Tousled-like kinases stabilize replication forks and show synthetic lethality with checkpoint and PARP inhibitors

DNA sequence and epigenetic information embedded in chromatin must be faithfully duplicated and transmitted to daughter cells during cell division. However, how chromatin assembly and DNA replication are integrated remains unclear. We examined the contribution of the Tousled-like kinases 1 and 2 (TLK1/TLK2) to chromatin assembly and maintenance of replication fork integrity. We show that TLK activity is required for DNA replication and replication-coupled nucleosome assembly and that lack of TLK activity leads to replication fork stalling and the accumulation of single-stranded DNA, a phenotype distinct from ASF1 depletion. Consistent with these results, sustained TLK depletion gives rise to replication-dependent DNA damage and p53-dependent cell cycle arrest in G1. We find that deficient replication-coupled de novo nucleosome assembly renders replication forks unstable and highly dependent on the ATR and CHK1 checkpoint kinases, as well as poly(adenosine 5′-diphosphate–ribose) polymerase (PARP) activity, to avoid collapse. Human cancer data revealed frequent up-regulation of TLK genes and an association with poor patient outcome in multiple types of cancer, and depletion of TLK activity leads to increased replication stress and DNA damage in a panel of cancer cells. Our results reveal a critical role for TLKs in chromatin replication and suppression of replication stress and identify a synergistic lethal relationship with checkpoint signaling and PARP that could be exploited in treatment of a broad range of cancers.</p

Ways to unwind with HROB, a new player in homologous recombination

Homologous recombination (HR) is an important route for repairing DNA double-strand breaks (DSBs). The early stages of HR are well understood, but later stages remain mysterious. In this issue of Genes &amp; Development, Hustedt and colleagues (pp. 1397-1415) reveal HROB as a new player in HR required for recruitment of the MCM8-9 complex, which is paralogous to the MCM2-7 replicative helicase. HROB functions closely with MCM8-9 to promote postsynaptic DNA repair synthesis. This study sheds valuable light on late events in HR and suggests that HROB may load MCM8-9 onto HR intermediates to facilitate the DNA unwinding required for DNA repair synthesis.</p

NuMA Assembles Into an Extensive Filamentous Structure When Expressed in the Cell Cytoplasm

NuMA is a 236 kDa protein that participates in the organization of the mitotic spindle despite its strict localization in the nucleus during interphase. To test how cells progress through mitosis when NuMA is localized in the cytoplasm instead of the nucleus, we have deleted the nuclear localization sequence of NuMA using site-directed mutagenesis and transiently expressed this mutant protein (NuMA-DeltaNLS) in BHK-21 cells. During interphase, NuMA-DeltaNLS accumulates in the cytoplasm as a large mass approximately the same size as the cell nucleus. When cells enter mitosis, NuMA-DeltaNLS associates normally with the mitotic spindle without causing any apparent deleterious effects on the progression of mitosis. Examination of the cytoplasmic mass formed by NuMA-DeltaNLS using transmission electron microscopy (TEM) revealed an extensive network of approximately 5 nm filaments that are further organized by the presence of dynamic microtubules into a dense web of solid, approximately 23 nm cables. Using flow cytometry, we have isolated the intact filamentous mass formed by NuMA-DeltaNLS from lysates of transiently transfected cells. These isolated structures are constructed of networks of interconnected 5 nm filaments and are composed exclusively of NuMA. These data demonstrate that NuMA is capable of assembling into an extensive filamentous structure supporting the possibility that NuMA serves a structural function either in the nucleus during interphase or at the polar ends of the mitotic spindle

Phosphorylation Regulates the Assembly of Numa in a Mammalian Mitotic Extract

NuMA is a 236 kDa nuclear protein that is required for the organization of the mitotic spindle. To determine how NuMA redistributes in the cell during mitosis, we have examined the behavior of NuMA in a mammalian mitotic extract under conditions conducive to the reassembly of interphase nuclei. NuMA is a soluble protein in mitotic extracts prepared from synchronized cultured cells, but forms insoluble structures when the extract becomes non-mitotic (as judged by the inactivation of cdc2/cyclin B kinase and the disappearance of mpm-2-reactive antigens). These NuMA-containing structures are irregularly shaped particles of 1–2 microm in diameter and their assembly is specific because other nuclear components such as the lamins remain soluble in the extract under these conditions. NuMA is dephosphorylated during this assembly process, and the assembly of these NuMA-containing structures is catalyzed by protein dephosphorylation because protein kinase inhibitors enhance their formation and protein phosphatase inhibitors block their formation. Finally, immunodepletion demonstrates that NuMA is an essential structural component of these insoluble particles, and electron microscopy shows that the particles are composed of a complex interconnected network of foci. These results demonstrate that phosphorylation regulates the solubility of NuMA in a mammalian mitotic extract, and the spontaneous assembly of NuMA into extensive structures upon dephosphorylation supports the conclusion that NuMA serves a structural function

Libro: Manual de Parasitología : Rina Girard de Kaminsky. Año: 2014. Páginas 185.. Edición: 3a. Honduras

El contenido del Manual se ha agrupado en diferentes partes: Introducción al trabajo en laboratorios de parasitología de atención primaria, Microscopio y microscopía, Parásitos intestinales luminares y tisulares, Parásitos trasmitidos por vectores, Fotografías y microfotografias, Algoritmos y acceso a paginas web. Su objetivo fundamental es constituirse en un instrumento de trabajo, consulta y referencia para la ejecución de las técnicas necesarias en el diagnóstico parasitológico. Abarca desde la organización y registro de resultados hasta la utilización correcta de la aparatología necesaria, como el uso correcto del microscopio, su calibración, medición y cuidados del mismo.Asociación Parasitológica Argentin

BRUSHY1/TONSOKU/MGOUN3 is required for heat stress memory

Plants encounter biotic and abiotic stresses many times during their life cycle and this limits their productivity. Moderate heat stress (HS) primes a plant to survive higher temperatures that are lethal in the naïve state. Once temperature stress subsides, the memory of the priming event is actively retained for several days preparing the plant to better cope with recurring HS. Recently, chromatin regulation at different levels has been implicated in HS memory. Here we report that the chromatin protein BRUSHY1/TONSOKU/MGOUN3 (BRU1/TSK/MGO3) plays a role in the HS memory in Arabidopsis thaliana. BRU1 is also involved in transcriptional gene silencing and DNA damage repair. This corresponds with the functions of its mammalian orthologue TONSL/NFΚBIL2. During HS memory, BRU1 is required to maintain sustained induction of HS memory-associated genes, whereas it is dispensable for the acquisition of thermotolerance. In summary, we report that BRU1 is required for HS memory in A. thaliana, and propose a model where BRU1 mediates the epigenetic inheritance of chromatin states across DNA replication and cell division.</p

Integrating DNA damage repair with the cell cycle

Abstract DNA is labile and constantly subject to damage. In addition to external mutagens, DNA is continuously damaged by the aqueous environment, cellular metabolites and is prone to strand breakage during replication. Cell duplication is orchestrated by the cell division cycle and specific DNA structures are processed differently depending on where in the cell cycle they are detected. This is often because a specific structure is physiological in one context, for example during DNA replication, while indicating a potentially pathological event in another, such as interphase or mitosis. Thus, contextualising the biochemical entity with respect to cell cycle progression provides information necessary to appropriately regulate DNA processing activities. We review the links between DNA repair and cell cycle context, drawing together recent advances

PROTAC-mediated conditional degradation of the WRN helicase as a potential strategy for selective killing of cancer cells with microsatellite instability

Multiple studies have demonstrated that cancer cells with microsatellite instability (MSI) are intolerant to loss of the Werner syndrome helicase (WRN), whereas microsatellite-stable (MSS) cancer cells are not. Therefore, WRN represents a promising new synthetic lethal target for developing drugs to treat cancers with MSI. Given the uncertainty of how effective inhibitors of WRN activity will prove in clinical trials, and the likelihood of tumours developing resistance to WRN inhibitors, alternative strategies for impeding WRN function are needed. Proteolysis-targeting chimeras (PROTACs) are heterobifunctional small molecules that target specific proteins for degradation. Here, we engineered the WRN locus so that the gene product is fused to a bromodomain (Bd)-tag, enabling conditional WRN degradation with the AGB-1 PROTAC specific for the Bd-tag. Our data revealed that WRN degradation is highly toxic in MSI but not MSS cell lines. In MSI cells, WRN degradation caused G2/M arrest, chromosome breakage and ATM kinase activation. We also describe a multi-colour cell-based platform for facile testing of selective toxicity in MSI versus MSS cell lines. Together, our data show that a degrader approach is a potentially powerful way of targeting WRN in MSI cancers and paves the way for the development of WRN-specific PROTAC compounds

Does needle calibre affect pain and complication rates in patients undergoing transperineal prostate biopsy? A prospective, randomized trial

Transperineal prostate biopsy is a procedure that can be used to obtain histological samples from the prostate. To improve both the quality of the biopsy core samples and prostate cancer detection, we are currently performing a prospective, randomized trial comparing prostate biopsy samples obtained using an 18 G-needle to those obtained using a 16 G needle. The aim of this preliminary study was to evaluate pain and complication rates in both groups in order to assess whether performing a prostate biopsy with a larger calibre needle is a feasible procedure. One hundred and eighty-seven patients undergoing transperineal prostate biopsy were prospectively evaluated and divided into two groups. The first group (94 patients, Group A) received a transperineal prostate biopsy using a 16 G-needle and the second group (93 patients, Group B) underwent transperineal prostate biopsy with an 18 G-needle. Anaesthesia was obtained with a single perineal injection at the prostatic apex in all subjects. A visual analogue scale (VAS) and facial expression scale (FES) were used to assess pain during multiple steps of the procedure in each group. A detailed questionnaire was used to obtain information about drug use because it could potentially influence the pain and complications that patients experienced. Two weeks after the procedure, early and late complications were evaluated. Statistical analysis was carried out using non-parametric tests. Prostate Specific Antigen (PSA) and drug use were similar at baseline between the two groups. Pain during prostate biopsy, which was measured with both the VAS and FES instruments, did not differ significantly between the 18- and 16 G-needle groups, and no significant differences were found in early or late complication rates between the groups. Transperineal prostate biopsy with a 16 G-needle is a feasible procedure in terms of pain and complication rates. Further studies with larger patient populations are required to assess whether or not this procedure can improve prostate cancer detection rates