Joint Molecules Research Articles

Development of Ion Conductive Monolithic Polymers

Polymer monoliths composed with macroporous sizes are one of the potential candidates in separation technology and for efficient and stable separators in batteries and fuel cells [1]. Recently, thiol-ene "click" reaction has been received extensive attention in the field of monolith preparation owing to its exceptional versatility and propensity for the quantitative conversions under mild conditions along with extending the choice of versatile categories of functional monomers. In this work we report a wide variety of joint-linker type porous polymers induced by phase separation during polymerization, by utilizing thiol-ene reaction of multi-functional thiol, 1,3,4-Tris(3-mercaptobutyloxethyl)-1,3,5-triazine-2,4,6- (1H,3H,5H)-trione (3T), pentaerythritol tetrakis(3-mercaptobutylate) (PTMB) etc. as the joint molecules, and poly (ethylene glycol) diacrylate-1000 (PEGDA-1000) as the linker molecules in ethanol. Ion conducting phenomenon of these monoliths has been introduced by swelling them in a wide variety of Ionic liquids (IL). We have studied the concentration effect of joint molecules and temperature on the physical features of these monolith polymers. These microporous monoliths exhibited diverse mechanical prosperities and morphology. In contrary to ionic gels [3] the monolithic structure with entrapped ILs exhibited high ionic conductivity due to the loosely bound ions within the porous polymer network. Physical properties of these IL entrapped monoliths have been investigated to optimize suitable IL-monolith combination for best performance in application of separator membranes for batteries and for separation technology. References Sakakibara, K., Kagata, H., Ishizuka, N., Sato, T., Tsujii, Y. Fabrication of surface skinless membranes of epoxy resin-based mesoporous monoliths toward advanced separators for lithium ion batteries. J. Mater. Chem. A, 5, 6866–6873 (2017).Liu, Z., Ou, J., Zou, H., Click polymerization for preparation of monolithic columns for liquid chromatography. Trends Anal. Chem., 82, 89–99 (2016).Ahmed, K., Naga, N., Kawakami, M. & Furukawa, H. Development of ionic gels using thiol-based monomers in ionic liquid. 9800, 980005 (2016).

The conserved LEM-3/Ankle1 nuclease is involved in the combinatorial regulation of meiotic recombination repair and chromosome segregation in Caenorhabditis elegans.

Homologous recombination is essential for crossover (CO) formation and accurate chromosome segregation during meiosis. It is of considerable importance to work out how recombination intermediates are processed, leading to CO and non-crossover (NCO) outcome. Genetic analysis in budding yeast and Caenorhabditis elegans indicates that the processing of meiotic recombination intermediates involves a combination of nucleases and DNA repair enzymes. We previously reported that in C. elegans meiotic joint molecule resolution is mediated by two redundant pathways, conferred by the SLX-1 and MUS-81 nucleases, and by the HIM-6 Bloom helicase in conjunction with the XPF-1 endonuclease, respectively. Both pathways require the scaffold protein SLX-4. However, in the absence of all these enzymes, residual processing of meiotic recombination intermediates still occurs and CO formation is reduced but not abolished. Here we show that the LEM-3 nuclease, mutation of which by itself does not have an overt meiotic phenotype, genetically interacts with slx-1 and mus-81 mutants, the respective double mutants displaying 100% embryonic lethality. The combined loss of LEM-3 and MUS-81 leads to altered processing of recombination intermediates, a delayed disassembly of foci associated with CO designated sites, and the formation of univalents linked by SPO-11 dependent chromatin bridges (dissociated bivalents). However, LEM-3 foci do not colocalize with ZHP-3, a marker that congresses into CO designated sites. In addition, neither CO frequency nor distribution is altered in lem-3 single mutants or in combination with mus-81 or slx-4 mutations. Finally, we found persistent chromatin bridges during meiotic divisions in lem-3; slx-4 double mutants. Supported by the localization of LEM-3 between dividing meiotic nuclei, this data suggest that LEM-3 is able to process erroneous recombination intermediates that persist into the second meiotic division.

Regulated Crossing-Over Requires Inactivation of Yen1/GEN1 Resolvase during Meiotic Prophase I

During meiosis, crossover recombination promotes the establishment of physical connections between homologous chromosomes, enabling their bipolar segregation. To ensure that persistent recombination intermediates are disengaged prior to the completion of meiosis, the Yen1(GEN1) resolvase is strictly activated at the onset of anaphase II. Whether controlled activation of Yen1 is important for meiotic crossing-over is unknown. Here, we show that CDK-mediated phosphorylation of Yen1 averts its pervasive recruitment to recombination intermediates during prophase I. Yen1 mutants that are refractory to phosphorylation resolve DNA joint molecules prematurely and form crossovers independently of MutLγ, the central crossover resolvase during meiosis. Despite bypassing the requirement for MutLγ in joint molecule processing and promoting crossover-specific resolution, unrestrained Yen1 impairs the spatial distribution of crossover events, genome-wide. Thus, active suppression of Yen1 function, and by inference also of Mus81-Mms4(EME1) and Slx1-Slx4(BTBD12) resolvases, avoids precocious resolution of recombination intermediates to enable meiotic crossover patterning.

Multi-Invasion-Induced Rearrangements as a Pathway for Physiological and Pathological Recombination.

Cells mitigate the detrimental consequences of DNA damage on genome stability by attempting high fidelity repair. Homologous recombination templates DNA double-strand break (DSB) repair on an identical or near identical donor sequence in a process that can in principle access the entire genome. Other physiological processes, such as homolog recognition and pairing during meiosis, also harness the HR machinery using programmed DSBs to physically link homologs and generate crossovers. A consequence of the homology search process by a long nucleoprotein filament is the formation of multi-invasions (MI), a joint molecule in which the damaged ssDNA has invaded more than one donor molecule. Processing of MI joint molecules can compromise the integrity of both donor sites and lead to their rearrangement. Here, two mechanisms for the generation of rearrangements as a pathological consequence of MI processing are detailed and the potential relevance for non-allelic homologous recombination discussed. Finally, it is proposed that MI-induced crossover formation may be a feature of physiological recombination.

Massive crossover elevation via combination of HEI10 and recq4a recq4b during Arabidopsis meiosis

During meiosis, homologous chromosomes undergo reciprocal crossovers, which generate genetic diversity and underpin classical crop improvement. Meiotic recombination initiates from DNA double-strand breaks (DSBs), which are processed into single-stranded DNA that can invade a homologous chromosome. The resulting joint molecules can ultimately be resolved as crossovers. In Arabidopsis, competing pathways balance the repair of ∼100-200 meiotic DSBs into ∼10 crossovers per meiosis, with the excess DSBs repaired as noncrossovers. To bias DSB repair toward crossovers, we simultaneously increased dosage of the procrossover E3 ligase gene HEI10 and introduced mutations in the anticrossovers helicase genes RECQ4A and RECQ4B As HEI10 and recq4a recq4b increase interfering and noninterfering crossover pathways, respectively, they combine additively to yield a massive meiotic recombination increase. Interestingly, we also show that increased HEI10 dosage increases crossover coincidence, which indicates an effect on interference. We also show that patterns of interhomolog polymorphism and heterochromatin drive recombination increases distally towards the subtelomeres in both HEI10 and recq4a recq4b backgrounds, while the centromeres remain crossover suppressed. These results provide a genetic framework for engineering meiotic recombination landscapes in plant genomes.

A meiotic XPF-ERCC1-like complex recognizes joint molecule recombination intermediates to promote crossover formation.

Meiotic crossover formation requires the stabilization of early recombination intermediates by a set of proteins and occurs within the environment of the chromosome axis, a structure important for the regulation of meiotic recombination events. The molecular mechanisms underlying and connecting crossover recombination and axis localization are elusive. Here, we identified the ZZS (Zip2-Zip4-Spo16) complex, required for crossover formation, which carries two distinct activities: one provided by Zip4, which acts as hub through physical interactions with components of the chromosome axis and the crossover machinery, and the other carried by Zip2 and Spo16, which preferentially bind branched DNA molecules in vitro. We found that Zip2 and Spo16 share structural similarities to the structure-specific XPF-ERCC1 nuclease, although it lacks endonuclease activity. The XPF domain of Zip2 is required for crossover formation, suggesting that, together with Spo16, it has a noncatalytic DNA recognition function. Our results suggest that the ZZS complex shepherds recombination intermediates toward crossovers as a dynamic structural module that connects recombination events to the chromosome axis. The identification of the ZZS complex improves our understanding of the various activities required for crossover implementation and is likely applicable to other organisms, including mammals.

RecA requires two molecules of Mg2+ ions for its optimal strand exchange activity in vitro.

Mg2+ 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 Mg2+ and the strand exchange reaction itself, we investigated the interaction of RecA with Mg2+ 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 Mg2+ ions with KD ≈ 2 mM and 5 mM. Deletion of the C-terminal acidic tail of RecA made its thermal stability and fluorescence characteristics insensitive to Mg2+ and similar to those of full-length RecA in the presence of saturating Mg2+. 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 Mg2+ to the tail prevents these interactions and releases RecA from inhibition. We observed that binding of the first Mg2+ 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.

Monitoring Recombination During Meiosis in Budding Yeast.

Homologous recombination is fundamental to sexual reproduction, facilitating accurate segregation of homologous chromosomes at the first division of meiosis, and creating novel allele combinations that fuel evolution. Following initiation of meiotic recombination by programmed DNA double-strand breaks (DSBs), homologous pairing and DNA strand exchange form joint molecule (JM) intermediates that are ultimately resolved into crossover and noncrossover repair products. Physical monitoring of the DNA steps of meiotic recombination in Saccharomyces cerevisiae (budding yeast) cultures undergoing synchronous meiosis has provided seminal insights into the molecular basis of meiotic recombination and affords a powerful tool for dissecting the molecular roles of recombination factors. This chapter describes a suit of electrophoretic and Southern hybridization techniques used to detect and quantify the DNA intermediates of meiotic recombination at recombination hotspots in budding yeast. DSBs and recombination products (crossovers and noncrossovers) are resolved using one-dimensional electrophoresis and distinguished by restriction site polymorphisms between the parental chromosomes. Psoralen cross-linking is used to stabilize branched JMs, which are resolved from linear species by native/native two-dimensional electrophoresis. Native/denaturing two-dimensional electrophoresis is employed to determine the component DNA strands of JMs and to measure the processing of DSBs. These techniques are generally applicable to any locus where the frequency of recombination is high enough to detect intermediates by Southern hybridization.

Dissecting the Recombination Mediator Activity of BRCA2 Using Biochemical Methods.

Homologous recombination (HR) is an essential pathway to restart stalled replication forks, repair spontaneous DNA double-strand breaks, and generate genetic diversity. Together with genetic studies in model organisms, the development of purification protocols and biochemical assays has allowed investigators to begin to understand how the complex machinery of HR functions. At the core of the HR process is the recombination enzyme RecA in bacteria or RAD51 and DMC1 in eukaryotes. The main steps of HR can be reconstituted in vitro and involve: (1) The formation of a ssDNA-RAD51 complex into a helical structure termed the nucleoprotein filament after one DNA strand has been resected at the site of the break. (2) The homologous DNA pairing with an intact copy of the damaged chromatid to form a joint molecule also called displacement loop (D-loop). (3) The exchange of DNA strands and de novo DNA synthesis to restore the damaged/lost DNA. (4) The resolution of joint molecules by nucleolytic cleavage. The human tumor suppressor BRCA2 is a mediator of HR as it actively facilitates the DNA transactions of the recombination proteins RAD51 and DMC1 in a variety of ways: It stabilizes ssDNA-RAD51/DMC1 nucleoprotein filaments. It limits the assembly of RAD51 on dsDNA. It facilitates the replacement of replication protein A by RAD51. The result of these activities is a net increase of DNA strand exchange products as observed in vitro. Here, we describe some of the biochemical assays used to dissect the mediator activities of BRCA2.

Reconstituting the 4-Strand DNA Strand Exchange.

Proteins of the Rad51 family play a key role in homologous recombination by carrying out DNA strand exchange. Here, we present the methodology and the protocols for the 4-strand exchange between gapped circular DNA and homologous linear duplex DNA promoted by human Rad51 and Escherichia coli RecA orthologs. This reaction includes formation of joint molecules and their extension by branch migration in a polar manner. The presented methodology may be used for reconstitution of the medial-to-late stages of homologous recombination in vitro as well as for investigation of the mechanisms of branch migration by helicase-like proteins, e.g., Rad54, BLM, or RecQ1.

Unresolved recombination intermediates lead to ultra-fine anaphase bridges, chromosome breaks and aberrations.

The resolution of joint molecules that link recombining sister chromatids is essential for chromosome segregation. Here, we determine the fate of unresolved recombination intermediates that arise in GEN1-/- knock-out cells depleted for MUS81, the two nucleases required for resolution. We find that intermediates persist until mitosis where they form a distinct class of anaphase bridges, which we term homologous recombination ultra-fine bridges, or HR-UFBs. The HR-UFBs are distinct from replication stress-associated UFBs, that arise at common fragile sites, and from centromeric UFBs. HR-UFBs are processed by BLM helicase to generate single-stranded RPA-coated bridges that are broken at mitosis. In the next cell cycle, DNA breaks activate the DNA damage checkpoint and chromosome fusions arise by non-homologous end joining. Consequently, the cells undergo a cell cycle delay and massive cell death. These results lead us to present a model detailing how unresolved recombination intermediates can promote DNA damage and chromosomal instability.

Break-induced replication promotes formation of lethal joint molecules dissolved by Srs2

Break-induced replication (BIR) is a DNA double-strand break repair pathway that leads to genomic instabilities similar to those observed in cancer. BIR proceeds by a migrating bubble where asynchrony between leading and lagging strand synthesis leads to accumulation of long single-stranded DNA (ssDNA). It remains unknown how this ssDNA is prevented from unscheduled pairing with the template, which can lead to genomic instability. Here, we propose that uncontrolled Rad51 binding to this ssDNA promotes formation of toxic joint molecules that are counteracted by Srs2. First, Srs2 dislodges Rad51 from ssDNA preventing promiscuous strand invasions. Second, it dismantles toxic intermediates that have already formed. Rare survivors in the absence of Srs2 rely on structure-specific endonucleases, Mus81 and Yen1, that resolve toxic joint-molecules. Overall, we uncover a new feature of BIR and propose that tight control of ssDNA accumulated during this process is essential to prevent its channeling into toxic structures threatening cell viability.

Modification of meiotic recombination by natural variation in plants.

Meiosis is a specialized cell division that produces haploid gametes required for sexual reproduction. During the first meiotic division, homologous chromosomes pair and undergo reciprocal crossing over, which recombines linked sequence variation. Meiotic recombination frequency varies extensively both within and between species. In this review, we will examine the molecular basis of meiotic recombination rate variation, with an emphasis on plant genomes. We first consider cis modification caused by polymorphisms at the site of recombination, or elsewhere on the same chromosome. We review cis effects caused by mismatches within recombining joint molecules, the effect of structural hemizygosity, and the role of specific DNA sequence motifs. In contrast, trans modification of recombination is exerted by polymorphic loci encoding diffusible molecules, which are able to modulate recombination on the same and/or other chromosomes. We consider trans modifiers that act to change total recombination levels, hotspot locations, or interactions between homologous and homeologous chromosomes in polyploid species. Finally, we consider the significance of genetic variation that modifies meiotic recombination for adaptation and evolution of plant species.

The Nucleoporin Nup2 Contains a Meiotic-Autonomous Region that Promotes the Dynamic Chromosome Events of Meiosis.

Meiosis is a specialized cellular program required to create haploid gametes from diploid parent cells. Homologous chromosomes pair, synapse, and recombine in a dynamic environment that accommodates gross chromosome reorganization and significant chromosome motion, which are critical for normal chromosome segregation. In Saccharomyces cerevisiae, Ndj1 is a meiotic telomere-associated protein required for physically attaching telomeres to proteins embedded in the nuclear envelope. In this study, we identified additional proteins that act at the nuclear periphery from meiotic cell extracts, including Nup2, a nonessential nucleoporin with a known role in tethering interstitial chromosomal loci to the nuclear pore complex. We found that deleting NUP2 affects meiotic progression and spore viability, and gives increased levels of recombination intermediates and products. We identified a previously uncharacterized 125aa region of Nup2 that is necessary and sufficient for its meiotic function, thus behaving as a meiotic autonomous region (MAR). Nup2-MAR forms distinct foci on spread meiotic chromosomes, with a subset overlapping with Ndj1 foci. Localization of Nup2-MAR to meiotic chromosomes does not require Ndj1, nor does Ndj1 localization require Nup2, suggesting these proteins function in different pathways, and their interaction is weak or indirect. Instead, several severe synthetic phenotypes are associated with the nup2Δ ndj1Δ double mutant, including delayed turnover of recombination joint molecules, and a failure to undergo nuclear divisions without also arresting the meiotic program. These data suggest Nup2 and Ndj1 support partially overlapping functions that promote two different levels of meiotic chromosome organization necessary to withstand a dynamic stage of the eukaryotic life cycle.

Expression and clinical relevance of SPOPL in medulloblastoma.

Medulloblastoma (MB) is a type of malignant brain tumor in children. Although knowledge of MB is increasing and the survival rate of patients with MB has improved in previous years, the long-term treatment-associated complications remain unfavorable. Early diagnosis and treatment is critical for patients with MB, but effective molecular markers for MB remain elusive. The Speckle-type POZ protein (SPOP) is a member of the MATH-BTB protein family and is a type of joint molecule for Cullin-3. SPOP inhibits tumor growth. However, the SPOP-like (SPOPL) gene, which is a SPOP paralog gene and shares an overall 85% sequence identity with SPOP, has not been explored in cancer studies at present. The results of the present study demonstrate that the SPOPL expression is decreased in MB cells and tissues compared with normal cells and tissues at the protein and mRNA levels. Immunohistochemical analysis revealed decreased expression of SPOPL in 42/56 (75%) paraffin-embedded archival MB biopsies, and SPOPL expression may be associated with the MB differentiation level (P=0.011). Patients with increased SPOPL expression exhibit improved survival rates compared with those with decreased SPOPL expression, and the SPOPL gene may be a potentially valuable molecular marker of MB.

Roles of Budding Yeast Hrr25 in Recombination and Sporulation.

Hrr25, a casein kinase 1 δ/ε homolog in budding yeast, is essential to set up mono-orientation of sister kinetochores during meiosis. Hrr25 kinase activity coordinates sister chromatid cohesion via cohesin phosphorylation. Here, we investigated the prophase role of Hrr25 using the auxin-inducible degron system and by ectopic expression of Hrr25 during yeast meiosis. Hrr25 mediates nuclear division in meiosis I but does not affect DNA replication. We also found that initiation of meiotic double-strand breaks as well as joint molecule formation were normal in HRR25-deficient cells. Thus, Hrr25 is essential for termination of meiotic division but not homologous recombination.

Synthesis of Network Polymers Containing Si-Vinylene Units by Mizoroki-Heck Reaction

Network polymers containing Si-vinylene units have been synthesized by Mizoroki-Heck reaction of cyclic siloxane or cubic silsesquioxane compounds with vinyl groups, as joint molecules, and dibromo aryl compounds, as linker molecules, using a Pd catalyst. The reaction of 1,3,5,7-tetravinyltetramethylcyclotetrasiloxane (TVMCTS) or octavinyloctasilsesquioxane (PVOSS) with o, m, p-dibromobenzene (DBB) or 4,4'-dibromobiphenyl (DBBP) yielded the corresponding network polymers, except TVMCTS-oDBB system. Optical properties of the network polymers were studied by UV-vis and photoluminescence spectroscopy, and absorption and emission derived from s-p conjugation of the Si-vinylene units were detected. Copolymerization of TVMCTS-DBB/bromobenzene yielded CHCl3 soluble fraction due to formation of the network fragments. The network polymers of TVMCTS or PVOSS with 2,7-dibromofluorene (BFl) or 9,9-dihexyl-2,7-dibromofluorene were synthesized by the same procedures. Scanning electron microscope image of TVMCTS-BFl network polymer indicated formation of porous structure. The network polymers have been also synthesized by the reactions of multifunctional aryl bromide, as the joint molecules, with divinyl or dially silane compounds, as the linker molecules. The molecular structure of the multifunctional aryl bromide affected the emission wavelengths of the network polymers. In the case of the 2,2’,7,7’-tetrabromo-9,9’-spirobifluorene-based network polymers, the emission spectra widely ranged from 450 to 500 nm, which were sensitive to molecular structure of the divinyl or dially silane compounds.

RecG controls DNA amplification at double‐strand breaks and arrested replication forks

DNA amplification is a powerful mutational mechanism that is a hallmark of cancer and drug resistance. It is therefore important to understand the fundamental pathways that cells employ to avoid over‐replicating sections of their genomes. Recent studies demonstrate that, in the absence of RecG, DNA amplification is observed at sites of DNA double‐strand break repair (DSBR) and of DNA replication arrest that are processed to generate double‐strand ends. RecG also plays a role in stabilising joint molecules formed during DSBR. We propose that RecG prevents a previously unrecognised mechanism of DNA amplification that we call reverse‐restart, which generates DNA double‐strand ends from incorrect loading of the replicative helicase at D‐loops formed by recombination, and at arrested replication forks.

Differential requirement of Srs2 helicase and Rad51 displacement activities in replication of hairpin-forming CAG/CTG repeats.

Trinucleotide repeats are a source of genome instability, causing replication fork stalling, chromosome fragility, and impaired repair. Specialized helicases play an important role in unwinding DNA structures to maintain genome stability. The Srs2 helicase unwinds DNA hairpins, facilitates replication, and prevents repeat instability and fragility. However, since Srs2 is a multifunctional protein with helicase activity and the ability to displace Rad51 recombinase, it was unclear which functions were required for its various protective roles. Here, using SRS2 separation-of-function alleles, we show that in the absence of Srs2 recruitment to PCNA or in helicase-deficient mutants, breakage at a CAG/CTG repeat increases. We conclude that Srs2 interaction with PCNA allows the helicase activity to unwind fork-blocking CAG/CTG hairpin structures to prevent breaks. Independently of PCNA binding, Srs2 also displaces Rad51 from nascent strands to prevent recombination-dependent repeat expansions and contractions. By 2D gel electrophoresis, we detect two different kinds of structured intermediates or joint molecules (JMs). Some JMs are Rad51-independent and exhibit properties of reversed forks, including being processed by the Exo1 nuclease. In addition, in a helicase-deficient mutant, Rad51-dependent JMs are detected, probably corresponding to recombination between sisters. These results clarify the many roles of Srs2 in facilitating replication through fork-blocking hairpin lesions.

Dbf4‐dependent kinase and the Rtt107 scaffold promote Mus81‐Mms4 resolvase activation during mitosis

DNA repair by homologous recombination is under stringent cell cycle control. This includes the last step of the reaction, disentanglement of DNA joint molecules (JMs). Previous work has established that JM resolving nucleases are activated specifically at the onset of mitosis. In case of budding yeast Mus81‐Mms4, this cell cycle stage‐specific activation is known to depend on phosphorylation by CDK and Cdc5 kinases. Here, we show that a third cell cycle kinase, Cdc7‐Dbf4 (DDK), targets Mus81‐Mms4 in conjunction with Cdc5—both kinases bind to as well as phosphorylate Mus81‐Mms4 in an interdependent manner. Moreover, DDK‐mediated phosphorylation of Mms4 is strictly required for Mus81 activation in mitosis, establishing DDK as a novel regulator of homologous recombination. The scaffold protein Rtt107, which binds the Mus81‐Mms4 complex, interacts with Cdc7 and thereby targets DDK and Cdc5 to the complex enabling full Mus81 activation. Therefore, Mus81 activation in mitosis involves at least three cell cycle kinases, CDK, Cdc5 and DDK. Furthermore, tethering of the kinases in a stable complex with Mus81 is critical for efficient JM resolution.