Joint Molecules Research Articles

Concurrent D-loop cleavage by Mus81 and Yen1 yields half-crossover precursors.

Homologous recombination involves the formation of branched DNA molecules that may interfere with chromosome segregation. To resolve these persistent joint molecules, cells rely on the activation of structure-selective endonucleases (SSEs) during the late stages of the cell cycle. However, the premature activation of SSEs compromises genome integrity, due to untimely processing of replication and/or recombination intermediates. Here, we used a biochemical approach to show that the budding yeast SSEs Mus81 and Yen1 possess the ability to cleave the central recombination intermediate known as the displacement loop or D-loop. Moreover, we demonstrate that, consistently with previous genetic data, the simultaneous action of Mus81 and Yen1, followed by ligation, is sufficient to recreate the formation of a half-crossover precursor in vitro. Our results provide not only mechanistic explanation for the formation of a half-crossover, but also highlight the critical importance for precise regulation of these SSEs to prevent chromosomal rearrangements.

Purification of Saccharomyces cerevisiae recombinant Crp1

A complex Mus81-Mm4 is a DNA structure–specific endonuclease in Saccharomyces cerevisiae. Mus81-Mms4 functions in processing of recombination intermediates that could arise during the repair of stalled and blocked replication forks and double stranded breaks. Mus81-Mms4 works with many proteins involved in DNA repair, replication fork stability, and joint molecule formation/resolution during homologous recombination repair. A biochemical screening of protein(s) that enhances the Mus81-Mms4 endonuclease activity on its preferable substrates in vitro revealed that Crp1, a cruciform DNA-recognizing protein, which can specifically bind to DNA four-way junction structures like Holliday junctions could be the potential factor. To further demonstrate that Crp1 interacts functionally with Mus81-Mms4 in vitro, we carried out the purification of recombinant Crp1 using Escherichia coli system. Our results showed that the purified Crp1 was highly homogenous and active that is ready for biochemical use.

Heterozygosity alters Msh5 binding to meiotic chromosomes in the baker's yeast.

Meiotic crossovers are initiated from programmed DNA double-strand breaks. The Msh4-Msh5 heterodimer is an evolutionarily conserved mismatch repair-related protein complex that promotes meiotic crossovers by stabilizing strand invasion intermediates and joint molecule structures such as Holliday junctions. In vivo studies using homozygous strains of the baker's yeast Saccharomyces cerevisiae (SK1) show that the Msh4-Msh5 complex associates with double-strand break hotspots, chromosome axes, and centromeres. Many organisms have heterozygous genomes that can affect the stability of strand invasion intermediates through heteroduplex rejection of mismatch-containing sequences. To examine Msh4-Msh5 function in a heterozygous context, we performed chromatin immunoprecipitation and sequencing (ChIP-seq) analysis in a rapidly sporulating hybrid S. cerevisiae strain (S288c-sp/YJM789, containing sporulation-enhancing QTLs from SK1), using SNP information to distinguish reads from homologous chromosomes. Overall, Msh5 localization in this hybrid strain was similar to that determined in the homozygous strain (SK1). However, relative Msh5 levels were reduced in regions of high heterozygosity, suggesting that high mismatch densities reduce levels of recombination intermediates to which Msh4-Msh5 binds. Msh5 peaks were also wider in the hybrid background compared to the homozygous strain (SK1). We determined regions containing heteroduplex DNA by detecting chimeric sequence reads with SNPs from both parents. Msh5-bound double-strand break hotspots overlap with regions that have chimeric DNA, consistent with Msh5 binding to heteroduplex-containing recombination intermediates.

A Method for Physical Analysis of Recombination Intermediates in Saccharomyces cerevisiae.

Meiosis is a process through which diploid cells divide into haploid cells, thus promoting genetic diversity. This diversity arises from the formation of genetic crossovers (COs) that repair DNA double-strand breaks (DSBs), through homologous recombination (HR). Deficiencies in HR can lead to chromosomal abnormality resulting from chromosomal nondisjunction, and genetic disorders. Therefore, investigating the mechanisms underlying effective HR is crucial for reducing genome instability. Budding yeast serves as an ideal model for studying HR mechanisms due to its amenability to gene modifications and the ease of inducing synchronized meiosis to yield four spores. During meiosis, at the DNA level, programmed DSBs are repaired as COs or non-crossovers (NCOs) through structural alterations in the nascent D-loop, involving single-end invasions(SEIs) and double-Holliday junctions(dHJs). This repair occurs using homologous templates rather than sister templates. This protocol, using Southern blotting, allows for the analysis and monitoring of changes in DNA structures in the recombination process. One-dimensional (1D) gel electrophoresis is employed to detect DSBs, COs, and NCOs, while two-dimensional (2D) gel electrophoresis is utilized to identify joint molecules (JMs). Therefore, physical analysis is considered the most effective method for investigating the HR mechanism. Our protocol provides more comprehensive information than previous reports by introducing conditions for obtaining a greater number of cells from synchronized yeast and a method that can analyze not only meiotic/mitotic recombination but also mitotic replication.

The FLIP-FIGNL1 complex regulates the dissociation of RAD51/DMC1 in homologous recombination and replication fork restart.

Recruitment of RAD51 and/or DMC1 recombinases to single-strand DNA is indispensable for homology search and strand invasion in homologous recombination (HR) and for protection of nascent DNA strands at stalled replication forks. Thereafter RAD51/DMC1 dissociate, actively or passively, from these joint molecules upon DNA repair or releasing from replication stress. However, the mechanism that regulates RAD51/DMC1 dissociation and its physiological importance remain elusive. Here, we show that a FLIP-FIGNL1 complex regulates RAD51 and DMC1 dissociation to promote meiotic recombination and replication fork restart in mammals. Mice lacking FLIP are embryonic lethal, while germline-specific deletion of FLIP leads to infertility in both males and females. FLIP-null meiocytes are arrested at a zygotene-like stage with massive RAD51 and DMC1 foci, which frequently co-localize with SHOC1 and TEX11. Furthermore, FLIP interacts with FIGNL1. Depletion of FLIP or FIGNL1 in cell lines destabilizes each other and impairs RAD51 dissociation. Thus, the active dissociation of RAD51/DMC1 by the FLIP-FIGNL1 complex is a crucial step required for HR and replication fork restart, and represents a conserved mechanism in somatic cells and germ cells.

Delineation of two multi-invasion-induced rearrangement pathways that differently affect genome stability.

Punctuated bursts of structural genomic variations (SVs) have been described in various organisms, but their etiology remains incompletely understood. Homologous recombination (HR) is a template-guided mechanism of repair of DNA double-strand breaks and stalled or collapsed replication forks. We recently identified a DNA break amplification and genome rearrangement pathway originating from the endonucleolytic processing of a multi-invasion (MI) DNA joint molecule formed during HR. Genome-wide approaches confirmed that multi-invasion-induced rearrangement (MIR) frequently leads to several repeat-mediated SVs and aneuploidies. Using molecular and genetic analysis and a novel, highly sensitive proximity ligation-based assay for chromosomal rearrangement quantification, we further delineate two MIR subpathways. MIR1 is a universal pathway occurring in any sequence context, which generates secondary breaks and frequently leads to additional SVs. MIR2 occurs only if recombining donors exhibit substantial homology and results in sequence insertion without additional breaks or SVs. The most detrimental MIR1 pathway occurs late on a subset of persisting DNA joint molecules in a PCNA/Polδ-independent manner, unlike recombinational DNA synthesis. This work provides a refined mechanistic understanding of these HR-based SV formation pathways and shows that complex repeat-mediated SVs can occur without displacement DNA synthesis. Sequence signatures for inferring MIR1 from long-read data are proposed.

FIRRM/C1orf112 is synthetic lethal with PICH and mediates RAD51 dynamics

Joint DNA molecules are natural byproducts of DNA replication and repair. Persistent joint molecules give rise to ultrafine DNA bridges (UFBs) in mitosis, compromising sister chromatid separation. The DNA translocase PICH (ERCC6L) has a central role in UFB resolution. A genome-wide loss-of-function screen is performed to identify the genetic context of PICH dependency. In addition to genes involved in DNA condensation, centromere stability, and DNA-damage repair, we identify FIGNL1-interacting regulator of recombination and mitosis (FIRRM), formerly known as C1orf112. We find that FIRRM interacts with and stabilizes the AAA+ ATPase FIGNL1. Inactivation of either FIRRM or FIGNL1 results in UFB formation, prolonged accumulation of RAD51 at nuclear foci, and impaired replication fork dynamics and consequently impairs genome maintenance. Combined, our data suggest that inactivation of FIRRM and FIGNL1 dysregulates RAD51 dynamics at replication forks, resulting in persistent DNA lesions and a dependency on PICH to preserve cell viability.

Stabilization of DNA fork junctions by Smc5/6 complexes revealed by single-molecule imaging.

SMC complexes play key roles in genome maintenance, where they ensure efficient genome replication and segregation. The SMC complex Smc5/6 is a crucial player in DNA replication and repair, yet many molecular features that determine its roles are unclear. Here, we use single-molecule microscopy to investigate Smc5/6's interaction with DNA. We find that Smc5/6 forms oligomers that dynamically redistribute on dsDNA by 1D diffusion and statically bind to ssDNA. Using combined force manipulation and single-molecule microscopy, we generate ssDNA-dsDNA junctions that mimic structures present in DNA repair intermediates or replication forks. We show that Smc5/6 accumulates at these junction sites, stabilizes the fork, and promotes the retention of RPA. Our observations provide a model for the complex's enrichment at sites of replication stress and DNA lesions from where it coordinates the recruitment and activation of downstream repair proteins.

Multiple Roles of SMC5/6 Complex during Plant Sexual Reproduction.

Chromatin-based processes are essential for cellular functions. Structural maintenance of chromosomes (SMCs) are evolutionarily conserved molecular machines that organize chromosomes throughout the cell cycle, mediate chromosome compaction, promote DNA repair, or control sister chromatid attachment. The SMC5/6 complex is known for its pivotal role during the maintenance of genome stability. However, a dozen recent plant studies expanded the repertoire of SMC5/6 complex functions to the entire plant sexual reproductive phase. The SMC5/6 complex is essential in meiosis, where its activity must be precisely regulated to allow for normal meiocyte development. Initially, it is attenuated by the recombinase RAD51 to allow for efficient strand invasion by the meiosis-specific recombinase DMC1. At later stages, it is essential for the normal ratio of interfering and non-interfering crossovers, detoxifying aberrant joint molecules, preventing chromosome fragmentation, and ensuring normal chromosome/sister chromatid segregation. The latter meiotic defects lead to the production of diploid male gametes in Arabidopsis SMC5/6 complex mutants, increased seed abortion, and production of triploid offspring. The SMC5/6 complex is directly involved in controlling normal embryo and endosperm cell divisions, and pioneer studies show that the SMC5/6 complex is also important for seed development and normal plant growth in cereals.

Measuring the contributions of helicases to break-induced replication.

Break-Induced Replication (BIR) is a homologous recombination (HR) pathway that differentiates itself from all other HR pathways by involving extensive DNA synthesis of up to hundreds of kilobases. This DNA synthesis occurs in G2/M arrested cells by a mechanism distinct from regular DNA replication. BIR initiates by strand invasion of a single end of a DNA double-strand break (DSB) followed by extensive D-loop migration. The main replicative helicase Mcm2-7 is dispensable for BIR, however, Pif1 helicase and its PCNA interaction domain are required. Pif1 helicase was shown to be important for extensive repair-specific DNA synthesis at DSB in budding and fission yeasts, flies, and human cells, implicating conservation of the mechanism. Additionally, Mph1 helicase negatively regulates BIR by unwinding migrating D-loops, and Srs2 promotes BIR by eliminating the toxic joint molecules. Here, we describe the methods that address the following questions in studying BIR: (i) how to distinguish enzymes needed specifically for BIR from enzymes needed for other HR mechanisms that require short patch DNA synthesis, (ii) what are the phenotypes expected for mutants deficient in extensive synthesis during BIR, (iii) how to follow extensive DNA synthesis during BIR? Methods are described using yeast model organism and wild-type cells are compared side-by-side with Pif1 deficient cells.

A role for synaptonemal complex in meiotic mismatch repair.

A large subset of meiotic recombination intermediates form within the physical context of synaptonemal complex (SC), but the functional relationship between SC structure and homologous recombination remains obscure. Our prior analysis of strains deficient for SC central element proteins demonstrated that tripartite SC is dispensable for interhomolog recombination in Saccharomyces cerevisiae. Here, we report that while dispensable for recombination per se, SC proteins promote efficient mismatch repair at interhomolog recombination sites. Failure to repair mismatches within heteroduplex-containing meiotic recombination intermediates leads to genotypically sectored colonies (postmeiotic segregation events). We discovered increased postmeiotic segregation at THR1 in cells lacking Ecm11 or Gmc2, or in the SC-deficient but recombination-proficient zip1[Δ21-163] mutant. High-throughput sequencing of octad meiotic products furthermore revealed a genome-wide increase in recombination events with unrepaired mismatches in ecm11 mutants relative to wildtype. Meiotic cells missing Ecm11 display longer gene conversion tracts, but tract length alone does not account for the higher frequency of unrepaired mismatches. Interestingly, the per-nucleotide mismatch frequency is elevated in ecm11 when analyzing all gene conversion tracts, but is similar between wildtype and ecm11 if considering only those events with unrepaired mismatches. Thus, in both wildtype and ecm11 strains a subset of recombination events is susceptible to a similar degree of inefficient mismatch repair, but in ecm11 mutants a larger fraction of events fall into this inefficient repair category. Finally, we observe elevated postmeiotic segregation at THR1 in mutants with a dual deficiency in MutSγ crossover recombination and SC assembly, but not in the mlh3 mutant, which lacks MutSγ crossovers but has abundant SC. We propose that SC structure promotes efficient mismatch repair of joint molecule recombination intermediates, and that absence of SC is the molecular basis for elevated postmeiotic segregation in both MutSγ crossover-proficient (ecm11, gmc2) and MutSγ crossover-deficient (msh4, zip3) strains.

The synaptonemal complex central region modulates crossover pathways and feedback control of meiotic double-strand break formation.

The synaptonemal complex (SC) is a proteinaceous structure that mediates homolog engagement and genetic recombination during meiosis. In budding yeast, Zip-Mer-Msh (ZMM) proteins promote crossover (CO) formation and initiate SC formation. During SC elongation, the SUMOylated SC component Ecm11 and the Ecm11-interacting protein Gmc2 facilitate the polymerization of Zip1, an SC central region component. Through physical recombination, cytological, and genetic analyses, we found that ecm11 and gmc2 mutants exhibit chromosome-specific defects in meiotic recombination. CO frequencies on a short chromosome (chromosome III) were reduced, whereas CO and non-crossover frequencies on a long chromosome (chromosome VII) were elevated. Further, in ecm11 and gmc2 mutants, more double-strand breaks (DSBs) were formed on a long chromosome during late prophase I, implying that the Ecm11–Gmc2 (EG) complex is involved in the homeostatic regulation of DSB formation. The EG complex may participate in joint molecule (JM) processing and/or double-Holliday junction resolution for ZMM-dependent CO-designated recombination. Absence of the EG complex ameliorated the JM-processing defect in zmm mutants, suggesting a role for the EG complex in suppressing ZMM-independent recombination. Our results suggest that the SC central region functions as a compartment for sequestering recombination-associated proteins to regulate meiosis specificity during recombination.

SUMO fosters assembly and functionality of the MutSγ complex to facilitate meiotic crossing over.

Crossing over is essential for chromosome segregation during meiosis. Protein modification by SUMO is implicated in crossover control, but pertinent targets have remained elusive. Here we identify Msh4 as a target of SUMO-mediated crossover regulation. Msh4 and Msh5 constitute the MutSγ complex, which stabilizes joint-molecule (JM) recombination intermediates and facilitates their resolution into crossovers. Msh4 SUMOylation enhances these processes to ensure that each chromosome pair acquires at least one crossover. Msh4 is directly targeted by E2 conjugase Ubc9, initially becoming mono-SUMOylated in response to DNA double-strand breaks, then multi/poly-SUMOylated forms arise as homologs fully engage. Mechanistically, SUMOylation fosters interaction between Msh4 and Msh5. We infer that initial SUMOylation of Msh4 enhances assembly of MutSγ in anticipation of JM formation, while secondary SUMOylation may promote downstream functions. Regulation of Msh4 by SUMO is distinct and independent of its previously described stabilization by phosphorylation, defining MutSγ as a hub for crossover control.

Smc5/6 functions with Sgs1-Top3-Rmi1 to complete chromosome replication at natural pause sites

Smc5/6 is essential for genome structural integrity by yet unknown mechanisms. Here we find that Smc5/6 co-localizes with the DNA crossed-strand processing complex Sgs1-Top3-Rmi1 (STR) at genomic regions known as natural pausing sites (NPSs) where it facilitates Top3 retention. Individual depletions of STR subunits and Smc5/6 cause similar accumulation of joint molecules (JMs) composed of reversed forks, double Holliday Junctions and hemicatenanes, indicative of Smc5/6 regulating Sgs1 and Top3 DNA processing activities. We isolate an intra-allelic suppressor of smc6-56 proficient in Top3 retention but affected in pathways that act complementarily with Sgs1 and Top3 to resolve JMs arising at replication termination. Upon replication stress, the smc6-56 suppressor requires STR and Mus81-Mms4 functions for recovery, but not Srs2 and Mph1 helicases that prevent maturation of recombination intermediates. Thus, Smc5/6 functions jointly with Top3 and STR to mediate replication completion and influences the function of other DNA crossed-strand processing enzymes at NPSs.

Meiotic DNA break repair can utilize homolog-independent chromatid templates in C. elegans

During meiosis, the maintenance of genome integrity is critical for generating viable haploid gametes.1 In meiotic prophase I, double-strand DNA breaks (DSBs) are induced and a subset of these DSBs are repaired as interhomolog crossovers to ensure proper chromosome segregation. DSBs not resolved as crossovers with the homolog must be repaired by other pathways to ensure genome integrity.2 To determine if alternative repair templates can be engaged for meiotic DSB repair during oogenesis, we developed an assay to detect sister and/or intra-chromatid repair events at a defined DSB site during Caenorhabditis elegans meiosis. Using this assay, we directly demonstrate that the sister chromatid or the same DNA molecule can be engaged as a meiotic repair template for both crossover and noncrossover recombination, with noncrossover events being the predominant recombination outcome. We additionally find that the sister or intra-chromatid substrate is available as a recombination partner for DSBs induced throughout meiotic prophase I, including late prophase when the homolog is unavailable. Analysis of noncrossover conversion tract sequences reveals that DSBs are processed similarly throughout prophase I. We further present data indicating that the XPF-1 nuclease functions in late prophase to promote sister or intra-chromatid repair at steps of recombination following joint molecule processing. Despite its function in sister or intra-chromatid repair, we find that xpf-1 mutants do not exhibit severe defects in progeny viability following exposure to ionizing radiation. Overall, we propose that C.elegans XPF-1 may assist as an intersister or intrachromatid resolvase only in late prophase I.

ATRX and RECQ5 define distinct homologous recombination subpathways

Homologous recombination (HR) is an important DNA double-strand break (DSB) repair pathway that copies sequence information lost at the break site from an undamaged homologous template. This involves the formation of a recombination structure that is processed to restore the original sequence but also harbors the potential for crossover (CO) formation between the participating molecules. Synthesis-dependent strand annealing (SDSA) is an HR subpathway that prevents CO formation and is thought to predominate in mammalian cells. The chromatin remodeler ATRX promotes an alternative HR subpathway that has the potential to form COs. Here, we show that ATRX-dependent HR outcompetes RECQ5-dependent SDSA for the repair of most two-ended DSBs in human cells and leads to the frequent formation of COs, assessed by measuring sister chromatid exchanges (SCEs). We provide evidence that subpathway choice is dependent on interaction of both ATRX and RECQ5 with proliferating cell nuclear antigen. We also show that the subpathway usage varies among different cancer cell lines and compare it to untransformed cells. We further observe HR intermediates arising as ionizing radiation (IR)-induced ultra-fine bridges only in cells expressing ATRX and lacking MUS81 and GEN1. Consistently, damage-induced MUS81 recruitment is only observed in ATRX-expressing cells. Cells lacking BLM show similar MUS81 recruitment and IR-induced SCE formation as control cells. Collectively, these results suggest that the ATRX pathway involves the formation of HR intermediates whose processing is entirely dependent on MUS81 and GEN1 and independent of BLM. We propose that the predominant ATRX-dependent HR subpathway forms joint molecules distinct from classical Holliday junctions.

Single-molecule visualization of human RECQ5 interactions with single-stranded DNA recombination intermediates.

RECQ5 is one of five RecQ helicases found in humans and is thought to participate in homologous DNA recombination by acting as a negative regulator of the recombinase protein RAD51. Here, we use kinetic and single molecule imaging methods to monitor RECQ5 behavior on various nucleoprotein complexes. Our data demonstrate that RECQ5 can act as an ATP-dependent single-stranded DNA (ssDNA) motor protein and can translocate on ssDNA that is bound by replication protein A (RPA). RECQ5 can also translocate on RAD51-coated ssDNA and readily dismantles RAD51–ssDNA filaments. RECQ5 interacts with RAD51 through protein–protein contacts, and disruption of this interface through a RECQ5–F666A mutation reduces translocation velocity by ∼50%. However, RECQ5 readily removes the ATP hydrolysis-deficient mutant RAD51–K133R from ssDNA, suggesting that filament disruption is not coupled to the RAD51 ATP hydrolysis cycle. RECQ5 also readily removes RAD51–I287T, a RAD51 mutant with enhanced ssDNA-binding activity, from ssDNA. Surprisingly, RECQ5 can bind to double-stranded DNA (dsDNA), but it is unable to translocate. Similarly, RECQ5 cannot dismantle RAD51-bound heteroduplex joint molecules. Our results suggest that the roles of RECQ5 in genome maintenance may be regulated in part at the level of substrate specificity.

Implications of Metastable Nicks and Nicked Holliday Junctions in Processing Joint Molecules in Mitosis and Meiosis.

Joint molecules (JMs) are intermediates of homologous recombination (HR). JMs rejoin sister or homolog chromosomes and must be removed timely to allow segregation in anaphase. Current models pinpoint Holliday junctions (HJs) as a central JM. The canonical HJ (cHJ) is a four-way DNA that needs specialized nucleases, a.k.a. resolvases, to resolve into two DNA molecules. Alternatively, a helicase–topoisomerase complex can deal with pairs of cHJs in the dissolution pathway. Aside from cHJs, HJs with a nick at the junction (nicked HJ; nHJ) can be found in vivo and are extremely good substrates for resolvases in vitro. Despite these findings, nHJs have been neglected as intermediates in HR models. Here, I present a conceptual study on the implications of nicks and nHJs in the final steps of HR. I address this from a biophysical, biochemical, topological, and genetic point of view. My conclusion is that they ease the elimination of JMs while giving genetic directionality to the final products. Additionally, I present an alternative view of the dissolution pathway since the nHJ that results from the second end capture predicts a cross-join isomerization. Finally, I propose that this isomerization nicely explains the strict crossover preference observed in synaptonemal-stabilized JMs in meiosis.

Genetic evidence for the involvement of mismatch repair proteins, PMS2 and MLH3, in a late step of homologous recombination

Homologous recombination (HR) repairs DNA double-strand breaks using intact homologous sequences as template DNA. Broken DNA and intact homologous sequences form joint molecules (JMs), including Holliday junctions (HJs), as HR intermediates. HJs are resolved to form crossover and noncrossover products. A mismatch repair factor, MLH3 endonuclease, produces the majority of crossovers during meiotic HR, but it remains elusive whether mismatch repair factors promote HR in nonmeiotic cells. We disrupted genes encoding the MLH3 and PMS2 endonucleases in the human B cell line, TK6, generating null MLH3-/- and PMS2-/- mutant cells. We also inserted point mutations into the endonuclease motif of MLH3 and PMS2 genes, generating endonuclease death MLH3DN/DN and PMS2EK/EK cells. MLH3-/- and MLH3DN/DN cells showed a very similar phenotype, a 2.5-fold decrease in the frequency of heteroallelic HR-dependent repair of restriction enzyme-induced double-strand breaks. PMS2-/- and PMS2EK/EK cells showed a phenotype very similar to that of the MLH3 mutants. These data indicate that MLH3 and PMS2 promote HR as an endonuclease. The MLH3DN/DN and PMS2EK/EK mutations had an additive effect on the heteroallelic HR. MLH3DN/DN/PMS2EK/EK cells showed normal kinetics of γ-irradiation-induced Rad51 foci but a significant delay in the resolution of Rad51 foci and a 3-fold decrease in the number of cisplatin-induced sister chromatid exchanges. The ectopic expression of the Gen1 HJ re-solvase partially reversed the defective heteroallelic HR of MLH3DN/DN/PMS2EK/EK cells. Taken together, we propose that MLH3 and PMS2 promote HR as endonucleases, most likely by processing JMs in mammalian somatic cells.

Joint-Linker Type Ionic Gels Using Polymerizable Ionic Liquid as a Crosslinker via Thiol-Ene Click Reactions.

In this work, we report the synthesis of ion-conductive gels, or ionic gels, via thiol-ene click reactions. The novel gel systems consist of the multifunctional thiol monomers tris[(3-mercaptopropionyloxy)-ethyl]-isocyanurate (TEMPIC), pentaerythritol tetrakis(3-mercaptopropionate) (PEMP), and dipentaerythritol hexakis(3-mercaptopionate) (DPMP) as joint molecules and bifunctional allyl ionic liquid (IL) as a crosslinker. The thiol-ene reaction was carried out in lithium bis(trifluoromethanesulfonyl)imide (Li-TFSI) in a propylene carbonate (PC) (1 M) solvent system via a photopolymerization process. The chemical structure and mechanical, thermal, and conductive properties of the gels were investigated using Fourier transform infrared (FTIR) spectroscopy, thermogravimetric analysis (TGA), compression tests, and impedance spectroscopy, respectively. The mechanical and conductive properties of the ionic gels were found to be largely dependent on the monomer content and functionalities of the joint molecules. TGA revealed good thermal stability of the gels up to 100 °C. An ionic conductivity of 4.89 mS cm−1 was realized at room temperature (298 K) for low-functional thiol monomers, and a further increase in ionic conductivity was observed with an increase in Li+ ion content and temperature.