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The alternative PCNA loader containing CTF18-DCC1-CTF8 facilitates sister chromatid cohesion (SCC) by poorly defined mechanisms. Here we found that in DT40 cells, CTF18 acts complementarily with the Warsaw breakage syndrome DDX11 helicase in mediating SCC and proliferation. We uncover that the lethality and cohesion defects of ctf18 ddx11 mutants are associated with reduced levels of chromatin-bound cohesin and rescued by depletion of WAPL, a cohesin-removal factor. On the contrary, high levels of ESCO1/2 acetyltransferases that acetylate cohesin to establish SCC do not rescue ctf18 ddx11 phenotypes. Notably, the tight proximity of sister centromeres and increased anaphase bridges characteristic of WAPL-depleted cells are abrogated by loss of both CTF18 and DDX11. The results reveal that vertebrate CTF18 and DDX11 collaborate to provide sufficient amounts of chromatin-loaded cohesin available for SCC generation in the presence of WAPL-mediated cohesin-unloading activity. This process modulates chromosome structure and is essential for cellular proliferation in vertebrates.

A trisomy is a type of aneuploidy characterised by an additional chromosome. The additional chromosome theoretically accepts any kind of changes since it is not necessary for cellular proliferation. This advantage led us to apply two chromosome manipulation methods to autosomal trisomy in chicken DT40 cells. We first corrected chromosome 2 trisomy to disomy by employing counter-selection markers. Upon construction of cells carrying markers targeted in one of the trisomic chromosome 2s, cells that have lost markers integrated in chromosome 2 were subsequently selected. The loss of one of the chromosome 2s had little impacts on the proliferative capacity, indicating unsubstantial role of the additional chromosome 2 in DT40 cells. We next tested large-scale truncations of chromosome 2 to make a mini-chromosome for the assessment of chromosome stability by introducing telomere repeat sequences to delete most of p-arm or q-arm of chromosome 2. The obtained cell lines had 0.7 Mb mini-chromosome, and approximately 0.2% of mini-chromosome was lost per cell division in wild-type background while the rate of chromosome loss was significantly increased by the depletion of DDX11, a cohesin regulatory protein. Collectively, our findings propose that trisomic chromosomes are good targets to make unique artificial chromosomes.

Transcriptional regulation, a pivotal biological process by which cells adapt to environmental fluctuations, is achieved by the binding of transcription factors to target sequences in a sequence-specific manner. However, how transcription factors recognize the correct target from amongst the numerous candidates in a genome has not been fully elucidated. We here show that, in the fission-yeast fbp1 gene, when transcription factors bind to target sequences in close proximity, their binding is reciprocally stabilized, thereby integrating distinct signal transduction pathways. The fbp1 gene is massively induced upon glucose starvation by the activation of two transcription factors, Atf1 and Rst2, mediated via distinct signal transduction pathways. Atf1 and Rst2 bind to the upstream-activating sequence 1 region, carrying two binding sites located 45 bp apart. Their binding is reciprocally stabilized due to the close proximity of the two target sites, which destabilizes the independent binding of Atf1 or Rst2. Tup11/12 (Tup-family co-repressors) suppress independent binding. These data demonstrate a previously unappreciated mechanism by which two transcription-factor binding sites, in close proximity, integrate two independent-signal pathways, thereby behaving as a hub for signal integration.

Sister chromatid cohesion is essential for faithful chromosome segregation and genome duplication during cell division. Failure to establish cohesion during S phase by the ring-shaped multiprotein complex cohesin leads to genomic instability1-4. Replisome-associated proteins are required to generate cohesion by two independent pathways5. One mediates conversion of cohesins bound to unreplicated DNA ahead of replication forks into cohesive entities behind them, while the second promotes cohesin de novo loading onto newly-replicated DNAs6. The latter process depends on the cohesin loader Scc2/NIPBL and the alternative PCNA loader CTF18-RFC. However, the precise mechanism of de novo cohesin loading during replication is unknown. Here we show that PCNA physically recruits yeast cohesin loader Scc2 via its C-terminal PCNA-interacting protein motif. Binding to PCNA is crucial, as scc2-pip mutant deficient in Scc2-PCNA interaction is defective in cohesion when combined with replisome mutants of the cohesin conversion pathway. Moreover, scc2-pip mutant becomes inviable without its partner Scc4/MAU2 that localizes cohesin loader to centromeres. Importantly, the role of NIPBL recruitment to PCNA for cohesion generation is conserved in vertebrate cells. Our results demonstrate that PCNA, the maestro of replication-linked functions, is also crucially involved in the cohesion establishment through de novo cohesin loading onto replicated DNA.

Electron transport experiments for molecular devices have been evaluated by using single-molecule bridging nano- and/or sub-nanoscale junctions between two metallic electrodes. Thus, although sever...

Muscle mass and strength decrease with aging; however, habitual exercise can maintain muscle health. β-Hydroxy-β-methyl butyrate calcium (HMB) and black ginger (BG) improve muscle protein metabolism and energy production. Combining these two molecules, which have similar effects, may have a synergistic effect. Senescence-accelerated mouse-prone 8 (SAMP8) is a useful model of muscle aging. Therefore, we explored how the combination of habitual exercise, HMB, and BG affected muscle aging. We used 28-wk-old (28w) SAMP8 mice divided into six groups: 28 wk (28w), 44 wk (44w, Con), exercise (Ex), Ex+BG, Ex+HMB, and Ex+BG+HMB (Ex+Comb). Mice were required to run on a treadmill for 16 wk for 5 d per week. In 28w and 44w mice, grip strength tests and dissection were conducted. Muscle weight was measured, and qPCR and immunoblotting were conducted. Muscle mass and strength were declined in the 44w group. Exercise with HMB or BG alone had no effect, whereas muscle mass and strength were augmented in the Ex+Comb group. Similarly, levels of mitochondrial function- and biogenesis-related genes were increased. Autophagy-related protein (Atg3, 7, 16L1 and Beclin1) were altered in the Ex+Comb group. These results suggest that Ex+Comb affects autophagy. Overall, the combination of habitual exercise and HMB+BG may enhance muscle mass and strength by affecting the mitochondrial and autophagy systems in SAMP8.

(1) Background: Extracorporeal circulation (ECC) is indispensable for cardiac surgery. Despite the fact that ECC causes non-physiological damage to blood components, its pathophysiology has not been fully elucidated. In our previous study, we constructed a rat ECC system and observed a systemic inflammatory response during and after blood tests assessing ECC, while the damage per organ localization caused by ECC was not examined. In this study, we used a rat model to assess the gene expression of inflammatory cytokines in major organs during ECC. (2) Methods: The ECC system consisted of a membranous oxygenator, tubing line, and a small roller pump. Rats were divided into a SHAM (which received surgical preparation only, without ECC) group and an ECC group. Proinflammatory cytokines were measured using real-time PCR in major organs after ECC to evaluate local inflammatory responses in the organs. (3) Results: Interleukin (IL)-6 levels were significantly elevated in the ECC group compared to the SHAM group, especially in the heart and lungs. (4) Conclusions: This study suggests that ECC promotes organ damage and the inflammatory response, but the degree of gene expression of proinflammatory cytokines varies from organ to organ, suggesting that it does not uniformly cause organ damage.

Systemic inflammatory responses in patients undergoing extracorporeal membrane oxygenation (ECMO) contribute significantly to ECMO-associated morbidity and mortality. In recent years, the number of type 2 diabetes mellitus patients has increased, and the number of these patients undergoing ECMO has also increased. Type 2 diabetes mellitus is a high-risk factor for complications during ECMO. We studied the effects of ECMO on inflammatory response in a diabetic rat ECMO model. Twenty-eight rats were divided into 4 groups: normal SHAM group (normal rats: n = 7), diabetic SHAM group (diabetic rats: n = 7), normal ECMO group (normal rats: n = 7), and diabetic ECMO group (diabetic rats: n = 7). We measured the plasma levels of cytokines, tumor necrosis factor-α, and interleukin-6. Aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), blood urea nitrogen (BUN), creatinine (Cr), and liver-type fatty acid binding protein (L-FABP) were examined in the rat cardiopulmonary bypass model to ascertain organ damage. In addition, the lung wet-to-dry weight (W/D) ratio was measured as an index of pulmonary tissue edema. A pathologic evaluation of kidneys was conducted by hematoxylin-eosin (HE) and periodic-acid-methenamine-silver (PAM) staining. In the diabetic ECMO group, levels of cytokines, AST, ALT, LDH, and L-FABP increased significantly, reaching a maximum at the end of ECMO in comparison with other groups (p &lt, 0.05). In addition, hematoxylin-eosin and periodic acid-methenamine-silver staining of renal tissues showed marked injury in the ECMO group (normal ECMO and diabetic ECMO groups). Furthermore, when the normal ECMO and diabetic ECMO groups were compared, severe organ injury was seen in the diabetic ECMO group. There was remarkable organ injury in the diabetic ECMO group. These data demonstrate that diabetes enhances proinflammatory cytokine release, renal damage, and pulmonary edema during ECMO in an animal model.

Muscle mass and strength decrease with aging, but habitual exercise can maintain muscle health. β-Hydroxy-β-methyl butyrate calcium (HMB) and black ginger (BG) are anti-oxidants that have been reported to improve muscle protein metabolism and energy production; these molecules may have synergistic effects. The senescence-accelerated mouse-prone 8 (SAMP8) model is a useful model of muscle aging. Therefore, in this study, we explored how the combination of habitual exercise, HMB, and BG affected muscle aging. We used 28-week-old SAMP8 mice divided into five groups: control, exercise (Ex), Ex+BG, Ex+HMB, and Ex+BG+HMB (Ex+Comb). Mice were required to run on a treadmill for 16 weeks at 5 days per week. In 44-week-old mice, grip strength tests and dissection were conducted. Muscle weight was measured, and the gastrocnemius muscle was subjected to quantitative polymerase chain reaction and immunoblotting. Muscle mass and strength were preserved in the Ex+Comb group, and mitochondrial function was preserved through suppressing oxidative stress. Muscle protein synthesis signaling was improved in the Ex+Comb group. Autophagy and the ubiquitin system were normalized by Ex+Comb treatment. Overall, habitual exercise and HMB plus BG treatment maintained muscle health by suppressing oxidative stress, preserving mitochondrial function, and maintaining muscle protein metabolism in SAMP8 mice.

Loss of muscle mass and strength are progressing with aging. Exercise is a beneficial method to prevent physical disfunction and habitual exercise improve the muscle quality. Therefore, we evaluated the effects of a long-term habitual exercise on the senescence-accelerated mice prone8 (SAMP8). 27wk SAMP8 were used in this study. Mice were classified into 28 (28w) and 44 weeks old. The 44-week group was divided into the sedentary group (44w) and a group exercising for 16 weeks (44w+Ex). The 44w+Ex performed habitual exercise from 28 to 44 weeks. Additionally, grip strength tests were performed with mice aged 28 and 44 weeks. Mice were dissected and collected muscle samples and measured muscle weight at 44w. Gastrocnemius was decreased in 44w but were unchanged in 44w+Ex. Grip strength in 44w was lower trend, but there was no change in 44w+Ex. The phosphorylation levels of Akt and p70S6K as a protein synthesis marker were decreased in 44w. Cytochrome c oxidase subunit IV(COXIV) mRNA and protein levels decreased in 44w. These results suggested that long-term habitual exercise attenuated muscle mass and strength decline through improving muscle protein synthesis and mitochondrial function. In conclusion, long-term habitual exercise attenuated muscle mass and strength decline.

Long noncoding RNAs (lncRNAs) transcribed across gene promoters have been detected. These regulate transcription by mechanisms that have not been fully elucidated. We herein show that the chromatin configuration is altered into an accessible state within 290 bp downstream from the initiation site of metabolic-stress-induced lncRNAs (mlonRNAs) in the promoter of the fission yeast fbp1 gene, whose transcription is massively induced upon glucose starvation. Chromatin upstream from fbp1 is progressively altered into an open configuration, as a cascade of transcription of three overlapping mlonRNA species (-a, -b and -c in order) occurs with transcriptional initiation sites progressing 5′ to 3′ upstream of the fbp1 promoter. Initiation of the shortest mlonRNA (mlonRNA-c) induces chromatin remodeling around a transcription factor-binding site and subsequent massive induction of fbp1. We identify the cis-element required for mlonRNA-c initiation, and by changing the distance between mlonRNA-initiation site and the transcription factor-binding site, we show that mlonRNA-initiation effectively induces chromatin remodeling in a limited distance within 290 bp. These results indicate that mlonRNAs are transcribed across the fbp1 promoter as a short-range inducer for local chromatin alterations, and suggest that strict chromatin modulation is archived via stepwise mlonRNA-initiations.

Numerous noncoding RNA transcripts are detected in eukaryotic cells. Noncoding RNAs transcribed across gene promoters are involved in the regulation of mRNA transcription via chromatin modulation. This function of noncoding RNA transcription was first demonstrated for the fission yeast fbp1 gene, where a cascade of noncoding RNA transcription events induces chromatin remodeling to facilitate transcription factor binding. We recently demonstrated that the noncoding RNAs from the fbp1 upstream region facilitate binding of the transcription activator Atf1 and thereby promote histone acetylation. Histone acetylation by histone acetyl transferases (HATs) and ATP-dependent chromatin remodelers (ADCRs) are implicated in chromatin remodeling, but the interplay between HATs and ADCRs in this process has not been fully elucidated. Here, we examine the roles played by two distinct ADCRs, Snf22 and Hrp3, and by the HAT Gcn5 in the transcriptional activation of fbp1. Snf22 and Hrp3 redundantly promote disassembly of chromatin in the fbp1 upstream region. Gcn5 critically contributes to nucleosome eviction in the absence of either Snf22 or Hrp3, presumably by recruiting Hrp3 in snf22∆ cells and Snf22 in hrp3∆ cells. Conversely, Gcn5-dependent histone H3 acetylation is impaired in snf22∆/hrp3∆ cells, suggesting that both redundant ADCRs induce recruitment of Gcn5 to the chromatin array in the fbp1 upstream region. These results reveal a previously unappreciated interplay between ADCRs and histone acetylation in which histone acetylation facilitates recruitment of ADCRs, while ADCRs are required for histone acetylation.

ESCO1/2 acetyltransferases mediating SMC3 acetylation and sister chromatid cohesion (SCC) are differentially required for genome integrity and development. Here we established chicken DT40 cell lines with mutations in ESCO1/2, SMC3 acetylation, and the cohesin remover WAPL. Both ESCO1 and ESCO2 promoted SCC, while ESCO2 was additionally and specifically required for proliferation and centromere integrity. ESCO1 overexpression fully suppressed the slow proliferation and centromeric separation phenotypes of esco2 cells but only partly suppressed its chromosome arm SCC defects. Concomitant inactivation of ESCO1 and ESCO2 caused lethality owing to compromised mitotic chromosome segregation. Neither wapl nor acetyl-mimicking smc3-QQ mutations rescued esco1 esco2 lethality. Notably, esco1 esco2 wapl conditional mutants showed very severe proliferation defects associated with catastrophic mitoses and also abnormal interphase chromatin organization patterns. The results indicate that cohesion establishment by vertebrate ESCO1/2 is linked to interphase chromatin architecture formation, a newly identified function of cohesin acetyltransferases that is both fundamentally and medically relevant.

We previously characterized a 177-kDa allergen, M-177, from Dermatophagoides farinae. Thereafter, a counterpart to M-177 for Euroglyphus maynei was cloned as Eur m 14, and its sequence revealed that two environmental allergens, Mag 1 and Mag 3, are digested fragments of M-177. The aims of this study were to clone the cDNA of Der f 14 corresponding to M-177 and to elucidate the allergenic capacities of the N-terminal fragment of Der f 14 (Der f 14-N). Recombinant allergens were produced as trigger-factor-fused proteins in Escherichia coli. Der f 14-N showed the highest IgE-binding frequency among Der f 14-derived fragments in patients allergic to house dust mite by enzyme-linked immunosorbent assay. Der f 14-N showed the highest capacity to induce cell proliferation in murine lymphocyte and human peripheral mononuclear cells among Der f 14-derived fragments. Der f 14-N induced IL-13, IFN-γ and IL-17 production more than Der f 1 and Der f 2 in mouse, and induced IL-5 and IFN-γ production at levels comparable to those of Der f 1 and Der f 2 in some patients. The high prevalence of IgE binding to the Der f 14-N indicates that it could be an important mite allergen.

Transcription factors (TFs) determine the transcription activity of target genes and play a central role in controlling the transcription in response to various environmental stresses. Three dimensional genome structures such as local loops play a fundamental role in the regulation of transcription, although the link between such structures and the regulation of TF binding to cis-regulatory elements remains to be elucidated. Here, we show that during transcriptional activation of the fission yeast fbp1 gene, binding of Rst2 (a critical C2H2 zinc-finger TF) is mediated by a local loop structure. During fbp1 activation, Rst2 is first recruited to upstream-activating sequence 1 (UAS1), then it subsequently binds to UAS2 (a critical cis-regulatory site located approximately 600 base pairs downstream of UAS1) through a loop structure that brings UAS1 and UAS2 into spatially close proximity. Tup11/12 (the Tup-family corepressors) suppress direct binding of Rst2 to UAS2, but this suppression is counteracted by the recruitment of Rst2 at UAS1 and following delivery to UAS2 through a loop structure. These data demonstrate a previously unappreciated mechanism for the recruitment and expansion of TF-DNA interactions within a promoter mediated by local three-dimensional genome structures and for timely TF-binding via counteractive regulation by the Tup-family corepressors.

Information on the role of termites in the tropical rain forest ecosystem is very scarce in comparison with that in the savannah ecosystem. Richness in the number and biomass of soil feeders seems to be one of the most important characteristics of the termites in the tropical rain forest. The role of fungi cultivated by other species on their fungus combs was discussed in relation to the nutrition of termites and decomposition of leaf litter with special reference to their nitrogen metabolism by T. Matsumoto. The fact that soil-feeding termites are most numerous in the tropical rain forest among various habitats of termites is interesting from this viewpoint. In Pasoh Forest about 65% of termites are fungus-growers in biomass and most of them carry much soil into wood. To estimate the consumption rate of leaf litter by termites, newly fallen leaves were marked and distributed on the forest floor. The loss of leaf area due to termites was determined photometrically.

SMC5/6 function in genome integrity remains elusive. Here, we show that SMC5 dysfunction in avian DT40 B cells causes mitotic delay and hypersensitivity toward DNA intra‐ and inter‐strand crosslinkers (ICLs), with smc5 mutants being epistatic to FANCC and FANCM mutations affecting the Fanconi anemia (FA) pathway. Mutations in the checkpoint clamp loader RAD17 and the DNA helicase DDX11, acting in an FA‐like pathway, do not aggravate the damage sensitivity caused by SMC5 dysfunction in DT40 cells. SMC5/6 knockdown in HeLa cells causes MMC sensitivity, increases nuclear bridges, micronuclei, and mitotic catastrophes in a manner similar and non‐additive to FANCD2 knockdown. In both DT40 and HeLa systems, SMC5/6 deficiency does not affect FANCD2 ubiquitylation and, unlike FANCD2 depletion, RAD51 focus formation. SMC5/6 components further physically interact with FANCD2‐I in human cells. Altogether, our data suggest that SMC5/6 functions jointly with the FA pathway to support genome integrity and DNA repair and may be implicated in FA or FA‐related human disorders., The structural maintenance complex SMC5/6 interacts with and functions jointly with key components of the Fanconi anemia pathway to facilitate DNA repair and preserve genome stability.

The replicative polymerase δ (Polδ), consisting of four subunits, plays a pivotal role in chromosomal replication. Pold4, the smallest subunit of Polδ, is believed to contribute to the regulation of replication by facilitating repair in response to DNA damage. However, that contribution has not been fully elucidated. We here show that Pold4 contributes to the suppression of gene conversion in immunoglobulin-variable (IgV) gene diversification in the chicken DT40 lymphocyte cell line, where gene conversion diversifies the IgV gene through intragenic homologous recombination (HR) between diverged pseudo-V segments. IgV gene conversion is initiated by activation-induced cytidine deaminase-mediated uracil formation in the IgV gene, which in turn converts into an abasic site, leading to replication arrest. POLD4−/− cells exhibited an increased rate of IgV gene conversion. Moreover, the gene-conversion tract was lengthened and the usage of pseudo-V segments was altered, showing a preference, to use the diverged sequence as a donor in POLD4−/− cells. These data suggest that Pold4 is involved in the regulation of HR-mediated gene conversion in IgV diversification. By contrast, the rate in HR-mediated, sister-chromatid exchange and gene-targeting induced by an I-SceI endonclease-mediated DNA double-strand break exhibited by POLD4−/− cells was indistinguishable from that by wild-type cells. These findings indicate that the functionality of general HR is preserved in POLD4−/− cells. In conclusion, Pold4 is involved in the suppression of IgV-gene conversion without affecting the general functionality of HR.

Azo dyes, including Sudan I, Orange II and Orange G, are industrial dyes that are assumed to have genotoxic potential. However, neither the type of DNA damage induced nor the structural features responsible for toxicity have been determined. We used a panel of DNA-repair-pathway-deficient mutants generated from chicken DT40 cells to evaluate the ability of these azo dyes to induce DNA damage and to identify the type of DNA damage induced. We compared the structurally related azo dyes Sudan I, Orange II and Orange G to identify the structural features responsible for genotoxicity. Compared with wild type cells, the double-strand break repair defective RAD54-/-/KU70-/- cells were significantly more sensitive to Sudan I, but not to Orange II or Orange G. The quantum-chemical calculations revealed that Sudan I, but not Orange II or Orange G, has a complete planar aromatic ring structure. These suggest that the planar feature of Sudan I is critical to the inducing of double-strand breaks. In summary, we used a DNA-repair mutant panel in combination with quantum-chemical calculations to provide a clue to the chemical structure responsible for genotoxicity.

The high prevalence of house dust mite (HDM) allergy is a growing health problem worldwide, and the characterization of clinically important HDM allergens is a prerequisite for the development of diagnostic and therapeutic strategies. Here, we report a novel HDM allergen that belongs structurally to the highly conserved Rid/YjgF/YER057c/UK114 family (Rid family) with imine deaminase activity. Isolated HDM cDNA, named der f 34, encodes 128 amino acids homologous to Rid-like proteins. This new protein belongs to the Rid family and has seven conserved residues involved in enamine/imine deaminase activity. Indeed, we demonstrated that purified Der f 34 had imine deaminase activity that preferentially acted on leucine and methionine. Native Der f 34 showed a high IgE binding frequency as revealed by two-dimensional immunoblotting (62.5%) or ELISA (68%), which was comparable with those of a major HDM allergen Der f 2 (77.5 and 79%, respectively). We also found that Der f 34 showed cross-reactivity with another prominent indoor allergen source, Aspergillus fumigatus. This is the first report showing that the Rid family imine deaminase represents an additional important pan-allergen that is conserved across organisms.

Zinc oxide (ZnO) materials are fine ceramics with non-linear electrical properties. Their non-linear electrical properties appear from a double Schottky barrier formed in the grain boundaries (GBs). These microparticles are not easy to measure directly with electrical microprobes due to their small size (i.e., 50-100 µm). We developed a direct measurement process with two Cu cables of small diameter. In this paper, we have developed an amplified method of the previous measurement system, which can measure not only one ZnO microvaristor directly but also a group of microvaristors in a series connection. The I-V characteristics of the microvaristors were measured with the modified method, and we found non-linear properties in each particle while measuring I-V characteristics. Their fine structures were also investigated, and the non-linear I-V characteristics showed a direct relationship with the number of the GBs of the samples. Moreover, varistor voltage was calculated for a single GB for the I-V measurements for ZnO microvaristors connected in a series connection.

DNA replication is an essential biochemical reaction in dividing cells that frequently stalls at damaged sites. Homologous/homeologous recombination (HR)-mediated template switch and translesion DNA synthesis (TLS)-mediated bypass processes release arrested DNA replication forks. These mechanisms are pivotal for replication fork maintenance and play critical roles in DNA damage tolerance (DDT) and gap-filling. The avian DT40 B lymphocyte cell line provides an opportunity to examine HR-mediated template switch and TLS triggered by abasic sites by sequencing the constitutively diversifying immunoglobulin light-chain variable gene (IgV). During IgV diversification, activation-induced deaminase (AID) converts dC to dU, which in turn is excised by uracil DNA glycosylase and yields abasic sites within a defined window of around 500 base pairs. These abasic sites can induce gene conversion with a set of homeologous upstream pseudogenes via the HR-mediated template switch, resulting in templated mutagenesis, or can be bypassed directly by TLS, resulting in non-templated somatic hypermutation at dC/dG base pairs. In this review, we discuss recent works unveiling IgV diversification mechanisms in avian DT40 cells, which shed light on DDT mode usage in vertebrate cells and tolerance of abasic sites.

The arrangement of nucleosomes in chromatin plays a role in transcriptional regulation by restricting the accessibility of transcription factors and RNA polymerase II to cis-acting elements and promoters. For gene activation, the chromatin structure is altered to an open configuration. The mechanism for this process has been extensively analyzed. However, the mechanism by which repressive chromatin is reconstituted to terminate transcription has not been fully elucidated. Here, we investigated the mechanisms by which chromatin is reconstituted in the fission yeast Schizosaccharomyces pombe fbp1 gene, which is robustly induced upon glucose starvation but tightly repressed under glucose-rich conditions. We found that the chromatin structure in the region upstream from fbp1 is closed by a two-step process. When cells are returned to glucose-rich medium following glucose starvation, changes in the nucleosome pattern alter the chromatin configuration at the transcription factor binding site to an inaccessible state, after which the nucleosome density upstream from fbp1 gradually increases via histone loading. Interestingly, this histone loading was observed in the absence of the Tup family corepressors Tup11 and Tup12. Analysis of strains carrying either gene disruptions or mutations affecting nine fission yeast histone chaperone genes demonstrated that the histone chaperone Asf1 induces nucleosome loading during glucose repression. These data establish a previously unappreciated chromatin reconstitution mechanism in fbp1 repression.

We propose adaptive viscoelasticity control which dynamically changes the elasticity and viscosity coefficients according to the network delay and the moving speed of a haptic interface device. We also investigate how large range the optimum viscosity coefficient has. We further obtain an equation that derives the optimum viscosity coefficient from the network delay and the moving speed by multiple regression analysis. Then, we examine the effectiveness of the control by QoE (Quality of Experience) assessment.

ALC1 (amplified in liver cancer 1), an SNF2 superfamily chromatin-remodeling factor also known as CHD1L (chromodomain helicase/ATPase DNA binding protein 1-like), is implicated in base-excision repair, where PARP (Poly(ADP-ribose) polymerase) mediated Poly(ADP-ribose) signaling facilitates the recruitment of this protein to damage sites. We here demonstrate the critical role played by ALC1 in the regulation of replication-fork progression in cleaved template strands. To analyze the role played by ALC1 as well as its functional relationship with PARP1, we generated ALC1-/-, PARP1-/-, and ALC1-/-/PARP1-/- cells from chicken DT40 cells. We then exposed these cells to camptothecin (CPT), a topoisomerase I poison that generates single-strand breaks and causes the collapse of replication forks. The ALC1-/- and PARP1-/- cells exhibited both higher sensitivity to CPT and an increased number of chromosome aberrations, compared with wild-type cells. Moreover, phenotypes were very similar across all three mutants, indicating that the role played by ALC1 in CPT tolerance is dependent upon the PARP pathway. Remarkably, inactivation of ALC1 resulted in a failure to slow replication-fork progression after CPT exposure, indicating that ALC1 regulates replication-fork progression at DNA-damage sites. We disrupted ATPase activity by inserting the E165Q mutation into the ALC1 gene, and found that the resulting ALC1-/E165Q cells displayed a CPT sensitivity indistinguishable from that of the null-mutant cells. This observation suggests that ALC1 contributes to cellular tolerance to CPT, possibly as a chromatin remodeler. This idea is supported by the fact that CPT exposure induced chromatin relaxation in the vicinity of newly synthesized DNA in wild-type but not in ALC1-/- cells. This implies a previously unappreciated role for ALC1 in DNA replication, in which ALC1 may regulate replication-fork slowing at CPT-induced DNA-damage sites.

Significance Warsaw breakage syndrome, a developmental disorder caused by mutations in the conserved DDX11/ChlR1 DNA helicase, shows features of genome instability partly overlapping with those of Fanconi anemia (FA). Here, using avian cellular models of DDX11 deficiency, we find that DDX11 functions as backup to the FA pathway and facilitates, jointly with the checkpoint clamp 9-1-1, a homologous recombination pathway of DNA bulky-lesion repair that does not affect replication fork speed and stalled fork stability. DDX11 also promotes diversification of the immunoglobulin-variable gene locus by facilitating hypermutation and gene conversion at programmed abasic sites that constitute endogenous replication blocks. The results suggest commonality between postreplicative gap filling and replication through abasic sites and pinpoint DDX11 as a critical player in both these processes., Warsaw breakage syndrome, a developmental disorder caused by mutations in the DDX11/ChlR1 helicase, shows cellular features of genome instability similar to Fanconi anemia (FA). Here we report that DDX11-deficient avian DT40 cells exhibit interstrand crosslink (ICL)-induced chromatid breakage, with DDX11 functioning as backup for the FA pathway in regard to ICL repair. Importantly, we establish that DDX11 acts jointly with the 9-1-1 checkpoint clamp and its loader, RAD17, primarily in a postreplicative fashion, to promote homologous recombination repair of bulky lesions, but is not required for intra-S checkpoint activation or efficient fork progression. Notably, we find that DDX11 also promotes diversification of the chicken Ig-variable gene, a process triggered by programmed abasic sites, by facilitating both hypermutation and homeologous recombination-mediated gene conversion. Altogether, our results uncover that DDX11 orchestrates jointly with 9-1-1 and its loader, RAD17, DNA damage tolerance at sites of bulky lesions, and endogenous abasic sites. These functions may explain the essential roles of DDX11 and its similarity with 9-1-1 during development.

A new class of ordered silica-based materials has been prepared by hydrogen bond-directed assembly of cage siloxanes modified with dimethylsilanol groups, providing a soft-chemical approach to crystalline silica materials with molecularly designed architectures.