Drosophila CENP-A Mutations Cause a BubR1- Dependent Early Mitotic Delay without Normal Localization of Kinetochore Components

The centromere/kinetochore complex plays an essential role in cell and organismal viability by ensuring chromosome movements during mitosis and meiosis. The kinetochore also mediates the spindle attachment checkpoint (SAC), which delays anaphase initiation until all chromosomes have achieved bipolar attachment of kinetochores to the mitotic spindle. CENP-A proteins are centromere-specific chromatin components that provide both a structural and a functional foundation for kinetochore formation. Here we show that cells in Drosophila embryos homozygous for null mutations in CENP-A (CID) display an early mitotic delay. This mitotic delay is not suppressed by inactivation of the DNA damage checkpoint and is unlikely to be the result of DNA damage. Surprisingly, mutation of the SAC component BUBR1 partially suppresses this mitotic delay. Furthermore, cid mutants retain an intact SAC response to spindle disruption despite the inability of many kinetochore proteins, including SAC components, to target to kinetochores. We propose that SAC components are able to monitor spindle assembly and inhibit cell cycle progression in the absence of sustained kinetochore localization.

[1]  C. Lehner,et al.  Genetic interactions of separase regulatory subunits reveal the diverged Drosophila Cenp-C homolog. , 2005, Genes & development.

[2]  Samantha G. Zeitlin,et al.  Xenopus CENP-A assembly into chromatin requires base excision repair proteins. , 2005, DNA repair.

[3]  P. Todd Stukenberg,et al.  Two Complexes of Spindle Checkpoint Proteins Containing Cdc20 and Mad2 Assemble during Mitosis Independently of the Kinetochore in Saccharomyces cerevisiae , 2005, Eukaryotic Cell.

[4]  T. Zerjal,et al.  CENP-A Is Required for Accurate Chromosome Segregation and Sustained Kinetochore Association of BubR1 , 2005, Molecular and Cellular Biology.

[5]  C. Sunkel,et al.  The Drosophila Bub3 protein is required for the mitotic checkpoint and for normal accumulation of cyclins during G2 and early stages of mitosis , 2005, Journal of Cell Science.

[6]  Helder Maiato,et al.  Stuck in division or passing through: what happens when cells cannot satisfy the spindle assembly checkpoint. , 2004, Developmental cell.

[7]  Viji M. Draviam,et al.  Timing and checkpoints in the regulation of mitotic progression. , 2004, Developmental cell.

[8]  J. Millar,et al.  Disruption of astral microtubule contact with the cell cortex activates a Bub1, Bub3, and Mad3-dependent checkpoint in fission yeast. , 2004, Molecular biology of the cell.

[9]  Elliot Botvinick,et al.  Dynamics of Centromere and Kinetochore Proteins Implications for Checkpoint Signaling and Silencing , 2004, Current Biology.

[10]  E. Salmon,et al.  Spindle Checkpoint Protein Dynamics at Kinetochores in Living Cells , 2004, Current Biology.

[11]  K. Yoda,et al.  Human CENP-I specifies localization of CENP-F, MAD1 and MAD2 to kinetochores and is essential for mitosis , 2003, Nature Cell Biology.

[12]  K. Sullivan,et al.  Centromeres and Kinetochores From Epigenetics to Mitotic Checkpoint Signaling , 2003, Cell.

[13]  D. Baker,et al.  Rae1 is an essential mitotic checkpoint regulator that cooperates with Bub3 to prevent chromosome missegregation , 2003, The Journal of cell biology.

[14]  G. Goshima,et al.  Human centromere chromatin protein hMis12, essential for equal segregation, is independent of CENP-A loading pathway , 2003, The Journal of cell biology.

[15]  E. Salmon,et al.  hNuf2 inhibition blocks stable kinetochore–microtubule attachment and induces mitotic cell death in HeLa cells , 2002, The Journal of cell biology.

[16]  A. Mikhailov,et al.  DNA Damage during Mitosis in Human Cells Delays the Metaphase/Anaphase Transition via the Spindle-Assembly Checkpoint , 2002, Current Biology.

[17]  E. Nigg,et al.  Role of Hec1 in Spindle Checkpoint Signaling and Kinetochore Recruitment of Mad1/Mad2 , 2002, Science.

[18]  D. Lydall,et al.  EXO1-dependent single-stranded DNA at telomeres activates subsets of DNA damage and spindle checkpoint pathways in budding yeast yku70Delta mutants. , 2002, Genes & development.

[19]  M.Mitchell Smith,et al.  Centromeres and variant histones: what, where, when and why? , 2002, Current opinion in cell biology.

[20]  J. Rine,et al.  Overlapping roles of the spindle assembly and DNA damage checkpoints in the cell-cycle response to altered chromosomes in Saccharomyces cerevisiae. , 2002, Genetics.

[21]  T. Ikemura,et al.  CENP-I is essential for centromere function in vertebrate cells. , 2002, Developmental cell.

[22]  Gary H Karpen,et al.  Conserved organization of centromeric chromatin in flies and humans. , 2002, Developmental cell.

[23]  E. Salmon,et al.  Cytoplasmic dynein/dynactin drives kinetochore protein transport to the spindle poles and has a role in mitotic spindle checkpoint inactivation , 2001, The Journal of cell biology.

[24]  G. Lucchini,et al.  Bub3 interaction with Mad2, Mad3 and Cdc20 is mediated by WD40 repeats and does not require intact kinetochores , 2001, The EMBO journal.

[25]  Tin Tin Su,et al.  mei-41 and bub1 block mitosis at two distinct steps in response to incomplete DNA replication in Drosophila embryos , 2001, Current Biology.

[26]  G. Chan,et al.  Checkpoint inhibition of the APC/C in HeLa cells is mediated by a complex of BUBR1, BUB3, CDC20, and MAD2 , 2001, The Journal of cell biology.

[27]  T. Ikemura,et al.  CENP‐H, a constitutive centromere component, is required for centromere targeting of CENP‐C in vertebrate cells , 2001, The EMBO journal.

[28]  G. Karpen,et al.  The role of Drosophila CID in kinetochore formation, cell-cycle progression and heterochromatin interactions , 2001, Nature Cell Biology.

[29]  K. Oegema,et al.  Functional Analysis of Kinetochore Assembly in Caenorhabditis elegans , 2001, The Journal of cell biology.

[30]  M. Roth,et al.  Hcp-4, a Cenp-C–Like Protein inCaenorhabditis elegans, Is Required for Resolution of Sister Centromeres , 2001, The Journal of cell biology.

[31]  F. Sprenger,et al.  Mitotic degradation of cyclin A is mediated by multiple and novel destruction signals , 2001, Current Biology.

[32]  D. Burke,et al.  The spindle checkpoint of the yeast Saccharomyces cerevisiae requires kinetochore function and maps to the CBF3 domain. , 2001, Genetics.

[33]  W. Gerald,et al.  MAD2 haplo-insufficiency causes premature anaphase and chromosome instability in mammalian cells , 2001, Nature.

[34]  J. Shah,et al.  Waiting for Anaphase Mad2 and the Spindle Assembly Checkpoint , 2000, Cell.

[35]  A. Murray,et al.  Visualization of Mad2 Dynamics at Kinetochores, along Spindle Fibers, and at Spindle Poles in Living Cells , 2000, The Journal of cell biology.

[36]  M. Yanagida,et al.  Requirement of Mis6 centromere connector for localizing a CENP-A-like protein in fission yeast. , 2000, Science.

[37]  F. Winston,et al.  Histone H2A is required for normal centromere function in Saccharomyces cerevisiae , 2000, The EMBO journal.

[38]  K. Choo,et al.  Early disruption of centromeric chromatin organization in centromere protein A (Cenpa) null mice. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[39]  S. Henikoff,et al.  Heterochromatic deposition of centromeric histone H3-like proteins. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[40]  R. Saunders,et al.  The rough deal protein is a new kinetochore component required for accurate chromosome segregation in Drosophila. , 1999, Journal of cell science.

[41]  P. O’Farrell,et al.  Drosophila grapes/CHK1 mutants are defective in cyclin proteolysis and coordination of mitotic events , 1999, Current Biology.

[42]  C. Sunkel,et al.  Mutations in the Essential Spindle Checkpoint Gene bub1 Cause Chromosome Missegregation and Fail to Block Apoptosis in Drosophila , 1999, The Journal of cell biology.

[43]  C. Allis,et al.  Histone H3 phosphorylation is required for the initiation, but not maintenance, of mammalian chromosome condensation. , 1998, Journal of cell science.

[44]  D. Koshland,et al.  Cse4p Is a Component of the Core Centromere of Saccharomyces cerevisiae , 1998, Cell.

[45]  C. Allis,et al.  Mitosis-specific phosphorylation of histone H3 initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensation , 1997, Chromosoma.

[46]  K. Sullivan,et al.  Assembly of CENP-A into Centromeric Chromatin Requires a Cooperative Array of Nucleosomal DNA Contact Sites , 1997, The Journal of cell biology.

[47]  P. Bryant,et al.  The product of proliferation disrupter is concentrated at centromeres and required for mitotic chromosome condensation and cell proliferation in Drosophila. , 1997, Genes & development.

[48]  H. Bellen,et al.  Chromatid Segregation at Anaphase Requires the barren Product, a Novel Chromosome-Associated Protein That Interacts with Topoisomerase II , 1996, Cell.

[49]  T. Kaufman,et al.  The Drosophila homeotic target gene centrosomin (cnn) encodes a novel centrosomal protein with leucine zippers and maps to a genomic region required for midgut morphogenesis. , 1995, Development.

[50]  J. Sekelsky,et al.  The mei-41 gene of D. melanogaster is a structural and functional homolog of the human ataxia telangiectasia gene , 1995, Cell.

[51]  A Khodjakov,et al.  The checkpoint delaying anaphase in response to chromosome monoorientation is mediated by an inhibitory signal produced by unattached kinetochores , 1995, The Journal of cell biology.

[52]  D. Glover,et al.  The A‐ and B‐type cyclins of Drosophila are accumulated and destroyed in temporally distinct events that define separable phases of the G2‐M transition. , 1990, The EMBO journal.

[53]  Michael D. Weiss,et al.  Peer Reviewed Title: Mislocalization of the Drosophila centromere-specific histone CID promotes formation of functional ectopic kinetochores , 2006 .

[54]  W. Grady.  Genomic instability and colon cancer , 2004, Cancer and Metastasis Reviews.