohesin efficiently and topologically captures a second DNA, but only if that is single-stranded DNA (ssDNA). Like initial double-stranded DNA (dsDNA) embrace, second ssDNA capture is ATP-dependent, and it strictly requires the cohesin loader complex. Second-ssDNA capture is relatively labile but is converted into stable dsDNA-dsDNA cohesion through DNA synthesis. Our study illustrates second-DNA capture by an SMC complex and provides a molecular model for the establishment of sister chromatid cohesion., Graphical Abstract, Highlights • The recombinant cohesin complex mediates DNA-DNA interactions • Second-DNA capture by cohesin requires ssDNA • DNA replication stabilizes second-strand capture • Second-DNA capture at a replication fork could establish sister chromatid cohesion, Cohesin sets up the initial steps of chromosome cohesion by anchoring single-stranded DNA along to a region of duplex DNA.

International audience; Mg 2+ ion stimulates the DNA strand exchange reaction catalyzed by RecA, a key step in homologous recombination. To elucidate the molecular mechanisms underlying the role of Mg 2+ and the strand exchange reaction itself, we investigated the interaction of RecA with Mg 2+ and sought to determine which step of the reaction is affected. Thermal stability, intrinsic fluorescence, and native mass spectrometric analyses of RecA revealed that RecA binds at least two Mg 2+ ions with K D ≈ 2 mM and 5 mM. Deletion of the C-terminal acidic tail of RecA made its thermal stability and fluorescence characteristics insensitive to Mg 2+ and similar to those of full-length RecA in the presence of saturating Mg 2+. These observations, together with the results of a molecular dynamics simulation, support the idea that the acidic tail hampers the strand exchange reaction by interacting with other parts of RecA, and that binding of Mg 2+ to the tail prevents these interactions and releases RecA from inhibition. We observed that binding of the first Mg 2+ stimulated joint molecule formation, whereas binding of the second stimulated progression of the reaction. Thus, RecA is actively involved in the strand exchange step as well as bringing the two DNAs close to each other.

PURPOSE: We previously determined the pharmacokinetic (PK) parameters of landiolol in healthy male volunteers. In this study, we evaluated the usefulness of target-controlled infusion (TCI) of landiolol hydrochloride and determined PK parameters of landiolol in gynecologic patients. METHODS: Nine patients who were scheduled to undergo gynecologic surgery were enrolled. After inducing anesthesia, landiolol hydrochloride was administered at the target plasma concentrations of 500 and 1,000 ng/mL for each 30 min. A total of 126 data points of plasma concentration were collected from the patients and used for the population PK analysis. Furthermore, a population PK model was developed using the nonlinear mixed-effect modeling software. RESULTS: The patients had markedly decreased heart rates (HRs) at 2 min after the initiation of landiolol hydrochloride administration; however, their blood pressures did not markedly change from the baseline value. The concentration time course of landiolol was best described by a 2-compartment model with lag time. The estimate of PK parameters were total body clearance (CL) 34.0 mL/min/kg, distribution volume of the central compartment (V 1) 74.9 mL/kg, inter-compartmental clearance (Q) 70.9 mL/min/kg, distribution volume of the peripheral compartment (V 2) 38.9 mL/kg, and lag time (ALAG) 0.634 min. The predictive performance of this model was better than that of the previous model. CONCLUSION: TCI of landiolol hydrochloride is useful for controlling HR, and the PK parameters of landiolol in gynecologic patients were similar to those in healthy male volunteers and best described by a 2-compartment model with lag time.

High throughput screening using Automated Ligand Identification System (ALIS) resulted in the discovery of a new series of S-adenosyl-L-homocysteine hydrolase inhibitors based on non-adenosine analogs. The optimization campaign led to very potent and competitive compound 39 with a Ki value of 1.5 nM. Compound 39 could be a promising lead compound for research to reduce elevated homocysteine levels.

In this study, thermodynamic and molecular properties of a hydrated alanine dipeptide have been studied by the three-dimensional reference interaction site method (3D-RISM) theory. The excess chemi...

It is now recognized that intrinsically disordered proteins (IDPs) play important roles as hubs in intracellular networks, and their structural characterisation is of significance. However, due to their highly dynamic features, it is challenging to investigate the structures of IDPs solely by conventional methods. In the present study, we demonstrate a novel method to characterise protein complexes using electrospray ionization ion mobility mass spectrometry (ESI-IM-MS) in combination with small-angle X-ray scattering (SAXS). This method enables structural characterisation even of proteins that have difficulties in crystallisation. With this method, we have characterised the Schizosaccharomyces pombe Swi5-Sfr1 complex, which is expected to have a long disordered region at the N-terminal portion of Sfr1. ESI-IM-MS analysis of the Swi5-Sfr1 complex revealed that its experimental collision cross-section (CCS) had a wide distribution, and the CCS values of the most dominant ions were ∼56% of the theoretically calculated value based on the SAXS low-resolution model, suggesting a significant size reduction in the gas phase. The present study demonstrates that the newly developed method for calculation of the theoretical CCSs of the SAXS low-resolution models of proteins allows accurate evaluation of the experimental CCS values of IDPs provided by ESI-IM-MS by comparing with the low-resolution solution structures. Furthermore, it was revealed that the combination of ESI-IM-MS and SAXS is a promising method for structural characterisation of protein complexes that are unable to crystallise.

OBJECTIVE The prognosis for gastric carcinoma patients with liver metastasis is very poor. This retrospective study investigated the prognostic significance of MK-1 expression in gastric carcinoma patients with liver metastasis. METHODS Immunohistochemical staining using monoclonal antibody FU-MK-1 against MK-1 antigen was performed on paraffin-embedded tissues from 64 gastric carcinoma patients with liver metastasis. We attempted to determine the presence of any relationship between pathological prognostic factors and the expression of MK-1 in 64 gastric carcinoma patients with liver metastasis. RESULTS MK-1 expression was found in 43 (67%) of 64 tumor samples. MK-1 expression was significantly higher in the intestinal type (73%) than in the diffuse type carcinoma (33%, P = 0.049). Multivariate analysis showed that MK-1 expression and lymph node metastasis were significant factors for overall survival. The difference between overall survival rates with positive or negative MK-1 expression was statistically significant as shown by Kaplan-Meier survival analysis (P < 0.0001; log-rank). In addition, the difference between cumulative disease-free survival rates with positive or negative MK-1 expression in gastric carcinoma patients with metachronous liver metastasis was statistically significant as well, as shown by Kaplan-Meier survival analysis (P = 0.0006; log-rank). CONCLUSIONS The prognostic significance of MK-1 expression as a biological tumor marker was demonstrated in a series of gastric carcinoma patients with liver metastasis. MK-1 positivity may be a reliable marker for predicting and taking measures to control liver metastasis after curative gastrectomy for gastric carcinoma.

We have applied scanning tunnelling microscopy (STM) to the study of two proteins: pseudoazurin and apo-pseudoazurin. Both proteins adsorbed onto a Au (1 1 1) surface are visible to STM individually, forming into layers and multilayers, with currents from about 55 to 600 pA. The images reproduce well the expected dimensions laterally but not in the z direction. The apparent height of the proteins varies with the voltage polarity, being higher at negative sample voltages. The bias also affects their shape. Negative sample voltages of more than 1.5 V orient the proteins present on a gold terrace in parallel rows. The layer of water adsorbed on surfaces in ambient conditions can be related to our results to explain the reduced z dimensions, the asymmetry with the voltage polarity and the alignment of proteins at voltages more negative than −1.5 V.

SummaryRad51 forms a helical filament on single-stranded DNA and promotes strand exchange between two homologous DNA molecules during homologous recombination. The Swi5-Sfr1 complex interacts directly with Rad51 and stimulates strand exchange. Here we describe structural and functional aspects of the complex. Swi5 and the C-terminal core domain of Sfr1 form an essential activator complex with a parallel coiled-coil heterodimer joined firmly together via two previously uncharacterized leucine-zipper motifs and a bundle. The resultant coiled coil is sharply kinked, generating an elongated crescent-shaped structure suitable for transient binding within the helical groove of the Rad51 filament. The N-terminal region of Sfr1, meanwhile, has an interface for binding of Rad51. Our data suggest that the snug fit resulting from the complementary geometry of the heterodimer activates the Rad51 filament and that the N-terminal domain of Sfr1 plays a role in the efficient recruitment of the Swi5-Sfr1 complex to the Rad51 filaments.

Background: The Swi5-Sfr1 protein complex is an activator of Rad51 recombinase, which mediates DNA strand exchange in homologous recombination. Results: Swi5 and Sfr1 form a 1:1 complex, which exhibits an extremely elongated dogleg-shaped structure in solution. Conclusion: The Swi5-Sfr1 structure is suitable for binding within the helical groove of the Rad51 filament. Significance: A structural model will advance our understanding of the molecular mechanism of homologous recombination., In eukaryotes, DNA strand exchange is the central reaction of homologous recombination, which is promoted by Rad51 recombinases forming a right-handed nucleoprotein filament on single-stranded DNA, also known as a presynaptic filament. Accessory proteins known as recombination mediators are required for the formation of the active presynaptic filament. One such mediator in the fission yeast Schizosaccharomyces pombe is the Swi5-Sfr1 complex, which has been identified as an activator of Rad51 that assists in presynaptic filament formation and stimulates its strand exchange reaction. Here, we determined the 1:1 binding stoichiometry between the two subunits of the Swi5-Sfr1 complex using analytical ultracentrifugation and electrospray ionization mass spectrometry. Small-angle x-ray scattering experiments revealed that the Swi5-Sfr1 complex displays an extremely elongated dogleg-shaped structure in solution, which is consistent with its exceptionally high frictional ratio (f/f0) of 2.0 ± 0.2 obtained by analytical ultracentrifugation. Furthermore, we determined a rough topology of the complex by comparing the small-angle x-ray scattering-based structures of the Swi5-Sfr1 complex and four Swi5-Sfr1-Fab complexes, in which the Fab fragments of monoclonal antibodies were specifically bound to experimentally determined sites of Sfr1. We propose a model for how the Swi5-Sfr1 complex binds to the Rad51 filament, in which the Swi5-Sfr1 complex fits into the groove of the Rad51 filament, leading to an active and stable presynaptic filament.

Homologous recombination proceeds via the formation of several intermediates including Holliday junctions (HJs), which are important for creating crossover products. DNA strand exchange is a core reaction that produces these intermediates that is directly catalyzed by RecA family recombinases, of which there are two types in eukaryotes: universal Rad51 and meiosis-specific Dmc1. We demonstrated previously that Rad51 promotes four-strand exchange, mimicking the formation and branch migration of HJs. Here we show that Dmc1 from fission yeast has a similar activity, which requires ATP hydrolysis and is independent of an absolute requirement for the Swi5–Sfr1 complex. These features are critically different from three-strand exchange mediated by Dmc1, but similar to those of four-strand exchange mediated by Rad51, suggesting that strand exchange reactions between duplex–duplex and single-duplex DNAs are mechanistically different. Interestingly, despite similarities in protein structure and in reaction features, the preferential polarities of Dmc1 and Rad51 strand exchange are different (Dmc1 promotes exchange in the 5′-to-3′ direction and Rad51 promotes exchange in the 3′-to-5′ direction relative to the ssDNA region of the DNA substrate). The significance of the Dmc1 polarity is discussed within the context of the necessity for crossover production.

Combustion experiments of fuel droplet array in fuel vapor-air mixture were performed at microgravities to investigate growth mechanism of group combustion of fuel droplets. A 10-droplet array was inserted into the test section filled with a saturated fuel vapor-air mixture as a simple model of prevaporized sprays. Gas equivalence ratio of the fuel vapor-air mixture was regulated by the test section temperature. n -Decane droplets of 0.8 mm in the initial diameter were suspended at the crossing points of 10 sets of X-shaped suspenders. The first droplet was ignited by a hot wire to initiate flame spread along a fuel droplet array. Flame spread speed was obtained from the history of the leading edge position of a spreading flame. Effects of droplet spacing and gas equivalence ratio on the flame spreading behavior and the flame spread speed were examined. The droplet spacing and the gas equivalence ratio were varied from 1.6 to 10.2 mm and from 0.2 to 0.7, respectively. The gas equivalence ratio has little effect on the relationship between the flame spreading behavior and the droplet spacing. The flame spread speed increases as the increase in the gas equivalence ratio at all droplet spacings. The influence of the gas equivalence ratio on the flame spread speed becomes strong as the increase in the droplet spacings. The flame spread speed increases as the increase in the droplet spacing, and then decreases. The maximum flame spread speed appears in the range from 2.4 to 3 mm at all gas equivalence ratios.

application/pdf, The effect of ternary addition of the 3d elements Ti, V, Cr, Mn, Fe, Co and Ni to CuPt3 to form ternary CuMPt6 alloys on the thermal properties was investigated using high-temperature X-ray diffraction method. Data obtained were utilized to determine the lattice parameters, coefficients of thermal expansion, Debye temperature and mean-square displacements of an atom. It was found that the addition increases the lattice spacing, increases the degree of thermal expansion and lowers the Debye temperature. The results are discussed within the framework of the classical theory.

STM light emission (STML) has been shown to be a promising technique for nanometer scale analyses of electronic states of molecules on metals as the light emission due to the electronic transition between molecular levels is comparably intense with the plasmon induced light emission for noble metals. The energy forbidden light emission in the spectra observed for porphyrin molecules on Au has been discussed on the basis of strong coupling between molecules and plasmon field in STM nano-cavity. The substrate material dependence of plasmon enhancement is well reproduced by an electromagnetic calculation using the effective dielectric constant for the molecular film/substrate material.

Smoothing of an atomically rough vicinal surface of SrTiO 3 is studied by scanning tunneling microscope (STM) observation and by Monte Carlo (MC) simulation. A complex step pattern that resembles a two-dimensional phase separation pattern is observed on the surface. Analysis of the step pattern during annealing obtained by the STM in comparison with the MC simulation reveals an asymmetry of the relaxation pattern between islands and holes. The asymmetry is attributed to the difference of the mobility of an adatom and an atomic hole, and the asymmetry is enhanced by the step edge diffusion barrier. Values of an effective bond energy and an effective diffusion barrier as well as the surface diffusion coefficient are deduced from the relaxation pattern.

The Schizosaccharomyces pombe nip1(+)/ctp1(+) gene was previously identified as an slr (synthetically lethal with rad2) mutant. Epistasis analysis indicated that Nip1/Ctp1 functions in Rhp51-dependent recombinational repair, together with the Rad32 (spMre11)-Rad50-Nbs1 complex, which plays important roles in the early steps of DNA double-strand break repair. Nip1/Ctp1 was phosphorylated in asynchronous, exponentially growing cells and further phosphorylated in response to bleomycin treatment. Overproduction of Nip1/Ctp1 suppressed the DNA repair defect of an nbs1-s10 mutant, which carries a mutation in the FHA phosphopeptide-binding domain of Nbs1, but not of an nbs1 null mutant. Meiotic DNA double-strand breaks accumulated in the nip1/ctp1 mutant. The DNA repair phenotypes and epistasis relationships of nip1/ctp1 are very similar to those of the Saccharomyces cerevisiae sae2/com1 mutant, suggesting that Nip1/Ctp1 is a functional homologue of Sae2/Com1, although the sequence similarity between the proteins is limited to the C-terminal region containing the RHR motif. We found that the RxxL and CxxC motifs are conserved in Schizosaccharomyces species and in vertebrate CtIP, originally identified as a cofactor of the transcriptional corepressor CtBP. However, these two motifs are not found in other fungi, including Saccharomyces and Aspergillus species. We propose that Nip1/Ctp1 is a functional counterpart of Sae2/Com1 and CtIP.

We investigated scanning tunneling microscope (STM)-induced luminescence from Meso-tetrakis (3,5-di-tertiarybutyl-phenyl) porphyrin (H2TBPP) thin films on various metal substrates (copper, platinum and silver) under ambient conditions. Molecular fluorescence similar to the corresponding photoluminescence was observed from the H2TBPP/Ag and H2TBPP/Pt, but not H2TBPP/Cu, depending on the spectra of the surface plasmons of the substrates. The results support the mechanism that intense molecular fluorescence from porphyrin film on the noble metals is a result of enhancement of molecular excitation by substrate surface plasmons.

Study of STM-excited light emission (STML) from organic films is significant for the potential applications to organic photoelectronics devices in nanometer scale. In this note, practical tips for measurements are given, focusing on how to detect very weak light signals efficiently. The mechanism of STML from molecules is described, and the technique of surface-plasmons enhanced molecular fluorescence is utilized for observation of intense light emission at far field regions. Provided are experimental results of STML from spin cast organic films of porphyrin series such as H2TBPP, H2TPP and PhTPP and conjugate polymer MEH-PPV under atmospheric conditions as well as in UHV as the examples.

セラミックスやエレクトロニクス産業の分野で広く使用されている封着加工用鉛ガラスの無鉛化は急務な課題である．本研究ではわれわれの研究グループが報告した封着加工用鉛フリーガラスである，41.8 mol% V2O5–3.1 mol% ZnO–17.6 mol% BaO–37.5 mol% TeO2（以下VTEと略記）ガラスと53.0 mol% V2O5–4.0 mol% ZnO–22.4 mol% BaO–20.6 mol% P2O5（以下VPと略記）ガラスの2つの組成系の実用化に向けた評価試験の検討を行った．評価試験内容としては，平面蛍光管の封着加工後のサーマルショック試験，耐圧試験，連続点灯試験を行った．対象実験として従来の鉛ガラス（以下Pbと略記）で作製した平面蛍光管と比較した結果，サーマルショック試験および加圧試験では鉛ガラスで作製された平面蛍光管と同等の熱衝撃および圧力衝撃性を有していることがわかった．また，連続点灯試験においても鉛ガラスを使用したものと同等の発光特性を有することがわかった．

We have studied the desymmetrization of the polyhedral crystalline shape of tetragonal lysozyme crystals due to the growth rate differences of the equivalent {1 0 1} planes. Using atomic force microscopy, we have observed the evolution of the multifaceted structures composed of four equivalent {1 0 1} faces during growth. In our growth condition, lateral step flow, where a large density of dislocations acts as a source of steps, is the dominant growth mechanism. The measured step flow velocities are almost independent of the separation between the neighboring steps, revealing that the local face normal growth rate is determined by the local step density. By tracing the motion of the vertex surrounded by the {1 0 1} faces, we have found that the desymmetrization of the crystalline shape is due to the large fluctuation of the local face normal growth rate, which is comparable in magnitude to the average growth rate.

We studied the scanning tunneling microscope-induced luminescence (STML) of conjugated polymer poly[2-methoxy-5-(2′-ethyl-hexyloxy)-1,4-phenylene vinylene (MEH-PPV) spun from the solutions with various concentrations on metal substrates Ag and Au. Characteristic molecular fluorescence with the intrinsic molecular vibronic peaks associated with the HOMO–LUMO transition appears in addition to the curve based on plasmon-mediated light emission from the metal substrate Ag or Au. We found that it is a necessary condition to observe molecular fluorescence that the plasmon-mediated light from the metal substrate, which depends on the substrate, is intense in the absorption band of the molecule.

We have investigated the structural evolution of one-dimensional (1D) gratings with high aspect ratios on Si(001) surfaces by high-temperature annealing. Gratings 2 µm in depth and having different pitches from 0.8 to 4.0 µm were annealed at 1100 °C under an ultrahigh vacuum condition. On the basis of observing the cross-sectional profiles of the gratings by scanning electron microscopy, the structural evolution and its dependence the grating period are characterized. The observed decay of the periodic surface structures, in which the wave heights are larger than 0.2–0.3 µm, is faster than the well-known exponential amplitude decay. We have also performed numerical simulation of the profile evolution of 1D gratings based on Mullins' theory, showing that surface self-diffusion is the predominant mass transport mechanism for the observed structural evolution.

Tunneling electrons-induced molecular fluorescence in organic film is enhanced by the surface plasmons. The plasmon enhancement can be expected not only by the plasmons of the substrate but also by the noble metal tip of scanning tunneling microscope (STM). In this report we investigate the tip effect in photoluminescence of meso -tetrakis(3,5-di-tertiarybutyl-phenyl)porphyrin (H 2 TBPP) film on indium tin oxide (ITO) combined with a STM. The experimental result shows the PL of molecules is enhanced by an Ag tip. This enhancement factor is evaluated larger than 2000.

We have studied the effect of the plasmon field on the STM-induced luminescence from porphyrin (H2TBPP) film on Au substrate. We have measured the spectrally resolved photon intensity map for the STM-induced luminescence, and found the similarity of their contrast for the photon maps for different spectral regions with different spectral origins, i.e. molecular fluorescence and the local plasmon induced luminescence from the substrate. This fact indicates that the molecular fluorescence is closely related with the plasmon field of the substrate, and supports that the STM-induced fluorescence from porphyrin film on Au is enhanced by the local plasmon field of the Au substrate.

Several accessory proteins referred to as mediators are required for the full activity of the Rad51 (Rhp51 in fission yeast) recombinase. In this study, we analyzed in vivo functions of the recently discovered Swi5/Sfr1 complex from fission yeast. In normally growing cells, the Swi5-GFP protein localizes to the nucleus, where it forms a diffuse nuclear staining pattern with a few distinct foci. These spontaneous foci do not form in swi2Delta mutants. Upon UV irradiation, Swi5 focus formation is induced in swi2Delta mutants, a response that depends on Sfr1 function, and Sfr1 also forms foci that colocalize with damage-induced Rhp51 foci. The number of UV-induced Rhp51 foci is partially reduced in swi5Delta and rhp57Delta mutants and completely abolished in an swi5Delta rhp57Delta double mutant. An assay for products generated by HO endonuclease-induced DNA double-strand breaks (DSBs) reveals that Rhp51 and Rhp57, but not Swi5/Sfr1, are essential for crossover production. These results suggest that Swi5/Sfr1 functions as an Rhp51 mediator but processes DSBs in a manner different from that of the Rhp55/57 mediator.

We have studied the decay kinetics of nanoscale multilayer holes on SrTiO 3 (0 0 1) surfaces, using variable temperature scanning tunneling microscopy. We have performed real time observation of the decay of multilayer holes with diameters of 10 nm order at 750 °C. We have found that the hole decays, filling layer by layer from the bottom while expanding the periphery of the hole. We have performed numerical simulations of hole decay based on a step flow model. The observed decay kinetics is found to be diffusion limited with local mass conservation.