Publications by authors named "Laura A Díaz-Martínez"

31 Publications

Recommendations for Effective Integration of Ethics and Responsible Conduct of Research (E/RCR) Education into Course-Based Undergraduate Research Experiences: A Meeting Report.

CBE Life Sci Educ 2019 06;18(2):mr2

Department of Biological Sciences, University of Texas at El Paso, El Paso, TX 79968.

Advancement of the scientific enterprise relies on individuals conducting research in an ethical and responsible manner. Educating emergent scholars in the principles of ethics/responsible conduct of research (E/RCR) is therefore critical to ensuring such advancement. The recent impetus to include authentic research opportunities as part of the undergraduate curriculum, via course-based undergraduate research experiences (CUREs), has been shown to increase cognitive and noncognitive student outcomes. Because of these important benefits, CUREs are becoming more common and often constitute the first research experience for many students. However, despite the importance of E/RCR in the research process, we know of few efforts to incorporate E/RCR education into CUREs. The Ethics Network for Course-based Opportunities in Undergraduate Research (ENCOUR) was created to address this concern and promote the integration of E/RCR within CUREs in the biological sciences and related disciplines. During the inaugural ENCOUR meeting, a four-pronged approach was used to develop guidelines for the effective integration of E/RCR in CUREs. This approach included: 1) defining appropriate student learning objectives; 2) identifying relevant curriculum; 3) identifying relevant assessments; and 4) defining key aspects of professional development for CURE facilitators. Meeting outcomes, including the aforementioned E/RCR guidelines, are described herein.
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http://dx.doi.org/10.1187/cbe.18-10-0203DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6755229PMC
June 2019

Integration of RCR and Ethics Education into Course-Based Undergraduate Research Experiences in the Biological Sciences: A Needed Discussion.

J Microbiol Biol Educ 2017 1;18(2). Epub 2017 Sep 1.

Department of Biological Sciences, The University of Texas at El Paso, El Paso, TX 79968.

Course-based undergraduate research experiences (CUREs) have been identified as a promising vehicle to broaden novices' participation in authentic scientific opportunities. While recent studies in the bioeducation literature have focused on the influence of CUREs on cognitive and non-cognitive student outcomes (e.g., attitudes and motivation, science process skills development), few investigations have examined the extent to which the contextual features inherent in such experiences affect students' academic and professional growth. Central among these factors is that of ethics and the responsible conduct of research (RCR)-essential cornerstones of the scientific enterprise. In this article, we examine the intersectionality of ethics/RCR instruction within CURE contexts through a critical review of existing literature that details mechanisms for the integration of ethics/RCR education into undergraduate laboratory experiences in the science domains. Building upon this foundation, we propose a novel, evidence-based framework that seeks to illustrate posited interactions between core ethics/RCR principles and unique dimensions of CUREs. It is our intent that this framework will inform and encourage open dialogue around an often-overlooked aspect of CURE instruction-how to best prepare ethically responsible scholars for entrance into the global scientific workforce.
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http://dx.doi.org/10.1128/jmbe.v18i2.1344DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5577973PMC
September 2017

Visualizing chromosome segregation in live cells.

Cell Cycle 2016 07 10;15(14):1811. Epub 2016 May 10.

b Department of Genetics , Cell Biology and Development, University of Minnesota , Minneapolis , MN , USA.

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http://dx.doi.org/10.1080/15384101.2016.1185852DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4968893PMC
July 2016

Graded requirement for the spliceosome in cell cycle progression.

Cell Cycle 2015 ;14(12):1873-83

a Department of Physiology; University of Texas Southwestern Medical Center ; Dallas , TX , USA.

Genome stability is ensured by multiple surveillance mechanisms that monitor the duplication, segregation, and integrity of the genome throughout the cell cycle. Depletion of components of the spliceosome, a macromolecular machine essential for mRNA maturation and gene expression, has been associated with increased DNA damage and cell cycle defects. However, the specific role for the spliceosome in these processes has remained elusive, as different cell cycle defects have been reported depending on the specific spliceosome subunit depleted. Through a detailed cell cycle analysis after spliceosome depletion, we demonstrate that the spliceosome is required for progression through multiple phases of the cell cycle. Strikingly, the specific cell cycle phenotype observed after spliceosome depletion correlates with the extent of depletion. Partial depletion of a core spliceosome component results in defects at later stages of the cell cycle (G2 and mitosis), whereas a more complete depletion of the same component elicits an early cell cycle arrest in G1. We propose a quantitative model in which different functional dosages of the spliceosome are required for different cell cycle transitions.
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http://dx.doi.org/10.1080/15384101.2015.1039209DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4614359PMC
April 2016

The complexity of life and death decisions in mitosis.

Mol Cell Oncol 2015 Apr-Jun;2(2):e969658. Epub 2014 Dec 1.

Howard Hughes Medical Institute; Department of Pharmacology ; University of Texas Southwestern Medical Center ; Dallas, TX USA.

The anticancer drug taxol stabilizes microtubules and activates the spindle checkpoint, causing prolonged mitotic arrest in cancer cells. Our recent work suggests that the cellular decision to live or die following mitotic arrest is a complex process involving crosstalk between competing apoptotic and adaptation pathways.
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http://dx.doi.org/10.4161/23723548.2014.969658DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4904892PMC
June 2016

Sororin is tethered to Cohesin SA2.

Cell Cycle 2015 ;14(8):1133

a Department of Genetics, Cell Biology and Development ; University of Minnesota ; Minneapolis , MN USA.

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http://dx.doi.org/10.1080/15384101.2015.1018055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4614277PMC
December 2015

The Cdc20-binding Phe box of the spindle checkpoint protein BubR1 maintains the mitotic checkpoint complex during mitosis.

J Biol Chem 2015 Jan 10;290(4):2431-43. Epub 2014 Dec 10.

From the Department of Pharmacology, Howard Hughes Medical Institute, Department of Pharmacology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390

The spindle checkpoint ensures accurate chromosome segregation by monitoring kinetochore-microtubule attachment. Unattached or tensionless kinetochores activate the checkpoint and enhance the production of the mitotic checkpoint complex (MCC) consisting of BubR1, Bub3, Mad2, and Cdc20. MCC is a critical checkpoint inhibitor of the anaphase-promoting complex/cyclosome, a ubiquitin ligase required for anaphase onset. The N-terminal region of BubR1 binds to both Cdc20 and Mad2, thus nucleating MCC formation. The middle region of human BubR1 (BubR1M) also interacts with Cdc20, but the nature and function of this interaction are not understood. Here we identify two critical motifs within BubR1M that contribute to Cdc20 binding and anaphase-promoting complex/cyclosome inhibition: a destruction box (D box) and a phenylalanine-containing motif termed the Phe box. A BubR1 mutant lacking these motifs is defective in MCC maintenance in mitotic human cells but is capable of supporting spindle-checkpoint function. Thus, the BubR1M-Cdc20 interaction indirectly contributes to MCC homeostasis. Its apparent dispensability in the spindle checkpoint might be due to functional duality or redundant, competing mechanisms.
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http://dx.doi.org/10.1074/jbc.M114.616490DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4303692PMC
January 2015

Genome-wide siRNA screen reveals coupling between mitotic apoptosis and adaptation.

EMBO J 2014 Sep 14;33(17):1960-76. Epub 2014 Jul 14.

Department of Pharmacology, Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX, USA

The antimitotic anti-cancer drugs, including taxol, perturb spindle dynamics, and induce prolonged, spindle checkpoint-dependent mitotic arrest in cancer cells. These cells then either undergo apoptosis triggered by the intrinsic mitochondrial pathway or exit mitosis without proper cell division in an adaptation pathway. Using a genome-wide small interfering RNA (siRNA) screen in taxol-treated HeLa cells, we systematically identify components of the mitotic apoptosis and adaptation pathways. We show that the Mad2 inhibitor p31(comet) actively promotes mitotic adaptation through cyclin B1 degradation and has a minor separate function in suppressing apoptosis. Conversely, the pro-apoptotic Bcl2 family member, Noxa, is a critical initiator of mitotic cell death. Unexpectedly, the upstream components of the mitochondrial apoptosis pathway and the mitochondrial fission protein Drp1 contribute to mitotic adaption. Our results reveal crosstalk between the apoptosis and adaptation pathways during mitotic arrest.
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http://dx.doi.org/10.15252/embj.201487826DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4195789PMC
September 2014

Sgo1 as a guardian of chromosome stability.

Cell Cycle 2012 Feb;11(4):650-1

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http://dx.doi.org/10.4161/cc.11.4.19361DOI Listing
February 2012

Mutational inactivation of STAG2 causes aneuploidy in human cancer.

Science 2011 Aug;333(6045):1039-43

Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University School of Medicine, Washington, DC 20057, USA.

Most cancer cells are characterized by aneuploidy, an abnormal number of chromosomes. We have identified a clue to the mechanistic origins of aneuploidy through integrative genomic analyses of human tumors. A diverse range of tumor types were found to harbor deletions or inactivating mutations of STAG2, a gene encoding a subunit of the cohesin complex, which regulates the separation of sister chromatids during cell division. Because STAG2 is on the X chromosome, its inactivation requires only a single mutational event. Studying a near-diploid human cell line with a stable karyotype, we found that targeted inactivation of STAG2 led to chromatid cohesion defects and aneuploidy, whereas in two aneuploid human glioblastoma cell lines, targeted correction of the endogenous mutant alleles of STAG2 led to enhanced chromosomal stability. Thus, genetic disruption of cohesin is a cause of aneuploidy in human cancer.
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http://dx.doi.org/10.1126/science.1203619DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3374335PMC
August 2011

Timeless makes some time for itself.

Cell Cycle 2011 Jul 15;10(14):2254. Epub 2011 Jul 15.

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http://dx.doi.org/10.4161/cc.10.14.15853DOI Listing
July 2011

Cohesin is needed for bipolar mitosis in human cells.

Cell Cycle 2010 May 15;9(9):1764-73. Epub 2010 May 15.

Department of Genetics, Cell Biology & Development, University of Minnesota Medical School, Minneapolis, MN, USA.

Multi-polar mitosis is strongly linked with aggressive cancers and it is a histological diagnostic of tumor-grade. However, factors that cause chromosomes to segregate to more than two spindle poles are not well understood. Here we show that cohesins Rad21, Smc1 and Smc3 are required for bipolar mitosis in human cells. After Rad21 depletion, chromosomes align at the metaphase plate and bipolar spindles assemble in most cases, but in anaphase the separated chromatids segregate to multiple poles. Time-lapse microscopy revealed that the spindle poles often become split in Rad21-depleted metaphase cells. Interestingly, exogenous expression of non-cleavable Rad21 results in multi-polar anaphase. Since cohesins are present at the spindle poles in mitosis, these data are consistent with a non-chromosomal function of cohesin.
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http://dx.doi.org/10.4161/cc.9.9.11525DOI Listing
May 2010

Determinants of Rad21 localization at the centrosome in human cells.

Cell Cycle 2010 May 15;9(9):1759-63. Epub 2010 May 15.

Department of Genetics, Cell Biology & Development, University of Minnesota Medical School, Minneapolis, MN, USA.

Cohesin proteins help maintain the physical associations between sister chromatids that arise in S-phase and are removed in anaphase. Recent studies found that cohesins also localize to the centrosomes, the organelles that organize the mitotic bipolar spindle. We find that the cohesin protein Rad21 localizes to centrosomes in a manner that is dependent upon known regulators of sister chromatid cohesion as well as regulators of centrosome function. These data suggest that Rad21 functions at the centrosome and that the regulators of Rad21 coordinate the centrosome and chromosomal functions of cohesin.
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http://dx.doi.org/10.4161/cc.9.9.11523DOI Listing
May 2010

Rad21 is required for centrosome integrity in human cells independently of its role in chromosome cohesion.

Cell Cycle 2010 May 15;9(9):1774-80. Epub 2010 May 15.

Department of Genetics, Cell Biology & Development, University of Minnesota Medical School, Minneapolis, MN, USA.

Classically, chromosomal functions in DNA repair and sister chromatid association have been assigned to the cohesin proteins. More recent studies have provided evidence that cohesins also localize to the centrosomes, which organize the bipolar spindle during mitosis. Depletion of cohesin proteins is associated with multi-polar mitosis in which spindle pole integrity is compromised. However, the spindle pole defects after cohesin depletion could be an indirect consequence of a chromosomal cohesion defect which might impact centrosome integrity via alterations to the spindle microtubule network. Here we show that the cohesin Rad21 is required for centrosome integrity independently of its role as a chromosomal cohesin. Thus, Rad21 may promote accurate chromosome transmission not only by virtue of its function as a chromosomal cohesin, but also because it is required for centrosome function.
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http://dx.doi.org/10.4161/cc.9.9.11524DOI Listing
May 2010

Cohesin: a multi-purpose chromatin glue.

J Mol Cell Biol 2009 Dec 14;1(2):58-60. Epub 2009 Aug 14.

Department of Pharmacology, Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

Long thought to be the glue responsible for holding sister chromatids together, cohesin has been found to be stickier than previously thought. Recent discoveries point to cohesin having a role in transcription regulation by mediating long-distance intra-chromosomal interactions.
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http://dx.doi.org/10.1093/jmcb/mjp014DOI Listing
December 2009

Chromosome cohesion and the spindle checkpoint.

Cell Cycle 2009 Sep 30;8(17):2733-40. Epub 2009 Sep 30.

UT-Southwestern Medical Center, Department of Pharmacology, Dallas, TX 75390, USA.

Accurate chromosome segregation constitutes the basis of inheritance. Mistakes in chromosome segregation during mitosis lead to aneuploidy, a common feature of tumors. The accuracy of chromosome segregation is governed by a complex network of processes which ensure that each daughter cell receives the correct number of chromosomes. Herein we review recent developments in the understanding of chromosome segregation, focusing on the cohesion that holds the sister chromatids together and the spindle checkpoint which regulates anaphase onset.
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http://dx.doi.org/10.4161/cc.8.17.9403DOI Listing
September 2009

Multiple anaphase-promoting complex/cyclosome degrons mediate the degradation of human Sgo1.

J Biol Chem 2009 Jan 17;284(3):1772-80. Epub 2008 Nov 17.

Department of Pharmacology, Howard Hughes Medical Institute, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, 75390, USA.

Shugoshin 1 (Sgo1) protects centromeric sister-chromatid cohesion in early mitosis and, thus, prevents premature sister-chromatid separation. The protein level of Sgo1 is regulated during the cell cycle; it peaks in mitosis and is down-regulated in G1/S. Here we show that Sgo1 is degraded during the exit from mitosis, and its degradation depends on the anaphase-promoting complex/cyclosome (APC/C). Overexpression of Cdh1 reduces the protein levels of ectopically expressed Sgo1 in human cells. Sgo1 is ubiquitinated by APC/C bound to Cdh1 (APC/C(Cdh1)) in vitro. We have further identified two functional degradation motifs in Sgo1; that is, a KEN (Lys-Glu-Asn) box and a destruction box (D box). Although removal of either motif is not sufficient to stabilize Sgo1, Sgo1 with both KEN box and D box deleted is stable in cells. Surprisingly, mitosis progresses normally in the presence of non-degradable Sgo1, indicating that degradation of Sgo1 is not required for sister-chromatid separation or mitotic exit. Finally, we show that the spindle checkpoint kinase Bub1 contributes to the maintenance of Sgo1 steady-state protein levels in an APC/C-independent mechanism.
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http://dx.doi.org/10.1074/jbc.M807083200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2615523PMC
January 2009

Chromosome cohesion - rings, knots, orcs and fellowship.

J Cell Sci 2008 Jul;121(Pt 13):2107-14

Department of Pharmacology, UT-Southwestern Medical Center, 6001 Forest Park Rd, Dallas, TX75390, USA.

Sister-chromatid cohesion is essential for accurate chromosome segregation. A key discovery towards our understanding of sister-chromatid cohesion was made 10 years ago with the identification of cohesins. Since then, cohesins have been shown to be involved in cohesion in numerous organisms, from yeast to mammals. Studies of the composition, regulation and structure of the cohesin complex led to a model in which cohesin loading during S-phase establishes cohesion, and cohesin cleavage at the onset of anaphase allows sister-chromatid separation. However, recent studies have revealed activities that provide cohesion in the absence of cohesin. Here we review these advances and propose an integrative model in which chromatid cohesion is a result of the combined activities of multiple cohesion mechanisms.
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http://dx.doi.org/10.1242/jcs.029132DOI Listing
July 2008

Running on a treadmill: dynamic inhibition of APC/C by the spindle checkpoint.

Cell Div 2007 Jul 24;2:23. Epub 2007 Jul 24.

Department of Pharmacology, University of Texas Southwestern Medical Center, 6001 Forest Park Road, Dallas, TX 75390-9041, USA.

During mitosis, the genome duplicated during S-phase is synchronously and accurately segregated to the two daughter cells. The spindle checkpoint prevents premature sister-chromatid separation and mitotic exit. The anaphase-promoting complex/cyclosome (APC/C) is a key target of the spindle checkpoint. Upon checkpoint activation, the mitotic checkpoint complex (MCC) containing Mad2, Bub3, Mad3/BubR1 and Cdc20 inhibits APC/C. Two independent studies in budding yeast have now shed light on the mechanism by which MCC inhibits APC/C. These studies indicate that Mad3 binds to the mitotic activator of APC/C Cdc20 using peptide motifs commonly found in APC/C substrates and thus competes with APC/C substrates for APC/CCdc20 binding. In addition, Mad3 binding to APC/CCdc20 induces Cdc20 ubiquitination by APC/C, leading to the dissociation of MCC. Meanwhile, two other studies have shown that a deubiquitinating enzyme is required for the spindle checkpoint whereas APC/C-dependent ubiquitination is needed for checkpoint inactivation. Collectively, these studies suggest a dynamic model for APC/CCdc20 regulation by MCC in which APC/C- and Mad3-dependent ubiquitination of Cdc20 constitutes a self-regulated switch that rapidly inactivates the spindle checkpoint upon correct chromosome attachment.
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http://dx.doi.org/10.1186/1747-1028-2-23DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1947974PMC
July 2007

Cohesin is dispensable for centromere cohesion in human cells.

PLoS One 2007 Mar 28;2(3):e318. Epub 2007 Mar 28.

Department of Genetics, Cell Biology and Development, University of Minnesota Medical School, Minneapolis, Minnesota, United States of America.

Background: Proper regulation of the cohesion at the centromeres of human chromosomes is essential for accurate genome transmission. Exactly how cohesion is maintained and is then dissolved in anaphase is not understood.

Principal Findings: We have investigated the role of the cohesin complex at centromeres in human cells both by depleting cohesin subunits using RNA interference and also by expressing a non-cleavable version of the Rad21 cohesin protein. Rad21 depletion results in aberrant anaphase, during which the sister chromatids separate and segregate in an asynchronous fashion. However, centromere cohesion was maintained before anaphase in Rad21-depleted cells, and the primary constrictions at centromeres were indistinguishable from those in control cells. Expression of non-cleavable Rad21 (NC-Rad21), in which the sites normally cleaved by separase are mutated, resulted in delayed sister chromatid resolution in prophase and prometaphase, and a blockage of chromosome arm separation in anaphase, but did not impede centromere separation.

Conclusions: These data indicate that cohesin complexes are dispensable for sister cohesion in early mitosis, yet play an important part in the fidelity of sister separation and segregation during anaphase. Cleavage at the separase-sensitive sites of Rad21 is important for arm separation, but not for centromere separation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0000318PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1820851PMC
March 2007

Regulation of centromeric cohesion by sororin independently of the APC/C.

Cell Cycle 2007 Mar 1;6(6):714-24. Epub 2007 Mar 1.

Department of Genetics, Cell Biology and Development, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.

Regulated separation of sister chromatids is the key event of mitosis. Sister chromatids remain cohered from the moment of DNA duplication until anaphase. Two known factors account for cohesion: DNA catenations and cohesin complexes. Premature loss of centromeric cohesion is prevented by the spindle checkpoint. Here we show that sororin, a protein implicated in promoting cohesion through effects on cohesin complexes, is involved in maintenance of cohesion in response to the spindle checkpoint. Sororin-depleted cells reach prometaphase with cohered sister chromatids and are able to form metaphase plates. However, loss of cohesion in anaphase is asynchronous and cells are unresponsive to the spindle checkpoint, accumulating with separated sisters scattered throughout the cytoplasm. These phenotypes are similar to those seen after Shugoshin depletion, suggesting that sororin and Shugoshin might act in concert. Furthermore, sororin-depleted and Shugoshin-depleted cells lose cohesion independently of the APC/C. Therefore, sororin and Shugoshin protect centromeric cohesion in response to the spindle checkpoint, but prevent the removal of cohesion by a mechanism independent of the APC/C.
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http://dx.doi.org/10.4161/cc.6.6.3935DOI Listing
March 2007

PIASgamma is required for faithful chromosome segregation in human cells.

PLoS One 2006 Dec 20;1:e53. Epub 2006 Dec 20.

Department of Genetics, Cell Biology and Development, University of Minnesota Medical School, Minneapolis, Minnesota, United States of America.

Background: The precision of the metaphase-anaphase transition ensures stable genetic inheritance. The spindle checkpoint blocks anaphase onset until the last chromosome biorients at metaphase plate, then the bonds between sister chromatids are removed and disjoined chromatids segregate to the spindle poles. But, how sister separation is triggered is not fully understood.

Principal Findings: We identify PIASgamma as a human E3 sumo ligase required for timely and efficient sister chromatid separation. In cells lacking PIASgamma, normal metaphase plates form, but the spindle checkpoint is activated, leading to a prolonged metaphase block. Sister chromatids remain cohered even if cohesin is removed by depletion of hSgo1, because DNA catenations persist at centromeres. PIASgamma-depleted cells cannot properly localize Topoisomerase II at centromeres or in the cores of mitotic chromosomes, providing a functional link between PIASgamma and Topoisomerase II.

Conclusions: PIASgamma directs Topoisomerase II to specific chromosome regions that require efficient removal of DNA catenations prior to anaphase. The lack of this activity activates the spindle checkpoint, protecting cells from non-disjunction. Because DNA catenations persist without PIASgamma in the absence of cohesin, removal of catenations and cohesin rings must be regulated in parallel.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0000053PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1762334PMC
December 2006

Yeast UBL-UBA proteins have partially redundant functions in cell cycle control.

Cell Div 2006 Dec 4;1:28. Epub 2006 Dec 4.

Department of Genetics, Cell Biology and Development, University of Minnesota, 6-160 Jackson Hall, 321 Church Street SE, Minneapolis, USA.

Background: Proteins containing ubiquitin-like (UBL) and ubiquitin associated (UBA) domains have been suggested to shuttle ubiquitinated substrates to the proteasome for degradation. There are three UBL-UBA containing proteins in budding yeast: Ddi1, Dsk2 and Rad23, which have been demonstrated to play regulatory roles in targeting ubiquitinated substrates to the proteasome for degradation. An involvement of these proteins in cell cycle related events has also been reported. We tested whether these three proteins act redundantly in the cell cycle.

Results: Here we show that the UBL-UBA proteins are partially redundant for cell cycle related roles. RAD23 is redundant with DDI1 and DSK2, but DDI1 and DSK2 are not redundant with each other and the triple deletion shows a synthetic effect, suggesting the existence of at least two roles for RAD23 in cell cycle control. The rad23Deltaddi1Deltadsk2Delta triple deletion strain delays both in G2/M-phase and in mid-anaphase at high temperatures with duplicated spindle pole bodies. Cell cycle progression in the triple deletion strain can only be partially rescued by a rad23 allele lacking the c-terminal UBA domain, suggesting that RAD23 requires its c-terminal UBA domain for full function. In addition to their ability to bind ubiquitin and the proteasome, the UBL-UBA proteins also share the ability to homodimerize. Rad23 and Dsk2 dimerization requires their UBL and/or UBA domains whereas Ddi1 dimerization does not. Here we show that Ddi1 homodimerization is necessary for its cell cycle related functions.

Conclusion: The three yeast UBL-UBA proteins have partially redundant roles required for progression through mitosis.
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http://dx.doi.org/10.1186/1747-1028-1-28DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1697804PMC
December 2006

Topoisomerase II checkpoints: universal mechanisms that regulate mitosis.

Cell Cycle 2006 Sep 1;5(17):1925-8. Epub 2006 Sep 1.

Department of Genetics, Cell Biology & Development, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.

Checkpoint controls confer order to the cell cycle and help prevent genome instability. Here we discuss the Topoisomerase II (Decatenation) Checkpoint which functions to regulate mitotic progression so that chromosomes can be efficiently condensed in prophase and can be segregated with high fidelity in anaphase.
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http://dx.doi.org/10.4161/cc.5.17.3200DOI Listing
September 2006

A mitotic topoisomerase II checkpoint in budding yeast is required for genome stability but acts independently of Pds1/securin.

Genes Dev 2006 May;20(9):1162-74

Department of Genetics, Cell Biology and Development, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.

Topoisomerase II (Topo II) performs topological modifications on double-stranded DNA molecules that are essential for chromosome condensation, resolution, and segregation. In mammals, G2 and metaphase cell cycle delays induced by Topo II poisons have been proposed to be the result of checkpoint activation in response to the catenation state of DNA. However, the apparent lack of such controls in model organisms has excluded genetic proof that Topo II checkpoints exist and are separable from the conventional DNA damage checkpoint controls. But here, we define a Topo II-dependent G2/M checkpoint in a genetically amenable eukaryote, budding yeast, and demonstrate that this checkpoint enhances cell survival. Conversely, a lack of the checkpoint results in aneuploidy. Neither DNA damage-responsive pathways nor Pds1/securin are needed for this checkpoint. Unusually, spindle assembly checkpoint components are required for the Topo II checkpoint, but checkpoint activation is not the result of failed chromosome biorientation or a lack of spindle tension. Thus, compromised Topo II function activates a yeast checkpoint system that operates by a novel mechanism.
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http://dx.doi.org/10.1101/gad.1367206DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1472475PMC
May 2006

UBL/UBA ubiquitin receptor proteins bind a common tetraubiquitin chain.

J Mol Biol 2006 Mar 19;356(4):1027-35. Epub 2005 Dec 19.

Department of Biochemistry, Molecular Biology, University of Minnesota, Minneapolis, MN 55455, USA.

The ubiquitin-proteasome pathway is essential throughout the life cycle of a cell. This system employs an astounding number of proteins to ubiquitylate and to deliver protein substrates to the proteasome for their degradation. At the heart of this process is the large and growing family of ubiquitin receptor proteins. Within this family is an intensely studied group that contains both ubiquitin-like (UBL) and ubiquitin-associated (UBA) domains: Rad23, Ddi1 and Dsk2. Although UBL/UBA family members are reported to regulate the degradation of other proteins, their individual roles in ubiquitin-mediated protein degradation has proven difficult to resolve due to their overlapping functional roles and interaction with each other and other ubiquitin family members. Here, we use a combination of NMR spectroscopy and molecular biology to reveal that Rad23 and Ddi1 interact with each other by using UBL/UBA domain interactions in a manner that does not preclude their interaction with ubiquitin. We demonstrate that UBL/UBA proteins can bind a common tetraubiquitin molecule and thereby provide strong evidence for a model in which chains adopt an opened structure to bind multiple receptor proteins. Altogether our results suggest a mechanism through which UBL/UBA proteins could protect chains from premature de-ubiquitylation and unnecessary elongation during their transit to the proteasome.
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http://dx.doi.org/10.1016/j.jmb.2005.12.001DOI Listing
March 2006

Evidence that the yeast spindle assembly checkpoint has a target other than the anaphase promoting complex.

Cell Cycle 2005 Nov 5;4(11):1555-7. Epub 2005 Nov 5.

Department of Genetics, Cell Biology & Development, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.

The spindle assembly checkpoint monitors biorientation of chromosomes on the metaphase spindle and inhibits the Anaphase Promoting Complex (APC) specificity factor Cdc20. If APC-Cdc20 is the sole target of the spindle checkpoint, then cells lacking APC and its targets, B-type cyclin and securin, would lack spindle checkpoint function. We tested this hypothesis in yeast cells that are APC-null. Surprisingly, we find that such yeast cells are able to activate the spindle assembly checkpoint, delaying cell cycle progression in G2/M phase. These data suggest that the spindle checkpoint has a non-APC target that can restrain anaphase onset.
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http://dx.doi.org/10.4161/cc.4.11.2144DOI Listing
November 2005

Anaphase promoting complex or cyclosome?

Cell Cycle 2005 Nov 26;4(11):1585-92. Epub 2005 Nov 26.

Department of Genetics, Cell Biology & Development, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.

The anaphase promoting complex/cyclosome (APC/C) was initially described as a multi-subunit protein complex that ubiquitinates anaphase inhibitors thus targeting them for destruction by proteasomes to initiate loss of sister chromatid cohesion. However, recent studies have identified important new functions of the APC/C. Moreover, sister centromere separation can occur in the absence of APC/C activity in mammals, indicating that anaphase onset might be triggered by multiple factors. Here we discuss whether the APC/C functions primarily as the anaphase trigger, or whether it has more general properties, relevant for cell cycle control at multiple developmental and cell cycle stages. Additionally, we discuss the validity of the APC-dependent model for sister segregation in mammals.
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http://dx.doi.org/10.4161/cc.4.11.2143DOI Listing
November 2005

Proteasome activity is required for centromere separation independently of securin degradation in human cells.

Cell Cycle 2005 Nov 14;4(11):1558-60. Epub 2005 Nov 14.

Department of Genetics, Cell Biology & Development, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.

Loss of centromere cohesion during anaphase in human cells is regulated by the spindle assembly checkpoint and is thought to depend on a ubiquitin ligase, the Anaphase Promoting Complex/Cyclosome (APC). APC-Cdc20 adds ubiquitin chains to securin inducing its destruction by the proteasome and these events correlate with the loss of sister chromatid cohesion and the onset of anaphase. But whether securin destruction is necessary and sufficient for anaphase initiation is not clear. Therefore, we asked if proteasome activity is needed for anaphase onset in human cells that lack securin. We find that even in the absence of securin, a metaphase block with cohered sister centromeres can be enforced in the absence of proteasome activity. Therefore, other targets of the proteasome must be degraded to allow anaphase onset.
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http://dx.doi.org/10.4161/cc.4.11.2145DOI Listing
November 2005

Regulated separation of sister centromeres depends on the spindle assembly checkpoint but not on the anaphase promoting complex/cyclosome.

Cell Cycle 2005 Nov 7;4(11):1561-75. Epub 2005 Nov 7.

Department of Genetics, Cell Biology & Development, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.

Key to faithful genetic inheritance is the cohesion between sister centromeres that physically links replicated sister chromatids and is then abruptly lost at the onset of anaphase. Misregulated cohesion causes aneuploidy, birth defects and perhaps initiates cancers. Loss of centromere cohesion is controlled by the spindle checkpoint and is thought to depend on a ubiquitin ligase, the Anaphase Promoting Complex/Cyclosome (APC). But here we present evidence that the APC pathway is dispensable for centromere separation at anaphase in mammals, and that anaphase proceeds in the presence of cyclin B and securin. Arm separation is perturbed in the absence of APC, compromising the fidelity of segregation, but full sister chromatid separation is achieved after a delayed anaphase. Thereafter, cells arrest terminally in telophase with high levels of cyclin B. Extending these findings we provide evidence that the spindle checkpoint regulates centromere cohesion through an APC-independent pathway. We propose that this Centromere Linkage Pathway (CLiP) is a second branch that stems from the spindle checkpoint to regulate cohesion preferentially at the centromeres and that Sgo1 is one of its components.
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http://dx.doi.org/10.4161/cc.4.11.2146DOI Listing
November 2005