Gio Biosco (P’98) gets NIH Pioneer Award

Molecular and Cell Biology alum Giovanni Bosco Giovanni Bosco Ph.D. ’98, currently Associate Professor of Genetics at Dartmouth, recently received a Pioneer Award from the NIH.

Gio Bosco is a die-hard chromatin regulation guy who became interested in whether long-term changes in DNA structure are involved in long-term behavioral plasticity. Gio did his PhD work in Jim Haber’s lab and provided some of the earliest and strongest evidence for a critical DNA repair mechanism called break-induced replication, which plays an essential role in maintaining the integrity of chromosome ends when the normal end-addition of DNA by telomerase is absent.

In his postdoctoral work, Giovanni turned from using yeast as a model system to Drosophila.  In the lab of Terry Orr-Weaver at MIT he focused his attention on the role of DNA replication in regulating gene amplifications and became interested in the importance of post-translational modifications (acetylations and phosphorylations) of the histone proteins that wrap the DNA into chromatin.

Approximately 7 years ago Gio started contemplating the question of how these post-translational histone modifications change during behavior and learning. He returned to his Brandeis roots to develop tools and approaches to address this problem. He received an NIH K18 grant to fund a sabbatical in Leslie Griffith’s lab in 2010. He and his behavior group have remained connected to Brandeis since then through frequent joint group meeting visits.

We’ll be interested to hear more about the role of histone modifications in how learning and memory occurs in the context of social behavior, and in how social behavior can be inherited through multiple generations, as the result of the Bosco lab research funded by this award.


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”.

Now available: genome stability

genomestablilityProfessor of Biology Jim Haber‘s new book, Genome Stability: DNA Repair and Recombination, five years in the making, has appeared in print this month. The table of contents and a sample chapter can be checked out on the publisher’s website. As always, check Jim’s website (or PubMed) to see new research from the lab. New this month in Nature Structural & Molcular Biology: Dynamics of yeast histone H2A and H2B phosphorylation in response to a double-strand break.



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.


Haber 70th Bday Symposium on May 31/Jun 1

Jim Haber as a young professorWe’re holding a 70th Birthday Celebration for Jim Haber on Friday and Saturday this week (May 31 and June 1, 2013). Haber lab members past and present, as well of some of Jim’s colleagues and collaborators, will be giving talks to celebrate the occasion. Come learn about DNA repair and wish Jim a happy birthday! Talks will be held in the Shapiro Campus Auditorium, starting with keynote speaker Fred Alt at 1:15 Friday. The full speaker list is available.

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.

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