Looking for Fun(30)

A recent paper from the Haber lab by Eapen et al., “The Saccharomyces cerevisiae Chromatin Remodeler Fun30 Regulates DNA End Resection and Checkpoint Deactivation“, is the most read paper from the journal Molecular and Cell Biology for October 2012. Join the fun, read the paper!

Damaged DNA and self-eating (autophagy) in budding yeast.

Chromosome double-strand breaks (DSBs) threaten the integrity of the genome. Cells respond to DSBs by activating the DNA damage checkpoint that blocks cells prior to mitosis, allowing more time for the repair of damaged DNA. When the DSB can be repaired, the cell cycle checkpoint is turned off so that cells can resume cell cycle progression, a process termed recovery. If the DSB remains unrepaired, G2/M arrest persists for a long time but eventually cells adapt and – despite the persistent DNA damage – complete mitosis and divide. Much of our understanding of the DNA damage response has come from the study of the budding yeast Saccharomyces cerevisiae, where it is possible to create DSB damage synchronously in all cells of the population. This can be accomplished either by uncapping telomeres, exposing their normally protected ends or by creating a single, defined DSB by inducing the site-specific HO endonuclease. From such studies, it was possible to identify a highly evolutionarily conserved DNA damage sensing and signaling cascade that is initiated by Mec1, the yeast homolog of mammalian ATR protein kinase (reviewed in Ref. (1)). Yeast genetic approaches revealed a number of adaptation-defective mutants, a subset of which also are recovery-defective. Previous studies also demonstrated that triggering the DNA damage checkpoint affects not only mitosis and the efficiency of DNA repair within the nucleus; it also affects cytoplasmic responses (2, 3). In a new paper from the Haber lab published in PNAS, we uncovered mutations in the Golgi-Associated Retrograde Protein (GARP) complex that are adaptation-defective. We show that the defect in these mutants can be mimicked by activating the cytoplasm-to-vacuole (CVT) pathway of autophagy that prevents the nuclear accumulation of separase, Esp1, in the nucleus, thus preventing the cells both adapting and recovering from DSB damage.

In budding yeast, a single unrepaired double-strand break (DSB) triggers the Mec1-dependent cell cycle arrest prior to anaphase for 12-15 before they adapt. Adaptation is accompanied by the loss of hyperphosphorylation of Rad53, yeast’s Chk2 homolog.  Rad53 remains phosphorylated in a number of adaptation-defective mutations, including deletion of the two PP2C phosphatases, ptc2ptc3D, that normally dephosphorylate Rad53.  Adaptation is also blocked by ablating a number of proteins with diverse roles in DSB repair, including srs2D, rdh54D as well as by a mutation in yeast’s polo kinase cdc5-ad.

In our paper, we find that hyperactivation of the cytoplasm-to-vacuole (CVT) autophagy pathway causes the permanent G2/M arrest of cells with a single DSB that is reflected in the nuclear exclusion of both separase, Esp1, and its chaperone/inhibitor, securin, Pds1(See figure).  Autophagy in response to DNA damage can be induced in three different ways: (1) by deleting members of the Golgi-Associated Retrograde Protein complex (GARP) such as vps51D; (2) by adding rapamycin; or (3) by overexpressing a dominant-negative ATG13-8SA mutation.  The permanent checkpoint-mediated arrest in any of these three conditions can be overcome in three ways: (1) by blocking autophagy with mutations such as atg1D, atg5D or atg11D; (2) by deleting the vacuolar protease Prb1 or its activator, Pep4; or (3) by driving Esp1 into the nucleus with a SV40 nuclear localization signal.  In contrast, these same alterations fail to suppress the adaptation defects of ptc2ptc3D or cdc5-ad.  Transient accumulation of Pds1 in the vaucole is also seen in wild type cells lacking PEP4 after induction of a DSB.  Unlike other adaptation-defective mutations, G2/M arrest persists even as the DNA damage-dependent phosphorylation of Rad53 diminishes, suggesting that cells have become unable to activate separase to initiate anaphase after DNA damage.  In addition, we have found that cells fail to recover when VPS51 is deleted or when ATG13-8SA is overexpressed.

Increased autophagy causes the delocalization of both Pds1 (securin) and Esp1 (separase) from the nucleus in checkpoint-arrested budding yeast cells. A. GFP-tagged Pds1 and Esp1 localize to the nucleus at the neck of G2/M-arrested wild type (WT) cells that have suffered a single unrepaired chromosome double-strand break (DSB). Both rdh54Δ and vps51Δ prevent cells from adapting and resuming cell cycle progression, but only ablating Vps51 – part of the Golgi-associated retrograde protein (GARP) complex – causes the mislocalization of Pds1 and Esp1 and the partial degradation of Pds1 by vacuolar proteases. Preventing degradation of Pds1 (and possibly other mitotic regulators) results in the suppression of permanent arrest and the relocalization of sufficient Esp1 into the nucleus to release cells from their pre-anaphase arrest. A similar suppression of arrest in vps51Δ cells is obtained by disabling autophagy (not shown). B. Induction of autophagy by overexpression of ATG13-8SA (6) prevents adaptation in wild type cells. Expression of ATG13-SA was induced at the same time that a single, unrepairable DSB was created. Whereas normal cells adapt by 24 h, increased autophagy prevents cells from progressing beyond the G2/M stage of the cell cycle. Deletion of the PEP4 gene that activates vacuolar proteases or ATG1 that is required for autophagy suppresses the arrest and allows cells to divide and resume cell cycle progression.

Taken together with other recent results (4, 5), these observations emphasize that the DNA damage response can trigger the mislocalisation and cytoplasmic proteolysis of important nuclear proteins that regulate DNA repair and cell cycle progression. These results broaden our perspective on the ways in which cells respond to DNA damage and delay cell cycle progression while such damage persists.

Ex MCB grad Farokh Dotiwala, current MCB grad Vinay Eapen and ex-postdoc Jake Harrison were the co-first authors on this paper. Assistant professor Satoshi Yoshida also contributed significantly to this project.

Dotiwala F(*), Eapen VV(*), Harrison JC(*), Arbel-Eden A, Ranade V, Yoshida S & Haber JE (2012) DNA damage checkpoint triggers autophagy to regulate the initiation of anaphase, PNAS (Published online before print November 19, 2012, doi: 10.1073/pnas.1218065109)

1.         Harrison JC & Haber JE (2006) Surviving the breakup: the DNA damage checkpoint. Annu Rev Genet 40:209-235.
2.         Dotiwala F, Haase J, Arbel-Eden A, Bloom K, & Haber JE (2007) The yeast DNA damage checkpoint proteins control a cytoplasmic response to DNA damage. Proc Natl Acad Sci U S A 104(27):11358-11363.
3.         Smolka MB, et al. (2006) An FHA domain-mediated protein interaction network of Rad53 reveals its role in polarized cell growth. J Cell Biol 175(5):743-753.
4.         Robert T, et al. (2011) HDACs link the DNA damage response, processing of double-strand breaks and autophagy. Nature 471(7336):74-79.
5.         Dyavaiah M, Rooney JP, Chittur SV, Lin Q, & Begley TJ (2011) Autophagy-dependent regulation of the DNA damage response protein ribonucleotide reductase 1. Mol Cancer Res 9(4):462-475.
6.         Kamada Y (2010) Prime-numbered Atg proteins act at the primary step in autophagy: unphosphorylatable Atg13 can induce autophagy without TOR inactivation. Autophagy 6(3):415-416.

How yeast switch mating type and why we care

For grad students needing background on work in the Haber lab studying DNA recombination and repair, there are a couple new papers out to help you. A new review by Prof. Haber entitled Mating-type genes and MAT switching in Saccharomyces cerevisiae in Genetics provides a detailed introduction to literature. There’s a lot there… as Jim says in the Acknowledgements

The part of this work that derives from my own lab has been carried out for more than 30 years by an exceptional contingent of graduate students, postdoctoral fellows, technicians, and Brandeis University undergraduates […]

If methods papers are what you need instead, check out Sugawara & Haber (2012), Monitoring DNA Recombination Initiated by HO Endonuclease in Methods in Molecular Biology.

What a failed drug does (and is there hope for latrepirdine?)

Latrepirdine (Dimebon) was initially used as an antihistamine drug in Russia. It was later found to be neuroprotective, and entered phase II clinical trials in the US for both Alzheimer’s disease and Huntington’s disease. However, Dimebon failed in a US-based phase II replication trial of a prior successful Russian phase II trial of mild-to-moderate AD. Given the initial promise of the drug and split results,  as well as the lack of treatments for neurodegenerative diseases, there in is significant interest in understanding the underlying molecular mechanism(s) for the drug’s effects.

In a paper appearing this week in Molecular Psychiatry, Brandeis researchers in the Petsko-Ringe lab, including postdoc Shulin Ju and undergraduate Jessica Liken ’11, used yeast models of neurodegenerative disease associated proteins to show that Dimebon specifically protects yeast from the cytotoxiciy of α-synuclein, a protein involved in Parkinson’s disease. They further showed that protection is mediated through its up-regulation of autophagy pathway. In collaboration with Sam Gandy‘s group at Mount Sinai School of Medicine, these findings were further confirmed and validated in neuronal cell and animal models.

Given these observations, disparities in the contribution of α-synuclein to the neuropathology between the Russian and US Dimebon studies might also explain, at least in part, the inconsistency of the cognitive benefit in the two trials. If this speculation is correct, then it may be interesting to test for benefits of Dimebon in treating synucleinopathies such as Parkinson’s disease, Lewy body dementia, REM sleep disorder and/or multiple system atrophy.

see also: press release from Mt. Sinai Alzheimer’s Diesease Research Center

Steele JW (*), Ju S(*), Lachenmayer ML(*), Liken J, Stock A, Kim SH, Delgado LM, Alfaro IE, Bernales S, Verdile G, Bharadwaj P, Gupta V, Barr R, Friss A, Dolios G, Wang R, Ringe D, Protter AA, Martins RN, Ehrlich ME, Yue Z, Petsko GA, Gandy S. Latrepirdine stimulates autophagy and reduces accumulation of alpha-synuclein in cells and in mouse brain. Molecular psychiatry. 2012.

Steele JW(*), Lachenmayer ML(*), Ju S, Stock A, Liken J, Kim SH, Delgado LM, Alfaro IE, Bernales S, Verdile G, Bharadwaj P, Gupta V, Barr R, Friss A, Dolios G, Wang R, Ringe D, Fraser P, Westaway D, St George-Hyslop PH, Szabo P, Relkin NR, Buxbaum JD, Glabe CG, Protter AA, Martins RN, Ehrlich ME, Petsko GA, Yue Z, Gandy S. Latrepirdine improves cognition and arrests progression of neuropathology in an Alzheimer’s mouse model. Molecular psychiatry. 2012.

Yoshida kicks off Summer seminar series

Assistant Professor of Biology Satoshi Yoshida kicked off the Life Sciences Summer Research Seminar series today, describing his research on wound healing in yeast to a capacity crowd in Rosenstiel 118. Wound healing in cells has been a difficult problem to study, partly because methods to create defined wounds in cells in a genetic model system have been lacking Yoshida and co-workers discovered that after focusing laser light at sublethal dosages on a budding yeast cell, the yeast cell responses by reorganizing its actin cytoskeleton so that the focus of growth goes away from the bud and towards the wound site. Yoshida described experiments to then define the genetic requirements for this shift in focus, with key players including Rho GTPase, protein kinase C, and the formin Bni1. Yoshida discussed results from his recent Cell paper “Competition between Cell Polarization and Cellular Wound Healing” as well as more recent ongoing results from the lab. There was a lively discussion following the seminar with eager students suggesting all kinds of possible follow-up experiments.

The seminar series will continue next Monday, July 16 at noon in Rosenstiel 118, with presentations from Jerome Menet (Rosbash lab) and Adam Osborne (Wangh lab).

iBiomagazine and iBioseminars

Some video resources if you need to explain scientific topics to students (or need something explained to you!)

iBioMagazine.org features short (<15 min) talks that highlight the human side of research. iBioSeminars.org provides approximately hour-long seminars by high profile researchers.

Professor Emeritus of Biology Hugh Huxley discusses the sliding filament theory of muscle contraction in a November 2011 video from iBiomagazine.org

 

 

Professor of Biology Jim Haber discusses Mechanisms of DNA Repair in a 2009 video from iBioseminars.org

 

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