T-loops are thought to hide telomeres from DNA damage signaling and DSB repair pathways. T-loop formation requires the shelterin component TRF2, which represses ATM signaling and NHEJ. Here we ...establish that TRF2 alone, in the absence of other shelterin proteins can form t-loops. Mouse and human cells contain two isoforms of TRF2, one of which is uncharacterized. We show that both isoforms protect telomeres and form t-loops. The isoforms are not cell cycle regulated and t-loops are present in G1, S, and G2. Using the DNA wrapping deficient TRF2 Topless mutant, we confirm its inability to form t-loops and repress ATM. However, since the mutant is also defective in repression of NHEJ and telomeric localization, the role of topological changes in telomere protection remains unclear. Finally, we show that Rad51 does not affect t-loop frequencies or telomere protection. Therefore, alternative models for how TRF2 forms t-loops should be explored.
Telomeres are protected by shelterin, a six-subunit protein complex that represses the DNA damage response (DDR) at chromosome ends. Extensive data suggest that TRF2 in shelterin remodels telomeres ...into the t-loop structure, thereby hiding telomere ends from double-stranded break repair and ATM signaling, whereas POT1 represses ATR signaling by excluding RPA. An alternative protection mechanism was suggested recently by which shelterin subunits TRF1, TRF2, and TIN2 mediate telomeric chromatin compaction, which was proposed to minimize access of DDR factors. We performed superresolution imaging of telomeres in mouse cells after conditional deletion of TRF1, TRF2, or both, the latter of which results in the complete loss of shelterin. Upon removal of TRF1 or TRF2, we observed only minor changes in the telomere volume in most of our experiments. Upon codeletion of TRF1 and TRF2, the telomere volume increased by varying amounts, but even those samples exhibiting small changes in telomere volume showed DDR at nearly all telomeres. Upon shelterin removal, telomeres underwent 53BP1-dependent clustering, potentially explaining at least in part the apparent increase in telomere volume. Furthermore, chromatin accessibility, as determined by ATAC-seq (assay for transposase-accessible chromatin ATAC with high-throughput sequencing), was not substantially altered by shelterin removal. These results suggest that the DDR induced by shelterin removal does not require substantial telomere decompaction.
Significance Chromosomal double-strand breaks (DSBs) are cytotoxic forms of DNA damage that must be accurately repaired to maintain genome integrity. The conserved Mre11–Rad50–Xrs2/NBS1 ...nuclease/ATPase complex plays an important role in repair by functioning as a damage sensor and by regulation of DNA end processing to ensure repair by the most appropriate mechanism. Yeast Sae2 is known to function with Mre11 to process DNA ends, but its precise role is poorly understood. Here we show that it is the failure to remove Mre11 from DNA ends, leading to persistent DNA damage signaling and cell cycle arrest, that causes sensitivity of Sae2-deficient cells to DNA damaging agents.
The Mre11–Rad50–Xrs2/NBS1 (MRX/N) nuclease/ATPase complex plays structural and catalytic roles in the repair of DNA double-strand breaks (DSBs) and is the DNA damage sensor for Tel1/ATM kinase activation. Saccharomyces cerevisiae Sae2 can function with MRX to initiate 5′-3′ end resection and also plays an important role in attenuation of DNA damage signaling. Here we describe a class of mre11 alleles that suppresses the DNA damage sensitivity of sae2 Δ cells by accelerating turnover of Mre11 at DNA ends, shutting off the DNA damage checkpoint and allowing cell cycle progression. The mre11 alleles do not suppress the end resection or hairpin-opening defects of the sae2 Δ mutant, indicating that these functions of Sae2 are not responsible for DNA damage resistance. The purified M ᴾ¹¹⁰ᴸRX complex shows reduced binding to single- and double-stranded DNA in vitro relative to wild-type MRX, consistent with the increased turnover of Mre11 from damaged sites in vivo. Furthermore, overproduction of Mre11 causes DNA damage sensitivity only in the absence of Sae2. Together, these data suggest that it is the failure to remove Mre11 from DNA ends and attenuate Rad53 kinase signaling that causes hypersensitivity of sae2 Δ cells to clastogens.
Chromosomal double-strand breaks (DSBs) that have only one end with homology to a donor duplex undergo repair by strand invasion followed by replication to the chromosome terminus (break-induced ...replication, BIR). Using a transformation-based assay system, it was previously shown that BIR could occur by several rounds of strand invasion, DNA synthesis, and dissociation. Here we describe a modification of the transformation-based assay to facilitate detection of switching between donor templates during BIR by genetic selection in diploid yeast. In addition to the expected recovery of template switch products, we found a high frequency of recombination between chromosome homologs during BIR, suggesting transfer of the DSB from the transforming linear DNA to the donor chromosome, initiating secondary recombination events. The frequency of BIR increased in the mph1Δ mutant, but the percentage of template switch events was significantly decreased, revealing an important role for Mph1 in promoting BIR-associated template switching. In addition, we show that the Mus81, Rad1, and Yen1 structure-selective nucleases act redundantly to facilitate BIR.
Articular cartilage is a highly organized tissue that has a limited ability to heal. Tissue engineering is actively exploited for joint tissue reconstruction in numerous cases of articular cartilage ...degeneration associated with trauma, arthrosis, rheumatoid arthritis, and osteoarthritis. However, the optimal scaffolds for cartilage repair are not yet identified. Here we have directly compared five various scaffolds, namely collagen-I membrane, collagen-II membrane, decellularized cartilage, a cellulose-based implant, and commercially available Chondro-Gide
(Geistlich Pharma AG, Wolhusen, Switzerland) collagen membrane. The scaffolds were implanted in osteochondral full-thickness defects, formed on adult Wistar rats using a hand-held cutter with a diameter of 2.0 mm and a depth of up to the subchondral bone. The congruence of the articular surface was almost fully restored by decellularized cartilage and collagen type II-based scaffold. The most vivid restoration was observed 4 months after the implantation. The formation of hyaline cartilage was not detected in any of the groups. Despite cellular infiltration into scaffolds being observed in each group except cellulose, neither chondrocytes nor chondro-progenitors were detected. We concluded that for restoration of hyaline cartilage, scaffolds have to be combined either with cellular therapy or morphogens promoting chondrogenic differentiation.
Although t-loops protect telomeres, they are at risk of cleavage by Holliday junction (HJ) resolvases if branch migration converts the three-way t-loop junction into four-way HJs. T-loop cleavage is ...repressed by the TRF2 basic domain, which binds three- and four-way junctions and protects HJs in vitro. By replacing the basic domain with bacterial-protein domains binding three- and four-way junctions, we demonstrated the in vivo relevance of branched-DNA binding. Branched-DNA binding also repressed PARP1, presumably by masking the PARP1 site in the t-loop junction. Although PARP1 recruits HJ resolvases and promotes t-loop cleavage, PARP1 activation alone did not result in t-loop cleavage, thus suggesting that the basic domain also prevents formation of HJs. Concordantly, removal of HJs by BLM helicase mitigated t-loop cleavage in response to loss of the basic domain. We propose that TRF2 masks and stabilizes the t-loop three-way junction, thereby protecting telomeres from detrimental deletions and PARP1 activation.
Chitosan-
-oligolactide copolymers with relatively long oligolactide grafted chains of various stereochemical compositions have been synthetized via a solvent-free mechanochemical technique and ...tailored to fabricate three-dimensional hydrogels using two-photon induced microstereolithography. An effect of the characteristics of chitosan and oligolactide used for the synthesis on the grafting yield and copolymer's behavior were evaluated using fractional analysis, FTIR-spectroscopy, dynamic light scattering, and UV-spectrophotometry. The lowest copolymer yield was found for the system based on chitosan with higher molecular weight, while the samples consisting of low-molecular weight chitosan showed higher grafting degrees, which were comparable in both the cases of l,l- or l,d-oligolactide grafting. The copolymer processability in the course of two-photon stereolithography was evaluated as a function of the copolymer's characteristics and stereolithography conditions. The structure and mechanical properties of the model film samples and fabricated 3D hydrogels were studied using optical and scanning electron microscopy, as well as by using tensile and nanoindenter devices. The application of copolymer with oligo(l,d-lactide) side chains led to higher processability during two-photon stereolithography in terms of the response to the laser beam, reproduction of the digital model, and the mechanical properties of the fabricated hydrogels.
Bioprosthetic materials based on mammalian pericardium tissue are the gold standard in reconstructive surgery. Their application range covers repair of rectovaginal septum defects, abdominoplastics, ...urethroplasty, duraplastics, maxillofacial, ophthalmic, thoracic and cardiovascular reconstruction, etc. However, a number of factors contribute to the success of their integration into the host tissue including structural organization, mechanical strength, biocompatibility, immunogenicity, surface chemistry, and biodegradability. In order to improve the material's properties, various strategies are developed, such as decellularization, crosslinking, and detoxification. In this review, the existing issues and long‐term achievements in the development of bioprosthetic materials based on the mammalian pericardium tissue, aimed at a wide‐spectrum application in reconstructive surgery are analyzed. The basic technical approaches to preparation of biocompatible forms providing continuous functioning, optimization of biomechanical and functional properties, and clinical applicability are described.
The review describes the milestones in the development of bioprosthetic materials based on mammalian pericardium tissue. The basic technical approaches to preparation of biocompatible forms including decellularization, crosslinking, detoxification, and application range of pericardial biomeshes in reconstructive surgery are discussed.
The solvent-free synthesis of allyl-substituted chitosan derivatives through reactive co-extrusion of chitosan powder with allyl bromide at shear deformation was performed. For the structural ...characterization, FTIR and NMR methods were employed. The results were confirmed by chemical analysis. The total content of allyl substituents from 5 to 50 per 100 chitosan units as a function of the component ratio in the reactive mixtures was revealed. Carrying out the reaction without any additives leads to the selective formation of
N
-alkylated derivatives, whereas in the presence of alkali the ethers of chitosan were preferentially formed. The results suggest that the proposed approach allows significantly higher yield of products to be obtained at high process speeds and significantly lower reagent consumption as compared with the liquid-phase synthesis in organic medium. The synthesized unsaturated derivatives are promising photosensitive components for use in laser stereolithography for fabrication of three-dimensional biocompatible structures with well-defined architectonics.
The allyl chitosan derivatives are synthesized, characterized and evaluated as photosensitive components for creation of biomedical materials with well-defined architectonics.
Decellularized bovine pericardium (DBP)‐based biomeshes are the gold standard in reconstructive surgery. In order to prolong their stability after the transplantation, various chemical cross‐linking ...strategies are employed. However, structural and functional properties of the biomeshes differ in dependence on the cross‐linker used. Here, we performed a bottom‐up study of structural and functional alterations of DBP‐based biomeshes following cross‐linking with hexamethylene diisocyanate (HMDC), ethylene glycol diglycidyl ether (EGDE), 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide (EDC) and genipin. The in vitro cytotoxicity tests supported their clinical applicability. Their structural differences (eg roughness, fibre thickness, pore morphology) were evaluated using the two‐photon confocal laser scanning, atomic force, scanning electron and polarized light microscopies. HMDC and EDC samples appeared to be the roughest. Complex mechanical trials indicated the tendency to reduced Young’s Modulus and mechanical anisotropy values of DBP upon cross‐linking. The lowest mechanical anisotropy was found in EDC and genipin sample groups. In vitro collagenase susceptibility was the highest for EDC samples and the lowest for EGDE samples. The comparative analysis of the results allowed us to recognize the strengths and weaknesses of each cross‐linker in relation to a particular clinical application.
Cross‐linking‐assisted personalization of xenopericardial biomeshes for specific clinical purposes.
PDF
