Recombination-Mediated Telomere Maintenance in Saccharomyces cerevisiae Is Not Dependent on the Shu Complex

In cells lacking telomerase, telomeres shorten progressively during each cell division due to incomplete end-replication. When the telomeres become very short, cells enter a state that blocks cell division, termed senescence. A subset of these cells can overcome senescence and maintain their telomeres using telomerase-independent mechanisms. In Saccharomyces cerevisiae, these cells are called ‘survivors’ and are dependent on Rad52-dependent homologous recombination and Pol32-dependent break-induced replication. There are two main types of survivors: type I and type II. The type I survivors require Rad51 and maintain telomeres by amplification of subtelomeric elements, while the type II survivors are Rad51-independent, but require the MRX complex and Sgs1 to amplify the C1–3A/TG1–3 telomeric sequences. Rad52, Pol32, Rad51, and Sgs1 are also important to prevent accelerated senescence, indicating that recombination processes are important at telomeres even before the formation of survivors. The Shu complex, which consists of Shu1, Shu2, Psy3, and Csm2, promotes Rad51-dependent homologous recombination and has been suggested to be important for break-induced replication. It also promotes the formation of recombination intermediates that are processed by the Sgs1-Top3-Rmi1 complex, as mutations in the SHU genes can suppress various sgs1, top3, and rmi1 mutant phenotypes. Given the importance of recombination processes during senescence and survivor formation, and the involvement of the Shu complex in many of the same processes during DNA repair, we hypothesized that the Shu complex may also have functions at telomeres. Surprisingly, we find that this is not the case: the Shu complex does not affect the rate of senescence, does not influence survivor formation, and deletion of SHU1 does not suppress the rapid senescence and type II survivor formation defect of a telomerase-negative sgs1 mutant. Altogether, our data suggest that the Shu complex is not important for recombination processes at telomeres

Comparison of the chromosomal pattern of primary testicular nonseminomas and residual mature teratomas after chemotherapy

About 70 to 75% of patients with nonseminomatous testicular germ cell tumors (NSs) present with metastases. When these metastases are treated with chemotherapy, often residual mature teratoma (RMT) is left. RMT is composed of fully differentiated somatic tissue. Untreated metastases of NSs rarely consist exclusively of mature somatic tissue. Apparently, after chemotherapy treatment there is a shift towards higher degrees of differentiation. Investigating tumor progression and the mechanism(s) involved in therapy-related differentiation, we compared the cytogenetically abnormal karyotypes of a series of 70 NSs with those of 31 RMTs. In NSs and RMTs, the modal total chromosome number does not differ and is in the triploid range. Both the frequency and the average copy number of i(12p) are the same, and the pattern of chromosomal over-and underrepresentation and distribution of breakpoints do not differ significantly in these series. So, we found the chromosomal pattern of RMTs as abnormal as those of primary NSs. Based on cytogenetics, we found no indication that specific chromosomal alterations parallel metastasis and therapy-related differentiation of the metastases. The cytogenetic data suggest that both induction of differentiation of (selected) cells or selection of cells with capacity to differentiate are possible mechanisms for the therapy-related differentiation of RMTs. (C) Elsevier Science Inc., 1997

Impaired Coenzyme A metabolism affects histone and tubulin acetylation in Drosophila and human cell models of pantothenate kinase associated neurodegeneration

Pantothenate kinase-associated neurodegeneration (PKAN is a neurodegenerative disease with unresolved pathophysiology. Previously, we observed reduced Coenzyme A levels in a Drosophila model for PKAN. Coenzyme A is required for acetyl-Coenzyme A synthesis and acyl groups from the latter are transferred to lysine residues of proteins, in a reaction regulated by acetyltransferases. The tight balance between acetyltransferases and their antagonistic counterparts histone deacetylases is a well-known determining factor for the acetylation status of proteins. However, the influence of Coenzyme A levels on protein acetylation is unknown. Here we investigate whether decreased levels of the central metabolite Coenzyme A induce alterations in protein acetylation and whether this correlates with specific phenotypes of PKAN models. We show that in various organisms proper Coenzyme A metabolism is required for maintenance of histone- and tubulin acetylation, and decreased acetylation of these proteins is associated with an impaired DNA damage response, decreased locomotor function and decreased survival. Decreased protein acetylation and the concurrent phenotypes are partly rescued by pantethine and HDAC inhibitors, suggesting possible directions for future PKAN therapy development

Studying cell cycle checkpoints using Drosophila cultured cells

Drosophila cell lines are valuable tools to study a number of cellular processes, including DNA damage responses and cell cycle checkpoint control. Using an in vitro system instead of a whole organism has two main advantages: it saves time and simple and effective molecular techniques are available. It has been shown that Drosophila cells, similarly to mammalian cells, display cell cycle checkpoint pathways required to survive DNA damaging events (de Vries et al. 2005, Journal of Cell Science 118, 1833-1842; Bae et al. 1995, Experimental Cell Research 217, 541-545). Moreover, a number of proteins involved in checkpoint and cell cycle control in mammals are highly conserved among different species, including Drosophila (de Vries et al. 2005, Journal of Cell Science 118, 1833-1842; Bae et al. 1995, Experimental Cell Research 217, 541-545; LaRocque et al. 2007, Genetics 175, 1023-1033; Sibon et al. 1999, Current Biology 9, 302-312; Purdy et al. 2005, Journal of Cell Science 118, 3305-3315). Because of straightforward and highly efficient methods to downregulate specific transcripts in Drosophila cells, these cells are an excellent system for genome-wide RNA interference (RNAi) screens. Thus, the following methods, assays and techniques: Drosophila cell culture, RNAi, introducing DNA damaging events, determination of cell cycle arrest, and determination of cell cycle distributions described here may well be applied to identifying new players in checkpoint mechanisms and will be helpful to investigate the function of these new players in detail. Results obtained with studies using in vitro systems can subsequently be extended to studies in the complete organism as described in the chapters provided by the Su laboratory and the Takada laboratory

Cofilin/Twinstar Phosphorylation Levels Increase in Response to Impaired Coenzyme A Metabolism

Coenzyme A (CoA) is a pantothenic acid-derived metabolite essential for many fundamental cellular processes including energy, lipid and amino acid metabolism. Pantothenate kinase (PANK), which catalyses the first step in the conversion of pantothenic acid to CoA, has been associated with a rare neurodegenerative disorder PKAN. However, the consequences of impaired PANK activity are poorly understood. Here we use Drosophila and human neuronal cell cultures to show how PANK deficiency leads to abnormalities in F-actin organization. Cells with reduced PANK activity are characterized by abnormally high levels of phosphorylated cofilin, a conserved actin filament severing protein. The increased levels of phospho-cofilin coincide with morphological changes of PANK-deficient Drosophila S2 cells and human neuronal SHSY-5Y cells. The latter exhibit also markedly reduced ability to form neurites in culture - a process that is strongly dependent on actin remodeling. Our results reveal a novel and conserved link between a metabolic biosynthesis pathway, and regulation of cellular actin dynamics

Upregulation of dNTP Levels After Telomerase Inactivation Influences Telomerase-Independent Telomere Maintenance Pathway Choice in Saccharomyces cerevisiae

In 10–15% of cancers, telomere length is maintained by a telomerase-independent, recombination-mediated pathway called alternative lengthening of telomeres (ALT). ALT mechanisms were first seen, and have been best studied, in telomerase-null Saccharomyces cerevisiae cells called “survivors”. There are two main types of survivors. Type I survivors amplify Y′ subtelomeric elements while type II survivors, similar to the majority of human ALT cells, amplify the terminal telomeric repeats. Both types of survivors require Rad52, a key homologous recombination protein, and Pol32, a non-essential subunit of DNA polymerase δ. A number of additional proteins have been reported to be important for either type I or type II survivor formation, but it is still unclear how these two pathways maintain telomeres. In this study, we performed a genome-wide screen to identify novel genes that are important for the formation of type II ALT-like survivors. We identified 23 genes that disrupt type II survivor formation when deleted. 17 of these genes had not been previously reported to do so. Several of these genes (DUN1, CCR4, and MOT2) are known to be involved in the regulation of dNTP levels. We find that dNTP levels are elevated early after telomerase inactivation and that this increase favors the formation of type II survivors

Cdi and Ssh signaling is involved in the regulation of Twinstar in response to decreased levels of CoA.

<p>(<b>A</b>) <i>Drosophila</i> LIM kinases, dLimk, Center divider (Cdi), and Slingshot phosphates (Ssh) regulate levels of p-Tsr in S2 cells. Cells were treated with dsRNA against dLimk, Cdi, Ssh or with control dsRNA and the levels of Tsr and p-Tsr were assayed using Western blot analysis. Tubulin was used as a loading control. (<b>B</b>) dLimk, Cdi or Ssh were down-regulated for 3 days after which cells were subcultured and left untreated or HoPan was added to the cell culture medium. After additional 48 hr cell lysates were assayed for levels of Tsr and levels of p-Tsr. (<b>C</b>) Quantification of the levels of p-Tsr as illustrated in B. Fold change after HoPan addition is indicated above the bars, n.s: not significant.</p