Mitosis: One Polo controls it all

On November 6, 2014, Cell Cycle published a paper from the Yoshida lab entitled “The budding yeast Polo-like kinase Cdc5 is released from the nucleus in anaphase for timely mitotic exit.” This study was authored by Vladimir V. Botchkarev Jr., Valentina Rossio, and Satoshi Yoshida.

The cell cycle is one of the most fundamental biological processes whose ultimate goal is cell division with equal content of DNA in both daughter cells. The process of cell division is regulated by many intracellular events which must occur in a sequential order. These events include mitotic entry, faithful chromosome segregation, mitotic exit, and cytokinesis. Over the past 25 years, the Polo-like kinase (Polo) has been established to play important regulatory roles in each of these processes. Although many mitotic substrates of Polo have been discovered, the mechanism by which Polo can coordinate all of these mitotic events has remained largely elusive.

To understand the mechanism by which Polo can target its many substrates in a sequential order during mitosis, we decided to study the budding yeast Polo kinase Cdc5, which has high conservation with the human Polo-like kinase 1.

We found that Cdc5-GFP dynamically changes its localization during the cell cycle: Cdc5 is found in the cytoplasm in S- through early G2-phase, it accumulates in the nucleus at metaphase, and is released again to the cytoplasm in anaphase. Blocking nuclear import of Cdc5 in metaphase leads to a prolonged metaphase duration, suggesting that nuclear Cdc5 is required for chromosome segregation. In contrast, blocking nuclear release of Cdc5 in anaphase results in a prolonged anaphase duration, a defect in activation of the cytoplasmic Mitotic Exit Network, and a defect in cytokinesis. This indicates that Cdc5 is released from the nucleus to the cytoplasm in anaphase for timely mitotic exit and cytokinesis. We further found that activation of the Cdc14 phosphatase, a known nuclear substrate of Cdc5, is required for Cdc5 nuclear release in anaphase.

Collectively, our work reveals that the budding yeast Polo-like kinase Cdc5 controls the timing of mitotic events by dynamically changing its sub-cellular localization. Furthermore, our data suggests the existence of a positive feedback look between Cdc5 and Cdc14 to regulate timely mitotic exit. Read more

Chromosome Tethering in Yeast

On July 14, 2014, PLOS ONE  published a paper from the Haber and Kondev labs. The paper, Effect of chromosome tethering on nuclear organization in yeast, was authored by Baris Avsaroglu, Gabriel Bronk, Susannah Gordon-Messer, Jungoh Ham, Debra A. Bressan, James E. Haber, and Jane Kondev.

by Baris Avsaroglu

Chromosopone.0102474_350mes are folded into the cell nucleus in a non-random fashion. In yeast cells the Rabl model is used to describe the folded state of interphase chromosomes in terms of tethering interactions of the centromeres and the telomeres with the nuclear periphery. By combining theory and experiments, we assess the importance of chromosome tethering in determining the spatial location of genes within the interphase yeast nucleus. Using a well-established polymer model of yeast chromosomes to compute the spatial distributions of several genetic loci, we demonstrate that telomere tethering strongly affects the positioning of genes within the first 10 kb of the telomere. Further increasing the distance of the gene from the telomere reduces the effect of the attachment at the nuclear envelope exponentially fast with a characteristic distance of 20 kb. We test these predictions experimentally using fluorescently labeled genetic loci on chromosome III in wild type and in two mutant yeast strains with altered tethering interactions. For all the cases examined we find good agreement between theory and experiment. This study provides a quantitative test of the polymer model of yeast chromosomes, which can be used to predict long-ranged interactions between genetic loci relevant in transcription regulation and DNA recombination.

Patching Up Broken Chromosomes

Olga Tsaponina and James Haber’s recent paper “Frequent Interchromosomal Template Switches during Gene Conversion in S. cerevisiae” was published online by Molecular Cell on July 24, 2014.

by James Haber

“The process of copying DNA every time our cells divide is exceptionally accurate, but in copying 6,000,000,000 base pairs of the genome mistakes do occur, including both mutations and the formation of chromosome breaks. These breaks must be repaired to maintain the integrity of our chromosomes.  In our recent paper we have demonstrated that the mechanism of patching up a broken chromosome is associated with a surprisingly high level of alterations of the sequence.  Many of these changes result from “slippage” of the DNA polymerases copying the DNA during the repair process; for example in copying a sequence of 4 Gs, the polymerase occasionally jumps over one, to lose a base from the sequence (a frameshift mutation).

graphical_abstract_350In this paper we focused on more dramatic slippage events in which the copying machinery jumped from one chromosome to a related but divergent sequence on another chromosome and then jumped back, creating a chimeric sequence.  These interchromosomal template switches (ICTS) occur at a low rate when the distant sequence is only 71% identical, but if we make that segment 100% identical we could find such jumps 10,000 times more frequently, in about 1 in 300 events.  This result reveals how unstable the copying machinery in DNA repair is compared to normal DNA replication. This was very surprising and provides an explanation for many complex rearrangements associated with cancers.  In carrying out this work we identified the first protein that is needed to permit these frequent jumps: a chromatin remodeling enzyme known as Rdh54 that previously did not have a well-defined role in DNA repair in somatic cells.

Finally, we learned a new role for the proteins that survey the genome for mismatched bases that arise during replication and found that one of these proteins, Msh6, is required to specify which strand of DNA containing a mismatch is the “good one” that should be used as the template to correct the mismatch.

This work was supported by the National Institutes of Health General Medical Institute”.

Eapen wins HHMI International Student Research Fellowship

Vinay Eapen from the Haber Lab in Biology has been awarded an HHMI International Student Research Fellowship. These fellowships, highly sought-after, are among the few available to international students studying at major research universities in the US – there were only 42 recipients nationwide. Eapen is a graduate student entering his fourth year in the Molecular and Cell Biology PhD program at Brandeis, and already has 4 publications from Brandeis to his credit resulting from his studies of the DNA damage checkpoint and autophagy in yeast.

 

Rosenstiel Award 2012- Dr. Steven J. Elledge

The 2012 Rosenstiel award is being awarded to Dr. Steven J. Elledge of Harvard University and the Howard Hughes Medical Institute for his seminal contributions towards understanding the eukaryotic DNA damage response[1] [2] .
Cells are constantly challenged by damage to their DNA. It of no surprise therefore, that both prokaryotic and eukaryotic cells have evolved sophisticated and remarkably complex responses to deal with damaged DNA. It is for elucidating these mechanisms that Dr. Elledge is being honored with the Rosenstiel award this year.
Dr. Elledge’s interest with the DNA damage response began as a graduate student at MIT in the laboratory of Graham Walker, where he identified and cloned genes involved in DNA repair mechanism known as the SOS reponse in the bacterium E.coli[3, 4] . It was during this time that Dr. Elledge also invented an extremely useful molecular biology tool known as ‘phasmids’ which allowed for the ability to rapidly clone E.coli genes by packaging them in phages[5].
After MIT, Dr Elledge began his postdoctoral work at Stanford University where he discovered the Ribonucleotide reductase(RNR) genes in budding yeast[6, 7]. These genes are induced following DNA damage to promote the synthesis of deoxyribonucleotides which helps facilitate DNA repair. Dr. Elledge followed up on this work as a professor at Baylor University by a series of important papers that shed light on how cells arrest division after DNA damage. Most notably in 1994, his group identified the Rad53 checkpoint kinase that is activated after DNA damage and contributes to cell cycle arrest [8]. In 1998, his group also identified the mammalian homolog of Rad53 (Chk2) [9, 10].In 1999, the Elledge group reported that the DNA damage checkpoint in yeast occurs in two parallel pathways laying the foundation of our understanding of the DNA damage checkpoint[11]. More recently, work from the Elledge lab identified novel factors in the DNA damage response by performing a siRNA screen in mammalian cells[12].
Dr Steve Elledge has been an investigator of the Howard Hughes Medical Institute since 1993. In 2003 he moved to Harvard Medical School as Professor in the Departments of Genetics and as a Geneticist in the Department of Medicine, Brigham and Women’s Hospital. Dr. Elledge was elected in 2003 to both the U.S. National Academy of Sciences and the American Academy of Arts and Sciences. In addition to the Rosenstiel award, he has received the DAMD Breast Cancer Innovator Award (2003), the National Academy of Sciences Award in Molecular Biology (2002), the John B. Carter, Jr. Technology Innovation Award (2002), and the Paul Marks Prize for Cancer Research (2001[2]).

Editor’s Note: On Mar 20, 2013, Elledge was named to receive a 2013 Canada Gairdner International Award.

1. Brownlee, C., Biography of Stephen J. Elledge. Proc Natl Acad Sci U S A, 2004. 101(10): p. 3336-7.
2. Haber, J.E., The 2005 Genetics Society of America Medal. Steven J. Elledge. Genetics, 2005. 169(2): p. 506-7.
3. Elledge, S.J. and G.C. Walker, Proteins required for ultraviolet light and chemical mutagenesis. Identification of the products of the umuC locus of Escherichia coli. J Mol Biol, 1983. 164(2): p. 175-92.
4. Elledge, S.J. and G.C. Walker, The muc genes of pKM101 are induced by DNA damage. J Bacteriol, 1983. 155(3): p. 1306-15.
5. Elledge, S.J. and G.C. Walker, Phasmid vectors for identification of genes by complementation of Escherichia coli mutants. J Bacteriol, 1985. 162(2): p. 777-83.
6. Elledge, S.J. and R.W. Davis, Identification of the DNA damage-responsive element of RNR2 and evidence that four distinct cellular factors bind it. Mol Cell Biol, 1989. 9(12): p. 5373-86.
7. Elledge, S.J. and R.W. Davis, DNA damage induction of ribonucleotide reductase. Mol Cell Biol, 1989. 9(11): p. 4932-40.
8. Allen, J.B., et al., The SAD1/RAD53 protein kinase controls multiple checkpoints and DNA damage-induced transcription in yeast. Genes Dev, 1994. 8(20): p. 2401-15.
9. Hirao, A., et al., DNA damage-induced activation of p53 by the checkpoint kinase Chk2. Science, 2000. 287(5459): p. 1824-7.
10. Matsuoka, S., M. Huang, and S.J. Elledge, Linkage of ATM to cell cycle regulation by the Chk2 protein kinase. Science, 1998. 282(5395): p. 1893-7.
11. Sanchez, Y., et al., Control of the DNA damage checkpoint by chk1 and rad53 protein kinases through distinct mechanisms. Science, 1999. 286(5442): p. 1166-71.
12. Adamson, B., et al., A genome-wide homologous recombination screen identifies the RNA-binding protein RBMX as a component of the DNA-damage response. Nat Cell Biol, 2012. 14(3): p. 318-28.

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.

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