TIF1 Activates the Intra-S-Phase Checkpoint Response in the Diploid Micronucleus and Amitotic Polyploid Macronucleus of<i>Tetrahymena</i>

Yakisich, Juan Sebastián; Sandoval, Pamela Y.; Morrison, Tara L.; Kapler, Geoffrey M.

doi:10.1091/mbc.e06-05-0469

Cited by 21 publications

(36 citation statements)

References 35 publications

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“…To explore this possibility, we tested whether cell cycle arrest could be prevented by the addition of caffeine. Although caffeine has pleiotropic effects on cells, it is an effective inhibitor of ATM and ATR and has been used to abrogate DNA damage checkpoints in a variety of organisms, including Tetrahymena (24,26,48). For example, the addition of caffeine to Tetrahymena cells that have suffered DNA damage during S phase abrogates the intra-S-phase checkpoint and allows the cells to resume proliferation (48).…”

Section: Resultsmentioning

confidence: 99%

Tetrahymena POT1a Regulates Telomere Length and Prevents Activation of a Cell CycleCheckpoint

Jacob

Lescasse

Linger

et al. 2007

Molecular and Cellular Biology

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The POT1/TEBP telomere proteins are a group of single-stranded DNA (ssDNA)-binding proteins that have long been assumed to protect the G overhang on the telomeric 3 strand. We have found that the Tetrahymena thermophila genome contains two POT1 gene homologs, POT1a and POT1b. The POT1a gene is essential, but POT1b is not. We have generated a conditional POT1a cell line and shown that POT1a depletion results in a monster cell phenotype and growth arrest. However, G-overhang structure is essentially unchanged, indicating that POT1a is not required for overhang protection. In contrast, POT1a is required for telomere length regulation. After POT1a depletion, most telomeres elongate by 400 to 500 bp, but some increase by up to 10 kb. This elongation occurs in the absence of further cell division. The growth arrest caused by POT1a depletion can be reversed by reexpression of POT1a or addition of caffeine. Thus, POT1a is required to prevent a cell cycle checkpoint that is most likely mediated by ATM or ATR (ATM and ATR are protein kinases of the PI-3 protein kinase-like family). Our findings indicate that the essential function of POT1a is to prevent a catastrophic DNA damage response. This response may be activated when nontelomeric ssDNA-binding proteins bind and protect the G overhang.In order to maintain genome integrity, the telomeric DNA from cells with linear chromosomes is packaged into a protective nucleoprotein complex (10). In the absence of this complex, the telomeres are recognized as DNA damage and subject to repair by nonhomologous end joining (33). The resulting chromosome fusions lead to genome instability (36). The protective telomeric complex is composed of a series of unique telomere proteins that bind the double-stranded region of the telomeric DNA and/or the single-strand overhang on the 3Ј G-rich strand (37). Although the exact composition of the complex varies between species, vertebrates, yeasts, plants, and ciliates all use a series of structurally related proteins to protect their telomeres. The G-overhang binding proteins all bind Gstrand DNA through a conserved OB fold motif, while the double-stranded DNA-binding proteins bind via a conserved myb motif (23,37,46).In vertebrate cells, telomeres are packaged by a core complex of six proteins which function both in telomere protection and in telomere length regulation (10). This core complex (which is sometimes called shelterin) contains two doublestranded DNA-binding proteins, TRF1 and TRF2, which anchor the complex along the length of the telomere, and the TRF1/2 interaction partners Rap1, TIN2, and TPP1. The Gstrand binding protein POT1 is also a component of the complex. Although POT1 is secured into the complex through interactions with TPP1 (17, 31, 50), POT1 molecules are also thought to bind the G-strand overhang via their OB foldcontaining DNA-binding domain (45,46). Additional telomere-associated proteins include a number of DNA damage response factors (33). However, these factors appear to bind only transiently during replication of...

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Section: Resultsmentioning

confidence: 99%

Tetrahymena POT1a Regulates Telomere Length and Prevents Activation of a Cell CycleCheckpoint

Jacob

Lescasse

Linger

et al. 2007

Molecular and Cellular Biology

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“…TIF1-deficient cells are hypersensitive to genotoxic stress and are defective in the activation of the intra-S phase checkpoint mediated by the sensor/transducer kinase ATR (Yakisich et al, 2006). These studies suggest that a role of Whirlies in the maintenance of genome stability may be conserved between plants and ciliates.…”

Section: Possible Roles For Whirly Proteins In the Repair Of Dsbsmentioning

confidence: 88%

Crystal Structures of DNA-Whirly Complexes and Their Role in Arabidopsis Organelle Genome Repair

et al. 2010

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DNA double-strand breaks are highly detrimental to all organisms and need to be quickly and accurately repaired. Although several proteins are known to maintain plastid and mitochondrial genome stability in plants, little is known about the mechanisms of DNA repair in these organelles and the roles of specific proteins. Here, using ciprofloxacin as a DNA damaging agent specific to the organelles, we show that plastids and mitochondria can repair DNA double-strand breaks through an error-prone pathway similar to the microhomology-mediated break-induced replication observed in humans, yeast, and bacteria. This pathway is negatively regulated by the single-stranded DNA (ssDNA) binding proteins from the Whirly family, thus indicating that these proteins could contribute to the accurate repair of plant organelle genomes. To understand the role of Whirly proteins in this process, we solved the crystal structures of several Whirly-DNA complexes. These reveal a nonsequence-specific ssDNA binding mechanism in which DNA is stabilized between domains of adjacent subunits and rendered unavailable for duplex formation and/or protein interactions. Our results suggest a model in which the binding of Whirly proteins to ssDNA would favor accurate repair of DNA double-strand breaks over an error-prone microhomology-mediated break-induced replication repair pathway.

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“…It has been shown that the PI3K-related kinases ATM and particularly ATR are inhibited by wortmannin only at significantly higher concentrations than PI3Ks (Sarkaria et al, 1998;Liu et al, 2005;Knight et al, 2006). Accordingly, wortmannin at 250 nM impaired nuclear degradation in Tetrahymena , but it inhibited the-presumably-ATR-dependent intra-S-phase DNA damage checkpoint response only at 2 M (Yakisich et al, 2006). Here, we observed that concentrations below 2 M did not effectively inhibit MIC elongation (Supplemental Table S3), which is consistent with the suppression of ATM or ATR activity.…”

Section: In Caffeine-and Wortmannin-treated Meioses Mics Do Not Fullmentioning

confidence: 99%

“…A Tetrahymena orthologue of ATR (TTHERM_01008650) but not of ATM has been identified by a bioinformatics search (Yakisich et al, 2006), making the former the likely target of chemical inhibition.…”

Section: Knockout Of Atr1 Suppresses Mic Elongationmentioning

confidence: 99%

“…In vivo studies in human cell lines suggest that caffeine can block cell cycle checkpoint responses without inhibiting ATM or ATR activation (Cortez, 2003). On the other hand, Yakisich et al (2006) found evidence for a caffeine-sensitive intra-S-phase checkpoint activated by an ATR-like protein kinase (TTHERM_01008650) in Tetrahymena. Notably, Tif1p, a likely factor in the presumed checkpoint pathway, may also have a role in the response to meiotic DSBs since the TIF1-deficient partner in mating pairs of wild-type and tif1 mutant cells displayed reduced crescent formation (Morrison et al, 2005).…”

Section: Mic Elongation Is Elicited By a Caffeine-andmentioning

confidence: 99%

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TetrahymenaMeiotic Nuclear Reorganization Is Induced by a Checkpoint Kinase–dependent Response to DNA Damage

Loidl

Mochizuki

2009

MBoC

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In the ciliate Tetrahymena, meiotic micronuclei (MICs) undergo extreme elongation, and meiotic pairing and recombination take place within these elongated nuclei (the "crescents"). We have previously shown that elongation does not occur in the absence of Spo11p-induced DNA double-strand breaks (DSBs). Here we show that elongation is restored in spo11⌬ mutants by various DNA-damaging agents including ones that may not cause DSBs to a notable extent. MIC elongation following Spo11p-induced DSBs or artificially induced DNA lesions is probably a DNA-damage response mediated by a phosphokinase signal transduction pathway, since it is suppressed by the ATM/ATR kinase inhibitors caffeine and wortmannin and by knocking out Tetrahymena's ATR orthologue. MIC elongation occurs concomitantly with the movement of centromeres away from the telomeric pole of the MIC. This DNA damage-dependent reorganization of the MIC helps to arrange homologous chromosomes alongside each other but is not sufficient for exact pairing. Thus, Spo11p contributes to bivalent formation in two ways: by creating a favorable spatial disposition of homologues and by stabilizing pairing by crossovers. The polarized chromosome orientation inside the crescent resembles the conserved meiotic bouquet, and crescent and bouquet also share the putative function of aiding meiotic pairing. However, they are regulated differently because in Tetrahymena, DSBs are required for entering rather than exiting this stage.

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TIF1 Activates the Intra-S-Phase Checkpoint Response in the Diploid Micronucleus and Amitotic Polyploid Macronucleus ofTetrahymena

Cited by 21 publications

References 35 publications

Tetrahymena POT1a Regulates Telomere Length and Prevents Activation of a Cell CycleCheckpoint

Tetrahymena POT1a Regulates Telomere Length and Prevents Activation of a Cell CycleCheckpoint

Crystal Structures of DNA-Whirly Complexes and Their Role in Arabidopsis Organelle Genome Repair

TetrahymenaMeiotic Nuclear Reorganization Is Induced by a Checkpoint Kinase–dependent Response to DNA Damage

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