5,068 research outputs found

    Hydroxyurea-induced synchronisation of bloodstream stage Trypanosoma brucei

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    Synchronisation of the <i>Trypanosoma brucei</i> cell cycle proved elusive for many years. A recent report demonstrated that synchronisation of procyclic form cells was possible following treatment with hydroxyurea. Here, that work is extended to the disease-relevant, mammalian-infective bloodstream stage trypanosome. Treatment of bloodstream stage Lister 427 <i>T. Brucei</i> cells growing <i>in vitro</i> with 10 μg ml<sup>−1</sup> hydroxyurea for 6 h led to an enrichment of cells in S phase. Following removal of the drug, cells proceeded uniformly through one round of the cell cycle, providing a much needed tool to enrich for specific cell cycle stages, in a manner similar to hydroxyurea treatment of procyclic form <i>T. brucei.</i&gt

    Role of Schizosaccharomyces pombe RecQ homolog recombination and checkpoint genes in UV Damage tolerance

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    The cellular responses to DNA damage are complex and include direct DNA repair pathways that remove the damage and indirect damage responses which allow cells to survive DNA damage that has not been, or cannot be, removed. We have identified the gene mutated in the rad12.502 strain as a Schizosaccharomyces pombe recQ homolog. The same gene (designated rqh1) is also mutated in the hus2.22 mutant. We show that Rqh1 is involved in a DNA damage survival mechanism which prevents cell death when UV-induced DNA damage cannot be removed. This pathway also requires the correct functioning of the recombination machinery and the six checkpoint tad gene products plus the Cds1 kinase. Our data suggest that Rqh1 operates during S phase as part of a mechanism which prevents DNA damage causing cell lethality. This process may involve the bypass of DNA damage sites by the replication fork. Finally, in contrast with the reported literature, we do not find that rqh1 (rad12) mutant cells are defective in UV dimer endonuclease activity

    MRE11 facilitates the removal of human topoisomerase II complexes from genomic DNA

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    Topoisomerase II creates a double-strand break intermediate with topoisomerase covalently coupled to the DNA via a 5'-phosphotyrosyl bond. These intermediate complexes can become cytotoxic protein-DNA adducts and DSB repair at these lesions requires removal of topoisomerase II. To analyse removal of topoisomerase II from genomic DNA we adapted the trapped in agarose DNA immunostaining assay. Recombinant MRE11 from 2 sources removed topoisomerase IIalpha from genomic DNA in vitro, as did MRE11 immunoprecipitates isolated from A-TLD or K562 cells. Basal topoisomerase II complex levels were very high in A-TLD cells lacking full-length wild type MRE11, suggesting that MRE11 facilitates the processing of topoisomerase complexes that arise as part of normal cellular metabolism. In K562 cells inhibition of MRE11, PARP or replication increased topoisomerase IIalpha and beta complex levels formed in the absence of an anti-topoisomerase II dru

    Pulmonary hypertension in adolescents with sickle cell disease

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    Sickle cell disease consists of a group of disorders that have a similar mutation in at least one of the beta-globin chains of hemoglobin. This results in a change of the hemoglobin to sickle shaped cells when in the deoxygenated state. It is these sickled cells that lead to the symptoms and complications characteristic of sickle cell disease, including vaso-occlusion. The recurrence of sickling and polymerization in the blood vessels throughout the body ultimately results in tissue and organ damage that increases the mortality of patients. Pulmonary hypertension is the increase in blood pressure in the pulmonary vasculature causing less blood to reach the lungs. The right side of the heart has to pump harder to compensate for this, which can ultimately lead to right heart failure. With vaso-occlusion being the major complication in sickle cell disease and affecting many tissues and organs like the lungs, pulmonary hypertension has become a major risk factor in these patients, especially in children. Most research has been with adults so the seriousness of this condition is still being discovered in children. This review will cover the important aspects of both sickle cell disease and pulmonary hypertension then discuss how pulmonary hypertension has become a significant risk factor for death in those with sickle cell disease. From there the importance of this finding in children will be discussed and a plan for future endeavors will be proposed

    Ataxia-telangiectasia Mutated (ATM)-dependent Activation of ATR Occurs through Phosphorylation of TopBP1 by ATM

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    ATM (ataxia-telangiectasia mutated) is necessary for activation of Chk1 by ATR (ATM and Rad3-related) in response to double-stranded DNA breaks (DSBs) but not to DNA replication stress. TopBP1 has been identified as a direct activator of ATR. We show that ATM regulates Xenopus TopBP1 by phosphorylating Ser-1131 and thereby strongly enhancing association of TopBP1 with ATR. Xenopus egg extracts containing a mutant of TopBP1 that cannot be phosphorylated on Ser-1131 are defective in the ATR-dependent phosphorylation of Chk1 in response to DSBs but not to DNA replication stress. Thus, TopBP1 is critical for the ATM-dependent activation of ATR following production of DSBs in the genome

    Optimisation of the Schizosaccharomyces pombe urg1 expression system

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    The ability to study protein function in vivo often relies on systems that regulate the presence and absence of the protein of interest. Two limitations for previously described transcriptional control systems that are used to regulate protein expression in fission yeast are: the time taken for inducing conditions to initiate transcription and the ability to achieve very low basal transcription in the "OFF-state". In previous work, we described a Cre recombination-mediated system that allows the rapid and efficient regulation of any gene of interest by the urg1 promoter, which has a dynamic range of approximately 75-fold and which is induced within 30-60 minutes of uracil addition. In this report we describe easy-to-use and versatile modules that can be exploited to significantly tune down P urg1 "OFF-levels" while maintaining an equivalent dynamic range. We also provide plasmids and tools for combining P urg1 transcriptional control with the auxin degron tag to help maintain a null-like phenotype. We demonstrate the utility of this system by improved regulation of HO-dependent site-specific DSB formation, by the regulation Rtf1-dependent replication fork arrest and by controlling Rhp18(Rad18)-dependent post replication repair

    Anti-cancer Action of Metal Complexes: Electron Transfer and Oxidative Stress?

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    Evidence is presented in support of an electron transfer mechanism for various metal complexes possessing anti-neoplastic properties. Cyclic voltammetry was performed on several metallocenes, bis(acetato)bis(imidazole)Cu(II), and coordination compounds (Cu or Fe) of the anti-tumor agents, bipyridine, phenanthroline, hydroxyurea, diethyldithiocarbamate, and α, α1-bis(8-hydroxyquinolin-7-yl)-4-methoxytoluene. The favorable reduction potentials ranged from +0.5 to -0.5 V. Electrochemical behavior is correlated in some cases with structure and physiological activity. Relevant literature data are discussed

    Cell division promotes efficient retrotransposition in a stable L1 reporter cell line

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    Background: Long interspersed element type one (L1) actively modifies the human genome by inserting new copies of itself. This process, termed retrotransposition, requires the formation of an L1 ribonucleoprotein (RNP) complex, which must enter the nucleus before retrotransposition can proceed. Thus, the nuclear import of L1 RNP presents an opportunity for cells to regulate L1 retrotransposition post-translationally. The effect of cell division on L1 retrotransposition has been investigated by two previous studies, which observed varied degrees of inhibition in retrotransposition when primary cell strains or cancer cell lines were experimentally arrested in different stages of the cell cycle. However, seemingly divergent conclusions were reached. The role of cell division on retrotransposition remains highly debated. Findings: To monitor both L1 expression and retrotransposition quantitatively, we developed a stable dual-luciferase L1 reporter cell line, in which a bi-directional tetracycline-inducible promoter drives the expression of both a firefly luciferase-tagged L1 element and a Renilla luciferase, the latter indicative of the level of promoter induction. We observed an additional 10-fold reduction in retrotransposition in cell-cycle arrested cells even after retrotransposition had been normalized to Renilla luciferase or L1 ORF1 protein levels. In synchronized cells, cells undergoing two mitoses showed 2.6-fold higher retrotransposition than those undergoing one mitosis although L1 expression was induced for the same amount of time. Conclusions: Our data provide additional support for an important role of cell division in retrotransposition and argue that restricting the accessibility of L1 RNP to nuclear DNA could be a post-translational regulatory mechanism for retrotransposition

    Three DNA polymerases, recruited by different mechanisms, carry out NER repair synthesis in human cells

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    Nucleotide excision repair (NER) is the most versatile DNA repair system that deals with the major UV photoproducts in DNA, as well as many other DNA adducts. The early steps of NER are well understood, whereas the later steps of repair synthesis and ligation are not. In particular, which polymerases are definitely involved in repair synthesis and how they are recruited to the damaged sites has not yet been established. We report that, in human fibroblasts, approximately half of the repair synthesis requires both polκ and polδ, and both polymerases can be recovered in the same repair complexes. Polκ is recruited to repair sites by ubiquitinated PCNA and XRCC1 and polδ by the classical replication factor complex RFC1-RFC, together with a polymerase accessory factor, p66, and unmodified PCNA. The remaining repair synthesis is dependent on polɛ, recruitment of which is dependent on the alternative clamp loader CTF18-RFC