Search

Summary: Repair of DNA double-strand breaks by the non-homologous end-joining pathway is initiated by the binding of Ku to DNA ends. Multiple Ku proteins load onto linear DNAs in vitro. However, in cells, Ku loading is limited to ∼1–2 molecules per DNA end. The mechanisms enforcing this limit are currently unclear. Here, we show that the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), but not its protein kinase activity, is required to prevent excessive Ku entry into chromatin. Ku accumulation is further restricted by two mechanisms: a neddylation/FBXL12-dependent process that actively removes loaded Ku molecules throughout the cell cycle and a CtIP/ATM-dependent mechanism that operates in S phase. Finally, we demonstrate that the misregulation of Ku loading leads to impaired transcription in the vicinity of DNA ends. Together, our data shed light on the multiple mechanisms operating to prevent Ku from invading chromatin and interfering with other DNA transactions.

A major strategic priority of Barito Utara Regency is maternal and neonatal health issues since the maternal mortality rate (MMR) and infant mortality rate (IMR) have fluctuated with an increasing trend from 2016 to 2022. This case discusses innovation at Kandui Public Health Centre in Gunung Timang Subdistrict as the only thriving area that brought down IMR and MMR to zero for five consecutive years (2019-2023). Despite the benefits of JESAYA (Jembatan Bersalin Sayang Anak/Child-Friendly Maternity Bridge) innovation being recognised, its adoption rate by other public health centres remained low. This study aims to identify the intrinsic factors influencing the diffusion of JESAYA. To study these issues adequately, a qualitative case study with a phenomenological approach was selected using various data collection techniques, including interviews with nine research subjects, observation, and documentation. The results suggest that adopting innovation is not straightforward, and its intrinsic factors play a crucial role in its success. Additionally, it appears that the process of adopting JESAYA has a social component, which collegial interactions and discussions may facilitate.

Nicotinium Nitrate Monohydrate (NNM) was grown by slow evaporation method. The arrangment of the molecules and hydrogen bonding intractionsin the grown crystal were analyzed using single crystal XRD. The structure, functional groups (vibrational analysis) and electronic properties (Mulliken and HOMO-LUMO analysis) were calculated by the density functional theory (DFT)/B3LYP/wB97XD method using 6–311++G (d,p) basis set. Also, the binding energy and the effect of basis set superposition error (BSSE) on the ring motifs have been investigated. The intermolecular interactions and charge delocalization density of NNM crystal have been analyzed using NBO 3.1 program. The strength of intermolecular interaction was further analyzed by 3D Hirshfeld surface analysis using fingerprint plot. Improved solubility of the title crystal NNM shows an enhanced physio-chemical activity compared to pure vitamin B3 (Nicotinic acid). The antibacterial activity of NNM crystal increased against Staphylococcus aureus a gram-positive bacteria when compared with pure nicotinic acid.

Meiotic sex chromosome inactivation is an essential event in male germ cell development, which is directed by DNA damage response signaling independent of Xist RNA to silence the transcription activity of the sex chromosomes. However, the specific mechanism of establishment and maintenance of meiotic chromosome silencing is still unclear. Here we identify the HSF5 as a testicular specific protein and the expression of which was at the onset of meiosis pachytene stage to round sperm. When the function of the HSF5 was lost, meiosis sex chromosome remodeling and silencing fail, followed by activation of CHK2 checkpoint leads to germ cell apoptosis. Furthermore, we found that SMARCA4 in the linking the HSF5 to MSCI and uncover additional factors with meiotic sex chromosome remodeling. Together, our results demonstrate a requirement for HSF5 activity in spermatogenesis and suggest a role for the mammalian HSF5-SMARCA4 in programmed meiotic sex chromosome remodeling and silencing events that take place during meiosis.

Cagar budaya merupakan warisan budaya yang bisa bersifat kebendaaan dan perlu dilestarikan karena memiliki nilai penting bagi sejarah. Perkebunan Kopi De Karanganyar merupakan salah satu wisata cagar budaya yang terletak Kabupaten Blitar. Perkebunan ini didirikan tahun 1874 oleh warga Belanda bernama H.J. Velsink. Perkebunan kopi ini merupakan salah satu penghasil kopi hitam di Blitar. Perkebunan ini memiliki beberapa atraksi wisata didalamnya yang bisa dikunjungi oleh wisatawan. Atraksi wisata yang tersedia antara lain: Koffie Boomstaart, Roemah Lodji, Museum Nugroho, dan Pabrik. Perkebunan kopi ini merupakan salah satu destinasi wisata yang tersedia dalam paket wisata Saya Tour. Keberadaan wisata cagar budaya ini masih belum dikelola dengan optimal oleh Saya Tour, mitra kegiatan pengabdian. Berdasarkan kondisi mitra kegiatan pengabdian dan peluang yang bisa diberikan ke mitra, maka tujuan kegiatan pengabdian ini yaitu untuk memberikan pengetahuan dan wawasan terhadap mitra terkait dengan pemanfaatan teknologi digital untuk memetakan dan mengemas wisata cagar budaya. Metode yang digunaan dalam kegiatan pengabdian ini yaitu metode konsultasi dan pelatihan. Kegiatan telah dilaksanakan sesuai dengan target dan telah memberikan solusi atas permasalahan mitra dengan memberikan beberapa rekomendasi, antara lain pemetaan lokasi wisata berbasis digital, pengemasan wisata memanfaatkan teknologi multimedia dan virtual tourism

El artículo presenta los resultados de la evaluación realizada a estudiantes que ingresan a la carrera de Pedagogía en Educación General Básica, de dos instituciones universitarias regionales pertenecientes al Consejo de Rectores en Chile, en habilidades de razonamiento y conocimiento matemático y comprensión lectora. Corresponde a una evaluación diagnóstica de tipo descriptiva y comparativa de corte transversal, a una población compuesta por 80 alumnos, a quienes se les aplicó instrumentos debidamente validados con un Alfa de Cronbach α=0.82 en Matemática y de α=0.64 en Comprensión Lectora. Para el análisis de la información, se utilizaron métodos descriptivos univariados y multivariados. Los resultados evidencian, en general, un dominio descendido en ambas habilidades o por debajo de lo esperado al ingreso de la Educación Superior, especialmente en las habilidades matemáticas.Based on a comparative cross-assessment diagnosis conducted in two Council of Rectors (CRUCH) universities in southern Chile in 2009, this paper shows the results of Math reasoning and reading comprehension skills assessment of entrant students from the Primary School Teaching Program. Data for this paper comes from a test with Cronbach's Alpha reliability of 0,82 in Math and 0,64 in Reading Comprehension, which was applied to a sample of 80 students. Data analysis required one-variable and multivariable descriptive methods and the results dramatically showed either a low command of both skills or far lower than expected performance for entrant students from the Primary School Teaching Program, especially in Maths.

This paper considers schematically the various discourses through which popular music history is understood. My proposal is that five accounts of musical history (the business model, the musicological model, the sociological model, the historical model and the art history model) are commonly deployed in popular music discourse. One implies, superficially at least, that popular music evolves, gets better; four implies that, at least in the longer term, it does not. The concept of ′progress′ is shown to be problematic.

The classical Non-Homologous End Joining (c-NHEJ) pathway is the predominant process in mammals for repairing endogenous, accidental or programmed DNA Double-Strand Breaks. c-NHEJ is regulated by several accessory factors, post-translational modifications, endogenous chemical agents and metabolites. The metabolite inositol-hexaphosphate (IP6) stimulates c-NHEJ by interacting with the Ku70-Ku80 heterodimer (Ku). We report cryo-EM structures of apo- and DNA-bound Ku in complex with IP6, at 3.5 Å and 2.74 Å resolutions respectively, and an X-ray crystallography structure of a Ku in complex with DNA and IP6 at 3.7 Å. The Ku-IP6 interaction is mediated predominantly via salt bridges at the interface of the Ku70 and Ku80 subunits. This interaction is distant from the DNA, DNA-PKcs, APLF and PAXX binding sites and in close proximity to XLF binding site. Biophysical experiments show that IP6 binding increases the thermal stability of Ku by 2°C in a DNA-dependent manner, stabilizes Ku on DNA and enhances XLF affinity for Ku. In cells, selected mutagenesis of the IP6 binding pocket reduces both Ku accrual at damaged sites and XLF enrolment in the NHEJ complex, which translate into a lower end-joining efficiency. Thus, this study defines the molecular bases of the IP6 metabolite stimulatory effect on the c-NHEJ repair activity., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Nonhomologous end joining is a critical mechanism that repairs DNA double-strand breaks in human cells. In this work, we address the structural and functional role of the accessory protein PAXX [paralog of x-ray repair cross-complementing protein 4 (XRCC4) and XRCC4-like factor (XLF)] in this mechanism. Here, we report high-resolution cryo-electron microscopy (cryo-EM) and x-ray crystallography structures of the PAXX C-terminal Ku-binding motif bound to Ku70/80 and cryo-EM structures of PAXX bound to two alternate DNA-dependent protein kinase (DNA-PK) end-bridging dimers, mediated by either Ku80 or XLF. We identify residues critical for the Ku70/PAXX interaction in vitro and in cells. We demonstrate that PAXX and XLF can bind simultaneously to the Ku heterodimer and act as structural bridges in alternate forms of DNA-PK dimers. Last, we show that engagement of both proteins provides a complementary advantage for DNA end synapsis and end joining in cells.

Arginyl-tRNA-protein transferase 1 (ATE1) catalyses N-terminal protein arginylation, a post-translational modification implicated in cell migration, invasion and the cellular stress response. Herein, we report that ATE1 is overexpressed in NRAS-mutant melanomas, while it is downregulated in BRAF-mutant melanomas. ATE1 expression was higher in metastatic tumours, compared with primary tumours. Consistent with these findings, ATE1 depletion reduced melanoma cell viability, migration and colony formation. Reduced ATE1 expression also affected cell responses to mTOR and MEK inhibitors and to serum deprivation. Among putative ATE1 substrates is the tumour suppressor AXIN1, pointing to the possibility that ATE1 may fine-tune AXIN1 function in melanoma. Our findings highlight an unexpected role for ATE1 in melanoma cell aggressiveness and suggest that ATE1 constitutes a potential new therapeutic target., (© 2022 Federation of European Biochemical Societies.)

Two DNA repair pathways operate at DNA double strand breaks (DSBs): non-homologous end-joining (NHEJ), that requires two adjacent DNA ends for ligation, and homologous recombination (HR), that resects one DNA strand for invasion of a homologous duplex. Faithful repair of replicative single-ended DSBs (seDSBs) is mediated by HR, due to the lack of a second DNA end for end-joining. ATM stimulates resection at such breaks through multiple mechanisms including CtIP phosphorylation, which also promotes removal of the DNA-ends sensor and NHEJ protein Ku. Here, using a new method for imaging the recruitment of the Ku partner DNA-PKcs at DSBs, we uncover an unanticipated role of ATM in removing DNA-PKcs from seDSBs in human cells. Phosphorylation of DNA-PKcs on the ABCDE cluster is necessary not only for DNA-PKcs clearance but also for the subsequent MRE11/CtIP-dependent release of Ku from these breaks. We propose that at seDSBs, ATM activity is necessary for the release of both Ku and DNA-PKcs components of the NHEJ apparatus, and thereby prevents subsequent aberrant interactions between seDSBs accompanied by DNA-PKcs autophosphorylation and detrimental commitment to Lig4-dependent end-joining., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Poisons of topoisomerase II (TOP2) kill cancer cells by preventing religation of intermediate DNA breaks during the enzymatic process and thus by accumulating enzyme-drug-DNA complexes called TOP2 cleavage-complex (TOP2cc). F14512 is a highly cytotoxic polyamine-vectorized TOP2 inhibitor derived from etoposide and currently in clinical trials. It was shown in vitro that F14512 has acquired DNA-binding properties and that the stability of TOP2cc was strongly increased. Paradoxically, at equitoxic concentrations in cells, F14512 induced less DNA breaks than etoposide. Here, we directly compared etoposide and F14512 for their rates of TOP2cc production and resolution in human cells. We report that targeting of TOP2α and not TOP2β impacts cell killing by F14512, contrary to etoposide that kills cells through targeting both isoforms. Then, we show that despite being more cytotoxic, F14512 is less efficient than etoposide at producing TOP2α cleavage-complex (TOP2αcc) in cells. Finally, we report that compared with TOP2αcc mediated by etoposide, those generated by F14512 persist longer in the genome, are not dependent on TDP2 for cleaning break ends from TOP2α, are channeled to a larger extent to resection-based repair processes relying on CtIP and BRCA1 and promote RAD51 recruitment to damaged chromatin. In addition to the addressing of F14512 to the polyamine transport system, the properties uncovered here would be particularly valuable for a therapeutic usage of this new anticancer compound. More generally, the concept of increasing drug cytotoxicity by switching the repair mode of the induced DNA lesions via addition of a DNA-binding moiety deserves further developments. Mol Cancer Ther; 16(10); 2166-77. ©2017 AACR ., (©2017 American Association for Cancer Research.)

In humans, DNA double-strand breaks (DSBs) are repaired by two mutually-exclusive mechanisms, homologous recombination or end-joining. Among end-joining mechanisms, the main process is classical non-homologous end-joining (C-NHEJ) which relies on Ku binding to DNA ends and DNA Ligase IV (Lig4)-mediated ligation. Mostly under Ku- or Lig4-defective conditions, an alternative end-joining process (A-EJ) can operate and exhibits a trend toward microhomology usage at the break junction. Homologous recombination relies on an initial MRN-dependent nucleolytic degradation of one strand at DNA ends. This process, named DNA resection generates 3' single-stranded tails necessary for homologous pairing with the sister chromatid. While it is believed from the current literature that the balance between joining and recombination processes at DSBs ends is mainly dependent on the initiation of resection, it has also been shown that MRN activity can generate short single-stranded DNA oligonucleotides (ssO) that may also be implicated in repair regulation. Here, we evaluate the effect of ssO on end-joining at DSB sites both in vitro and in cells. We report that under both conditions, ssO inhibit C-NHEJ through binding to Ku and favor repair by the Lig4-independent microhomology-mediated A-EJ process., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

We previously identified the heterogeneous ribonucleoprotein SAF-A/hnRNP U as a substrate for DNA-PK, a protein kinase involved in DNA damage response (DDR). Using laser micro-irradiation in human cells, we report here that SAF-A exhibits a two-phase dynamics at sites of DNA damage, with a rapid and transient recruitment followed by a prolonged exclusion. SAF-A recruitment corresponds to its binding to Poly(ADP-ribose) while its exclusion is dependent on the activity of ATM, ATR and DNA-PK and reflects the dissociation from chromatin of SAF-A associated with ongoing transcription. Having established that SAF-A RNA-binding domain recapitulates SAF-A dynamics, we show that this domain is part of a complex comprising several mRNA biogenesis proteins of which at least two, FUS/TLS and TAFII68/TAF15, exhibit similar biphasic dynamics at sites of damage. Using an original reporter for live imaging of DNA:RNA hybrids (R-loops), we show a transient transcription-dependent accumulation of R-loops at sites of DNA damage that is prolonged upon inhibition of RNA biogenesis factors exclusion. We propose that a new component of the DDR is an active anti-R-loop mechanism operating at damaged transcribed sites which includes the exclusion of mRNA biogenesis factors such as SAF-A, FUS and TAF15., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Nonhomologous end joining is the primary deoxyribonucleic acid (DNA) double-strand break repair pathway in multicellular eukaryotes. To initiate repair, Ku binds DNA ends and recruits the DNA-dependent protein kinase (DNA-PK) catalytic subunit (DNA-PKcs) forming the holoenzyme. Early end synapsis is associated with kinase autophosphorylation. The XRCC4 (X4)-DNA Ligase IV (LIG4) complex (X4LIG4) executes the final ligation promoted by Cernunnos (Cer)-X4-like factor (XLF). In this paper, using a cell-free system that recapitulates end synapsis and DNA-PKcs autophosphorylation, we found a defect in both activities in human cell extracts lacking LIG4. LIG4 also stimulated the DNA-PKcs autophosphorylation in a reconstitution assay with purified components. We additionally uncovered a kinase autophosphorylation defect in LIG4-defective cells that was corrected by ectopic expression of catalytically dead LIG4. Finally, our data support a contribution of Cer-XLF to this unexpected early role of the ligation complex in end joining. We propose that productive end joining occurs by early formation of a supramolecular entity containing both DNA-PK and X4LIG4-Cer-XLF complexes on DNA ends.

In mammalian cells, the main pathway for DNA double-strand breaks (DSBs) repair is classical non-homologous end joining (C-NHEJ). An alternative or back-up NHEJ (B-NHEJ) pathway has emerged which operates preferentially under C-NHEJ defective conditions. Although B-NHEJ appears particularly relevant to genomic instability associated with cancer, its components and regulation are still largely unknown. To get insights into this pathway, we have knocked-down Ku, the main contributor to C-NHEJ. Thus, models of human cell lines have been engineered in which the expression of Ku70/80 heterodimer can be significantly lowered by the conditional induction of a shRNA against Ku70. On Ku reduction in cells, resulting NHEJ competent protein extracts showed a shift from C- to B-NHEJ that could be reversed by addition of purified Ku protein. Using a cellular fractionation protocol after treatment with a strong DSBs inducer followed by western blotting or immunostaining, we established that, among C-NHEJ factors, Ku is the main counteracting factor against mobilization of PARP1 and the MRN complex to damaged chromatin. In addition, Ku limits PAR synthesis and single-stranded DNA production in response to DSBs. These data support the involvement of PARP1 and the MRN proteins in the B-NHEJ route for the repair of DNA DSBs., (© The Author(s) 2011. Published by Oxford University Press.)

DNA-dependent protein kinase (DNA-PK) is involved in DNA double-strand break (DSB) signalling and repair. We report that DNA-PK is activated by mild hypoxia conditions (0.1-1% O₂) as shown by (1) its autophosphorylation on Ser2056, and (2) its mobilisation from a soluble nucleoplasmic compartment to a less extractable nuclear fraction. The recruitment of DNA-PK was not followed by activation and recruitment of the XRCC4-DNA-ligase-IV complex, suggesting that DSBs are not responsible for activation of DNA-PK. To unravel the mechanism of DNA-PK activation, we show that exposure of cells to trichostatin A, a histone deacetylase inhibitor, leads to DNA-PK autophosphorylation and relocalisation to DNA. Histone acetylation (mainly H3K14) is increased in hypoxic cells and treatment with anacardic acid, an inhibitor of histone acetyl transferase, prevented both histone modifications and DNA-PK activation in hypoxic conditions. Importantly, in using either silenced DNA-PK cells or cells exposed to a specific DNA-PK inhibitor (NU7026), we demonstrated that hypoxic DNA-PK activation positively regulates the key transcription factor HIF-1 and one subsequent target gene, GLUT1. Our results show that hypoxia initiates chromatin modification and consequently DNA-PK activation, which positively regulate cellular oxygen-sensing and oxygen-signalling pathways.