The inhibition of checkpoint activation by telomeres does not involve exclusion of dimethylation of histone H4 lysine 20 (H4K20me2) [version 2; referees: 2 approved, 1 not approved]

DNA double-strand breaks (DSBs) activate the DNA damage checkpoint machinery to pause or halt the cell cycle.  Telomeres, the specific DNA-protein complexes at linear eukaryotic chromosome ends, are capped DSBs that do not activate DNA damage checkpoints.  This “checkpoint privileged” status of telomeres was previously investigated in the yeast Schizosaccharomyces pombelacking the major double-stranded telomere DNA binding protein Taz1. Telomeric DNA repeats in cells lacking Taz1 are 10 times longer than normal and contain single-stranded DNA regions. DNA damage checkpoint proteins associate with these damaged telomeres, but the DNA damage checkpoint is not activated. This severing of the DNA damage checkpoint signaling pathway was reported to stem from exclusion of histone H4 lysine 20 dimethylation (H4K20me2) from telomeric nucleosomes in both wild type cells and cells lacking Taz1.  However, experiments to identify the mechanism of this exclusion failed, prompting our re-evaluation of H4K20me2 levels at telomeric chromatin.  In this short report, we used an extensive series of controls to identify an antibody specific for the H4K20me2 modification and show that the level of this modification is the same at telomeres and internal loci in both wild type cells and those lacking Taz1.  Consequently, telomeres must block activation of the DNA Damage Response by another mechanism that remains to be determined

Dbf4-dependent kinase (DDK)-mediated proteolysis of CENP-A prevents mislocalization of CENP-A in Saccharomyces cerevisiae

The evolutionarily conserved centromeric histone H3 variant (Cse4 in budding yeast, CENP-A in humans) is essential for faithful chromosome segregation. Mislocalization of CENP-A to non-centromeric chromatin contributes to chromosomal instability (CIN) in yeast, fly, and human cells and CENP-A is highly expressed and mislocalized in cancers. Defining mechanisms that prevent mislocalization of CENP-A is an area of active investigation. Ubiquitin-mediated proteolysis of overexpressed Cse4 (GALCSE4)byE3 ubiquitin ligases such as Psh1 prevents mislocalization of Cse4, and psh1D strains display synthetic dosage lethality (SDL) with GALCSE4. We previously performed a genome-wide screen and identified five alleles of CDC7 and DBF4 that encode the Dbf4-dependent kinase (DDK) complex, which regulates DNA replication initiation, among the top twelve hits that displayed SDL with GALCSE4. We determined that cdc7-7 strains exhibit defects in ubiquitin-mediated proteolysis of Cse4 and show mislocalization of Cse4. Mutation of MCM5 (mcm5-bob1) bypasses the requirement of Cdc7 for replication initiation and rescues replication defects in a cdc7-7 strain. We determined that mcm5-bob1 does not rescue the SDL and defects in proteolysis of GALCSE4 in a cdc7-7 strain, suggesting a DNA replication-independent role for Cdc7 in Cse4 proteolysis. The SDL phenotype, defects in ubiquitin-mediated proteolysis, and the mislocalization pattern of Cse4 in a cdc7-7 psh1D strain were similar to that of cdc7-7 and psh1D strains, suggesting that Cdc7 regulates Cse4 in a pathway that overlaps with Psh1. Our results define a DNA replication initiation-independent role of DDK as a regulator of Psh1-mediated proteolysis of Cse4 to prevent mislocalization of Cse4.Fil: Eisenstatt, Jessica R.. National Institutes of Health; Estados UnidosFil: Boeckmann, Lars. National Institutes of Health; Estados UnidosFil: Au, Wei Chun. National Institutes of Health; Estados UnidosFil: Garcia, Valerie. National Institutes of Health; Estados UnidosFil: Bursch, Levi. National Institutes of Health; Estados UnidosFil: Ocampo, Josefina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. National Instituto of Child Health & Human Development; Estados UnidosFil: Costanzo, Michael. National Institutes of Health; Estados Unidos. University of Toronto; CanadáFil: Weinreich, Michael. Van Andel Research Institute; Estados UnidosFil: Sclafani, Robert A.. University of Colorado; Estados UnidosFil: Baryshnikova, Anastasia. University of Princeton; Estados UnidosFil: Myers, Chad L.. University of Minnesota; Estados UnidosFil: Boone, Charles. University of Toronto; Canadá. National Institutes of Health; Estados UnidosFil: Clark, David J.. National Institutes of Health; Estados UnidosFil: Baker, Richard. University of Massachusetts; Estados UnidosFil: Basrai, Munira A.. National Institutes of Health; Estados Unido

Skp, Cullin, F-box (SCF)-Met30 and SCF-Cdc4-Mediated Proteolysis of CENP-A Prevents Mislocalization of CENP-A for Chromosomal Stability in Budding Yeast

Restricting the localization of the histone H3 variant CENP-A (Cse4 in yeast, CID in flies) tocentromeres is essential for faithful chromosome segregation. Mislocalization of CENP-Aleads to chromosomal instability (CIN) in yeast, fly and human cells. Overexpression andmislocalization of CENP-A has been observed in many cancers and this correlates withincreased invasiveness and poor prognosis. Yet genes that regulate CENP-A levels andlocalization under physiological conditions have not been defined. In this study we used agenome-wide genetic screen to identify essential genes required for Cse4 homeostasis toprevent its mislocalization for chromosomal stability. We show that two Skp, Cullin, Fbox(SCF) ubiquitin ligases with the evolutionarily conserved F-box proteins Met30 andCdc4 interact and cooperatively regulate proteolysis of endogenous Cse4 and prevent itsmislocalization for faithful chromosome segregation under physiological conditions. Theinteraction of Met30 with Cdc4 is independent of the D domain, which is essential for theirhomodimerization and ubiquitination of other substrates. The requirement for both Cdc4and Met30 for ubiquitination is specifc for Cse4; and a common substrate for Cdc4 andMet30 has not previously been described. Met30 is necessary for the interaction betweenCdc4 and Cse4, and defects in this interaction lead to stabilization and mislocalization ofCse4, which in turn contributes to CIN. We provide the first direct link between Cse4 mislocalizationto defects in kinetochore structure and show that SCF-mediated proteolysis ofPLOS Genetics Cse4 is a major mechanism that prevents stable maintenance of Cse4 at non-centromericregions, thus ensuring faithful chromosome segregation. In summary, we have identifiedessential pathways that regulate cellular levels of endogenous Cse4 and shown that proteolysisof Cse4 by SCF-Met30/Cdc4 prevents mislocalization and CIN in unperturbed cells.Fil: Au, Wei-Chun. National Institutes of Health; Estados UnidosFil: Zhang, Tianyi. National Institutes of Health; Estados UnidosFil: Mishra, Prashant K.. National Institutes of Health; Estados UnidosFil: Eisenstatt, Jessica R.. National Institutes of Health; Estados UnidosFil: Walker, Robert L.. National Institutes of Health; Estados UnidosFil: Ocampo, Josefina. National Institutes of Health; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Dawson, Anthony. National Institutes of Health; Estados UnidosFil: Warren, Jack. National Institutes of Health; Estados UnidosFil: Costanzo, Michael. University of Toronto; CanadáFil: Baryshnikova, Anastasia. California Life Company; Estados UnidosFil: Flick, Karin. University of California; Estados UnidosFil: Clark, David J.. National Institutes of Health; Estados UnidosFil: Meltzer, Paul S.. National Institutes of Health; Estados UnidosFil: Baker, Richard E.. University of Massachussets; Estados UnidosFil: Myers, Chad. University of Minnesota; Estados UnidosFil: Boone, Charles. University of Toronto; CanadáFil: Kaiser, Peter. University of California; Estados UnidosFil: Basrai, Munira A.. National Institutes of Health; Estados Unido

Identification of a Lifespan Extending Mutation in the <i>Schizosaccharomyces pombe</i> Cyclin Gene <i>clg1</i>⁺ by Direct Selection of Long-Lived Mutants

<div><p>Model organisms such as budding yeast, worms and flies have proven instrumental in the discovery of genetic determinants of aging, and the fission yeast <i>Schizosaccharomyces pombe</i> is a promising new system for these studies. We devised an approach to directly select for long-lived <i>S. pombe</i> mutants from a random DNA insertion library. Each insertion mutation bears a unique sequence tag called a bar code that allows one to determine the proportion of an individual mutant in a culture containing thousands of different mutants. Aging these mutants in culture allowed identification of a long-lived mutant bearing an insertion mutation in the cyclin gene <i>clg1</i>^<i>+</i>. Clg1p, like Pas1p, physically associates with the cyclin-dependent kinase Pef1p. We identified a third Pef1p cyclin, Psl1p, and found that only loss of Clg1p or Pef1p extended lifespan. Genetic and co-immunoprecipitation results indicate that Pef1p controls lifespan through the downstream protein kinase Cek1p. While Pef1p is conserved as Pho85p in <i>Saccharomyces cerevisiae</i>, and as cdk5 in humans, genome-wide searches for lifespan regulators in <i>S. cerevisiae</i> have never identified Pho85p. Thus, the <i>S. pombe</i> system can be used to identify novel, evolutionarily conserved lifespan extending mutations, and our results suggest a potential role for mammalian cdk5 as a lifespan regulator.</p> </div

Isolating long-lived mutants using an <i>S. pombe</i> bar code-tagged insertion mutant library.

<p>(A) The schematic diagram of the insertion vector DNA used to generate the bar code-tagged <i>S. pombe</i> insertion mutants. The gray boxes represent sequences that protect the bar code from degradation. The 5’ and 3’ primers are to amplify the bar code-containing DNA. (B) The flow chart of the selection procedure. Cells from ~3,600 bar code-tagged insertion mutants were pooled together and aged in the standard SD medium. Bar codes with flanking <i>Sfi</i>I recognition sequences were amplified by PCR from the surviving cells, digested with <i>Sfi</i>I and ligated to produce long bar code oligomers for cloning so that many bar codes can be sequenced in a single reaction. As the bar code was designed to exclude <i>Sfi</i>I sites, all bar codes can be recovered in this procedure. (C) A representative bar code oligomerization. Purified bar code DNA monomer is shown in lane 1. After ligation, the bar code DNA monomer is converted to a series of higher molecular-weight oligomers (lane 2).</p

Clg1p and Pef1p interact and regulate CLS through the same pathway.

<p>(A) The two-hybrid assay of Clg1p and Pef1p. Cells expressing both Gal4 DNA binding domain (DBD)- and Gal4 activation domain (AD)-fusion proteins were analyzed as described in Materials and Methods. Blue color is indicative of a positive interaction whereas white color means no detectable protein interaction. Clg1(N) p is the N-terminal fragment predicted to be made in the <i>clg1</i> insertion mutation, and encodes the first 590 bps of the <i>clg1</i>⁺ ORF. (B) Clg1p co-immunoprecipitates with Pef1p in <i>S. pombe</i> cells. Lysates prepared from cells expressing Pef1p-3HA and FLAG-Clg1p, or Pef1p-3HA alone, were immunoprecipitated with anti-FLAG antibody. The precipitated complexes and 100 µg of input lysate were analyzed by SDS-PAGE and Western blotting with anti-HA or anti-FLAG antibodies. (C) Deletion of <i>pef1</i>^<i>+</i> extends CLS in the same pathway as <i>clg1</i>^<i>+</i>. The CLS of the <i>pef1</i>Δ single deletion mutant and the <i>clg1</i>Δ <i>pef1</i>Δ double deletion mutant were determined in SD + 3% glucose medium along side the wild type and <i>clg1</i>Δ mutant strain (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069084#pone-0069084-g002" target="_blank">Figure 2C</a>). The lifespans of the two <i>pef1∆</i> mutants were longer than that of the wild type (p = 0.0045 for <i>pef1</i>Δ, p = 0.0029 for <i>clg1</i>Δ <i>pef1</i>Δ). The lifespan curve of the <i>clg1</i>Δ <i>pef1</i>Δ double deletion mutant was indistinguishable from the <i>pef1</i>Δ single deletion mutant, and the <i>clg1</i>Δ <i>pef1</i>Δ curve significantly overlapped with the <i>clg1</i>Δ mutant (p > 0.1 for all comparisons). Survival curves comparing each individual mutant with the wild type with error bars are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069084#pone.0069084.s005" target="_blank">Figure S5</a>.</p

Deletion of <i>ppk18</i>⁺ shortens CLS in both wild type and the <i>clg1</i>Δ strain.

<p>The CLS of the <i>ppk18</i>Δ and <i>clg1</i>Δ <i>ppk18</i>Δ mutants were shorter than that of the wild type (p = 0.002 for <i>ppk18</i>Δ; p = 0.001 for <i>clg1</i>Δ <i>ppk18</i>Δ). The <i>clg1</i>Δ <i>ppk18</i>Δ double deletion mutant had an intermediate lifespan, which is statistically different from that of <i>clg1</i>Δ (p = 0.001) and <i>ppk18</i>Δ (p = 0.0078 after Day 2). All four strains reached the same density by day 0 the lifespan (~5.7 x 10⁷ cells/ml). Survival curves comparing each individual mutant with the wild type with error bars are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069084#pone.0069084.s008" target="_blank">Figure S8</a>.</p

Pef1p interacts with Cek1p in fission yeast cells.

<p>Pef1p-3HA was expressed in cells with either FLAG-Cek1p or FLAG-Ppk18p. Cell lysates were subjected to immunoprecipitation with anti-FLAG antibody, and the immunoprecipitates and cell lysates were analyzed by Western blotting with anti-FLAG and anti-HA antibodies.</p

Insertion mutation or deletion of the <i>clg1</i>⁺ ORF extends <i>S. pombe</i> CLS.

<p>(A) In the mutant carrying bar codes 4031 and 4033 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069084#tab1" target="_blank">Table 1</a>), tandem integration of two vectors (black triangle) was identified in the <i>clg1</i>⁺ ORF and accompanied by a 4-bp deletion (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069084#tab2" target="_blank">Table 2</a>). (B, C) The CLS of the re-generated mutant bearing the same <i>clg1</i>^<i>-</i> insertion mutation in an isogenic wild type strain (KRP42, B), and a strain bearing a deletion of the <i>clg1</i>⁺ ORF (KRP138, C) were determined in SD + 3% glucose medium (Materials and Methods). Both mutant strains exhibited a longer CLS than the wild type strain (p = 0.0001 for KRP42; p = 0.003 for KRP138). Error bars show the range of the duplicate cultures of each strain.</p