scispace - formally typeset
Search or ask a question
Journal ArticleDOI

Hsp90 induces increased genomic instability toward DNA-damaging agents by tuning down RAD53 transcription.

01 Aug 2016-Molecular Biology of the Cell (American Society for Cell Biology)-Vol. 27, Iss: 15, pp 2463-2478
TL;DR: The model organism Saccharomyces cerevisiae is used to establish that a transient heat shock and particularly the concomitant induction of Hsp90 lead to increased genomic instability via transcriptional regulation of the major checkpoint kinase Rad53.
Abstract: It is well documented that elevated body temperature causes tumors to regress upon radiotherapy. However, how hyperthermia induces DNA damage sensitivity is not clear. We show that a transient heat shock and particularly the concomitant induction of Hsp90 lead to increased genomic instability under DNA-damaging conditions. Using Saccharomyces cerevisiae as a model eukaryote, we demonstrate that elevated levels of Hsp90 attenuate efficient DNA damage signaling and dictate preferential use of the potentially mutagenic double-strand break repair pathway. We show that under normal physiological conditions, Hsp90 negatively regulates RAD53 transcription to suppress DNA damage checkpoint activation. However, under DNA damaging conditions, RAD53 is derepressed, and the increased level of Rad53p triggers an efficient DNA damage response. A higher abundance of Hsp90 causes increased transcriptional repression on RAD53 in a dose-dependent manner, which could not be fully derepressed even in the presence of DNA damage. Accordingly, cells behave like a rad53 loss-of-function mutant and show reduced NHEJ efficiency, with a drastic failure to up-regulate RAD51 expression and manifestly faster accumulation of CLN1 and CLN2 in DNA-damaged G1, cells leading to premature release from checkpoint arrest. We further demonstrate that Rad53 overexpression is able to rescue all of the aforementioned deleterious effects caused by Hsp90 overproduction.
Citations
More filters
Journal ArticleDOI
TL;DR: The contributions of the molecular chaperone Hsp90, a protein that facilitates the folding of many key regulators of growth and development, to canalization of phenotype - and de-canalization in times of stress - are reviewed, drawing on studies in eukaryotes as diverse as baker's yeast, mouse ear cress, and blind Mexican cavefish.

70 citations

Journal ArticleDOI
29 Apr 2020
TL;DR: It is reported that induction of DNA damage and perturbation of replication forks by treatment with genotoxins, such as hydroxyurea, methyl methanesulfonate, and the clinically relevant fungistatic 5-fluorocytosine, cause filamentation in C. auris.
Abstract: The morphogenetic switching between yeast cells and filaments (true hyphae and pseudohyphae) is a key cellular feature required for full virulence in many polymorphic fungal pathogens, such as Candida albicans. In the recently emerged yeast pathogen Candida auris, occasional elongation of cells has been reported. However, environmental conditions and genetic triggers for filament formation have remained elusive. Here, we report that induction of DNA damage and perturbation of replication forks by treatment with genotoxins, such as hydroxyurea, methyl methanesulfonate, and the clinically relevant fungistatic 5-fluorocytosine, cause filamentation in C. auris. The filaments formed were characteristic of pseudohyphae and not parallel-sided true hyphae. Pseudohyphal growth is apparently signaled through the S phase checkpoint and, interestingly, is Tup1 independent in C. auris. Intriguingly, the morphogenetic switching capability is strain specific in C. auris, highlighting the heterogenous nature of the species as a whole. IMPORTANCECandida auris is a newly emerged fungal pathogen of humans. This species was first reported in 2009 when it was identified in an ear infection of a patient in Japan. However, despite intense interest in this organism as an often multidrug-resistant fungus, there is little knowledge about its cellular biology. During infection of human patients, fungi are able to change cell shape from ellipsoidal yeast cells to elongated filaments to adapt to various conditions within the host organism. There are different types of filaments, which are triggered by reactions to different cues. Candida auris fails to form filaments when exposed to triggers that stimulate yeast filament morphogenesis in other fungi. Here, we show that it does form filaments when its DNA is damaged. These conditions might arise when Candida auris cells interact with host immune cells or during growth in certain host tissues (kidney or bladder) or during treatment with antifungal drugs.

35 citations

Journal ArticleDOI
TL;DR: There is still a long journey ahead of us, before the authors fully understand this novel pathogen, but critically important is the development of molecular tools for C. auris to make this fungus genetically tractable and traceable.

28 citations

Posted ContentDOI
06 Nov 2019-bioRxiv
TL;DR: It is reported that induction of DNA damage and perturbation of replication forks by treatment with genotoxins, such as hydroxyurea, methyl methanesulfonate, and the clinically relevant fungistatic 5-fluorocytosine, causes filamentation in C. auris.
Abstract: The morphogenetic switching between yeast cells and filaments (true hyphae and pseudohyphae) is a key cellular feature required for full virulence in many polymorphic fungal pathogens, such as Candida albicans. In the recently emerged yeast pathogen Candida auris, occasional elongation of cells has been reported. However, environmental conditions and genetic triggers for filament formation have remained elusive. Here, we report that induction of DNA damage and perturbation of replication forks by treatment with genotoxins, such as hydroxyurea, methyl methanesulfonate, and the clinically relevant fungistatic 5-fluorocytosine, causes filamentation in C. auris. The filaments formed were characteristic of pseudohyphae and not parallel-sided true hyphae. Pseudohyphal growth is apparently signalled through the S phase checkpoint and, interestingly, is Tup1-independent in C. auris. Intriguingly, the morphogenetic switching capability is strain-specific in C. auris, highlighting the heterogenous nature of the species as a whole.

16 citations

Journal ArticleDOI
TL;DR: Protein aggregation and self-assembly has now been observed in multiple proteins that regulate the DNA damage response, highlighting the importance of these connections by disease models of both cancer and neurodegeneration.

13 citations

References
More filters
Journal ArticleDOI
01 Jul 1998-Yeast
TL;DR: A new set of plasmids that serve as templates for the PCR synthesis of fragments that allow a variety of gene modifications that should further facilitate the rapid analysis of gene function in S. cerevisiae.
Abstract: An important recent advance in the functional analysis of Saccharomyces cerevisiae genes is the development of the one-step PCR-mediated technique for deletion and modification of chromosomal genes This method allows very rapid gene manipulations without requiring plasmid clones of the gene of interest We describe here a new set of plasmids that serve as templates for the PCR synthesis of fragments that allow a variety of gene modifications Using as selectable marker the S cerevisiae TRP1 gene or modules containing the heterologous Schizosaccharomyces pombe his5 + or Escherichia coli kan r gene, these plasmids allow gene deletion, gene overexpression (using the regulatable GAL1 promoter), C- or N-terminal protein tagging [with GFP(S65T), GST, or the 3HA or 13Myc epitope], and partial N- or C-terminal deletions (with or without concomitant protein tagging) Because of the modular nature of the plasmids, they allow eYcient and economical use of a small number of PCR primers for a wide variety of gene manipulations Thus, these plasmids should further facilitate the rapid analysis of gene function in S cerevisiae ? 1998 John Wiley & Sons, Ltd

5,301 citations

Journal ArticleDOI
TL;DR: These checkpoints contain, as their most proximal signaling elements, sensor proteins that scan chromatin for partially replicated DNA, DNA strand breaks, or other abnormalities, and translate these DNA-derived stimuli into biochemical signals that modulate the functions of specific downstream target proteins.
Abstract: The genomes of eukaryotic cells are under continuous assault by environmental agents (e.g., UV light and reactive chemicals) as well as the byproducts of normal intracellular metabolism (e.g., reactive oxygen intermediates and inaccurately replicated DNA). Whatever the origin, genetic damage threatens cell survival, and, in metazoans, leads to organ failure, immunodeficiency, cancer, and other pathologic sequelae. To ensure that cells pass accurate copies of their genomes on to the next generation, evolution has overlaid the core cell-cycle machinery with a series of surveillance pathways termed cell-cycle checkpoints. The overall function of these checkpoints is to detect damaged or abnormally structured DNA, and to coordinate cell-cycle progression with DNA repair. Typically, cell-cycle checkpoint activation slows or arrests cell-cycle progression, thereby allowing time for appropriate repair mechanisms to correct genetic lesions before they are passed on to the next generation of daughter cells. In certain cell types, such as thymocytes, checkpoint proteins link DNA strand breaks to apoptotic cell death via induction of p53. Hence, loss of either of two biochemically connected checkpoint kinases, ATM or Chk2, paradoxically increases the resistance of immature (CD4CD8) T cells to ionizing radiation (IR)-induced apoptosis (Xu and Baltimore 1996; Hirao et al. 2000). In a broader context, cell-cycle checkpoints can be envisioned as signal transduction pathways that link the pace of key cell-cycle phase transitions to the timely and accurate completion of prior, contingent events. It is important to recognize that checkpoint surveillance functions are not confined solely to the happenings within the nucleus–extranuclear parameters, such as growth factor availability and cell mass accumulation, also govern the pace of the cell cycle (Stocker and Hafen 2000). However, for the purposes of this review we will focus exclusively on the subset of checkpoints that monitor the status and structure of chromosomal DNA during cell-cycle progression (Fig. 1). These checkpoints contain, as their most proximal signaling elements, sensor proteins that scan chromatin for partially replicated DNA, DNA strand breaks, or other abnormalities, and translate these DNA-derived stimuli into biochemical signals that modulate the functions of specific downstream target proteins. Despite the recent explosion of information regarding the molecular components of cell-cycle checkpoints in eukaryotic cells, we still have only a skeletal understanding of both the identities of the DNA damage sensors and the mechanisms through which they initiate and terminate the activation of checkpoints. However, members of the Rad group of checkpoint proteins, which include Rad17, Rad1, Rad9, Rad26, and Hus1 (nomenclature based on the Schizosaccharomyces pombe gene products) are widely expressed in all eukaryotic cells, and are prime suspects in the lineup of candidate DNA damage sensors (Green et al. 2000; O’Connell et al. 2000). Three of these Rad proteins, Rad1, Rad9, and Hus1, exhibit structural similarity to the proliferating cell nuclear antigen (PCNA), and accumulating evidence supports the idea that this similarity may extend to function as well (Thelen et al. 1999; Burtelow et al. 2000). During DNA replication, PCNA forms a homotrimeric complex that encircles DNA, creating a “sliding clamp” that tethers DNA polymerase to the DNA strand. Rad1, Rad9, and Hus1 are also found as a heterotrimeric complex in intact cells, and it has been postulated that the Rad1–Rad9–Hus1 complex encircles DNA at or near sites of damage to form a checkpoint sliding clamp (CSC) (O’Connell et al. 2000), which could serve as a nucleus for the recruitment of the checkpoint signaling machinery to broken or abnormally structured DNA. The analogy between PCNA and the Rad1–Rad9–Hus1 complex extends even further. The loading of the PCNA clamp onto DNA is controlled by the clamp loading complex, replication factor C (RFC). Interestingly, yet another member of the Rad family, Rad17, bears homology to the RFC subunits and, in fact, associates with RFC subunits to form a putative checkpoint clamp loading complex (CLC) that governs the interaction of the Rad1– Rad9–Hus1 CSC with damaged DNA (Green et al. 2000; O’Connell et al. 2000). Although this model is fascinating, rigorous biochemical evaluations of the interplay between the CLC and CSC complexes, and the interactions of both complexes with damaged chromatin, are needed before the model can be accepted without reservation. Present address: The Burnham Institute, 10901 N. Torrey Pines Road, La Jolla, CA 92037, USA E-MAIL abraham@burnham.org; FAX (858) 713-6268. Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/ gad.914401.

2,053 citations


"Hsp90 induces increased genomic ins..." refers background in this paper

  • ...cerevisiae (MRN complex in humans) (Abraham, 2001; Nakada et al., 2004; Falck et al., 2005; Nakada et al., 2003; Cuadrado et al., 2006; Jazayeri et al., 2006)....

    [...]

Journal ArticleDOI
31 Mar 2005-Nature
TL;DR: Findings reveal that recruitment of these PIKKs to DNA lesions occurs by common mechanisms through an evolutionarily conserved motif, and provide direct evidence that PIKK recruitment is required for PIKK-dependent DNA-damage signalling.
Abstract: Ataxia-telangiectasia mutated (ATM), ataxia-telangiectasia and Rad3-related (ATR) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) are members of the phosphoinositide-3-kinase-related protein kinase (PIKK) family, and are rapidly activated in response to DNA damage. ATM and DNA-PKcs respond mainly to DNA double-strand breaks, whereas ATR is activated by single-stranded DNA and stalled DNA replication forks. In all cases, activation involves their recruitment to the sites of damage. Here we identify related, conserved carboxy-terminal motifs in human Nbs1, ATRIP and Ku80 proteins that are required for their interaction with ATM, ATR and DNA-PKcs, respectively. These motifs are essential not only for efficient recruitment of ATM, ATR and DNA-PKcs to sites of damage, but are also critical for ATM-, ATR- and DNA-PKcs-mediated signalling events that trigger cell cycle checkpoints and DNA repair. Our findings reveal that recruitment of these PIKKs to DNA lesions occurs by common mechanisms through an evolutionarily conserved motif, and provide direct evidence that PIKK recruitment is required for PIKK-dependent DNA-damage signalling.

1,282 citations


"Hsp90 induces increased genomic ins..." refers background in this paper

  • ...cerevisiae (MRN complex in humans) (Abraham, 2001; Nakada et al., 2004; Falck et al., 2005; Nakada et al., 2003; Cuadrado et al., 2006; Jazayeri et al., 2006)....

    [...]

Journal ArticleDOI
TL;DR: It is shown that ATM and the nuclease activity of meiotic recombination 11 are required for the processing of DNA double-strand breaks (DSBs) to generate the replication protein A (RPA)-coated ssDNA that is needed for ATR recruitment and the subsequent phosphorylation and activation of Chk1.
Abstract: It is generally thought that the DNA-damage checkpoint kinases, ataxia-telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR), work independently of one another. Here, we show that ATM and the nuclease activity of meiotic recombination 11 (Mre11) are required for the processing of DNA double-strand breaks (DSBs) to generate the replication protein A (RPA)-coated ssDNA that is needed for ATR recruitment and the subsequent phosphorylation and activation of Chk1. Moreover, we show that efficient ATM-dependent ATR activation in response to DSBs is restricted to the S and G2 cell cycle phases and requires CDK kinase activity. Thus, in response to DSBs, ATR activation is regulated by ATM in a cell-cycle dependent manner.

1,097 citations


"Hsp90 induces increased genomic ins..." refers background in this paper

  • ...cerevisiae (MRN complex in humans) (Abraham, 2001; Nakada et al., 2004; Falck et al., 2005; Nakada et al., 2003; Cuadrado et al., 2006; Jazayeri et al., 2006)....

    [...]

Journal ArticleDOI
TL;DR: It is proposed that Sml1 inhibits dNTP synthesis posttranslationally by binding directly to Rnr1 and that Mec1 and Rad53 are required to relieve this inhibition.

762 citations