Browsing by Subject "Cell Cycle Proteins"
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Item CDK5RAP2 Regulates Centriole Licensing to Restrict Centriole Duplication in Mice(2009-09-04) Barrera, Jose Anselmo; Megraw, TimothyCells division is a highly coordinated series of events that must occur with extreme precision. Defects during segregation of genetic material (DNA) can have adverse effects on the health of the cell, surrounding tissue, organ, and the organism as a whole. Accurate assembly of the bipolar mitotic spindle apparatus is crucial for precise chromosome segregation. Centrosomes play a crucial role in establishment of the mitotic spindle and therefore are vital to the maintenance of genetic stability. Centrosomes are composed of two centrioles that arrange a specialized conglomerate of proteins into a pericentriolar matrix. Centrosomes are highly regulated throughout the cell cycle, and duplicate only once per cell cycle ensuring that each cell inherits one centrosome after mitotic exit, and contains only two centrosomes at the following mitosis. Truncating mutations in the Cyclin-Dependent Kinase 5 Regulatory Associated Protein 2 gene (CDK5RAP2), which encodes a centrosomal protein, result in autosomal recessive primary microcephaly (MCPH, [MIM 251200]) in humans. The major phenotypic manifestation of this rare genetic disorder is a small head. Affected individuals have head circumferences at least 4 standard deviations below sex- and age-matched individuals and suffer mental retardation. In order to investigate how mutations in CDK5RAP2 affect centrosome structure and regulation, and how this leads to MCPH, we derived two distinct mouse mutant lines with truncating mutations within the CDK5RAP2 locus similar to those found in affected humans. We show that centriole engagement and cohesion, two distinct centriole-binding processes, are disrupted in CDK5RAP2 mutant cells. Partial disruption of CDK5RAP2 affected centriole cohesion, whereas complete CDK5RAP2 disruption deregulated the centriole duplication cycle leading to centriole/centrosome amplification. During mitosis amplified centrosomes in CDK5RAP2 mutant cells were potent microtubule organizing centers that drove formation of multipolar spindles. Furthermore, cells formed multiple primary cilia from multiple centrioles inherited from the previous cell cycle. Together these results define a role for CDK5RAP2 in the regulation of centriole duplication and also provide a basis for the development of MCPH.Item Function And Recruitment Of Centromeric Heterochromatin Protein 1(2011-02-01T19:34:12Z) Chaudhary, Jaideep; Yu, HongtaoDuring early mitosis, the sister chromatids are held together by Cohesin, a protein complex composed of Smc3, Smc1, Scc1/Rad21 and Scc3. Cohesin is first released from the arms of chromosomes, leaving it intact at the centromere. At the metaphase – anaphase transition, centromeric cohesin is cleaved, allowing the chromatids to segregate to two daughter cells. Shugoshin (Sgo-1) is a known protector of cohesin at the centromere. It prevents phosphorylation of cohesin complex by Plk1 before the metaphase – anaphase transition, which would otherwise lead to cohesin release, causing the two chromatids to separate untimely. In this study we show that Sgo1 localizes on centromeres through HP1 during interphase in human cells. Also, Sgo1 binds all three forms of HP1 (i.e. alpha, beta, gamma) through its chromoshadow domain. We have determined the dissociation constant of this interaction to be in the sub-micromolar range. We have shown conclusively that Sgo1 binds to HP1 chromoshadow domain via one PxVxL motif. We have further shown that, in mitosis, HP1 is recruited to centromeres by Incenp, a subunit of the chromosomal passenger complex via the chromoshadow domain of HP1. This interaction is most likely at the HP1 CSD dimer interface, where PxVxL motifsbind. Hence, it seems that Incenp may provide competition to Sgo1 for HP1 binding.Item Functional Analysis of the Human SMC5/6 Complex in Homologous Recombination and Telomere Maintenance(2008-05-13) Potts, Patrick Ryan; Yu, HongtaoDNA repair is required for the genomic stability and well-being of an organism. The structural maintenance of chromosomes (SMC) family of proteins has been implicated in the repair of DNA double-strand breaks (DSBs) by homologous recombination (HR). The SMC1/3 cohesin complex promotes HR by localizing to DSBs where it holds sister chromatids in close proximity to allow HR-induced strand invasion and exchange. The SMC5/6 complex is also required for DNA repair, but the mechanism by which it accomplishes this has been unclear. We have characterized the role of the human SMC5/6 complex in HRmediated DNA damage repair. The yeast SMC5/6 complex has been shown to be composed of the SMC5-SMC6 heterodimer and six non-SMC element (NSE) proteins. We show that the human homolog of one of these NSE proteins, MMS21/NSE2, is a ligase for small ubiquitin-like modifier (SUMO). Depletion of MMS21 by RNA interference (RNAi) sensitizes cells toward DNA damageinduced apoptosis. This hypersensitization of MMS21-RNAi cells is not due to a defect in DNA damage-induced cell cycle checkpoint, but rather in the kinetics of DNA damage repair. Since the yeast SMC5/6 complex has been implicated in HR-mediated DSB repair, we investigated the role of the human SMC5/6 complex in HR-mediated DSB repair. RNAi-mediated knockdown of the SMC5/6 complex components specifically decreases sister chromatid HR, but not non-homologous end-joining (NHEJ) or intra-chromatid, homologue, or extrachromosomal HR. We show that one potential mechanism by which the SMC5/6 complex specifically promotes sister chromatid HR is by facilitating the recruitment of the SMC1/3 cohesin complex to DSBs. We next examined whether the SMC5/6 complex is also required for sister chromatid HR of telomeres. Specific types of cancer cells, known as alternative lengthening of telomeres (ALT) cells, rely on telomere recombination for telomere lengthening and unlimited replicative potential. We show that the SMC5/6 complex promotes telomere recombination and lengthening in ALT cells by MMS21-dependent sumoylation of telomere-binding proteins. Sumoylation of these telomere-binding proteins relocalizes telomeres to nuclear PML bodies where HR proteins facilitate telomere recombination. These studies identify the human SMC5/6 complex and SUMO modification as critical mediators of sister chromatid HR.Item Regulation and Mechanism of Bub1-Mediated Spindle Checkpoint Signaling(2006-12-20) Qi, Wei; Yu, HongtaoThe spindle checkpoint is a surveillance mechanism that ensures the fidelity of chromosome segregation during mitosis and meiosis. Bub1 is a highly conserved protein serine/threonine kinase that plays multiple roles in the spindle checkpoint. The regulation and mechanism of Bub1 in spindle checkpoint were investigated. Bub1 is degraded during mitotic exit and the degradation of it is mediated by APC/C in complex with its activator Cdh1 (APC/CCdh1). Overexpression of Cdh1 reduces the protein levels of ectopically expressed Bub1 whereas depletion of Cdh1 by RNA interference (RNAi) increases the level of the endogenous Bub1 protein. Two KEN-box motifs on Bub1 are required for its degradation in vivo and ubiquitination in vitro. A Bub1 mutant protein with both KEN-boxes mutated is stable in cells. Kinetochore is the origin of spindle checkpoint signal and contains the catalytic machinery for generating the signal. We identify an ATP-dependent APC/CCdc20 inhibitory activity on metaphase chromosomes with unattached kinetochores. The Cdc20-S153A that cannot be phosphorylated by Bub1 is not inhibited by metaphase chromosomes, suggesting Bub1 is likely responsible for the inhibitory activity. Bub1 on unattached kinetochores is hyperphosphorylated and activated. Furthermore, the kinase-dead mutant of Bub1 cannot restore spindle checkpoint in Bub1-RNAi cells, demonstrating that the kinase activity of Bub1 is required for the spindle checkpoint. Plk1 is required for the generation of the tension-sensing 3F3/2 kinetochore epitope and facilitates kinetochore localization of Mad2 and other spindle checkpoint proteins. We investigate the mechanism by which Plk1 is recruited to kinetochores. We show that Plk1 binds to Bub1 in mitotic cells. The Plk1-Bub1 interaction requires the polo-box domain (PBD) of Plk1 and is enhanced by Cdk1-mediated phosphorylation of Bub1 at T609. The PBD-dependent binding of Plk1 to Bub1 facilitates phosphorylation of Bub1 by Plk1 in vitro. Depletion of Bub1 in HeLa cells by RNAi diminishes the kinetochore localization of Plk1. Ectopic expression of the wild-type Bub1, but not the Bub1-T609A mutant, in Bub1-RNAi cells restores the kinetochore localization of Plk1. Our results suggest that phosphorylation of Bub1 at T609 by Cdk1 creates a docking site for the PBD of Plk1 and facilitates the kinetochore recruitment of Plk1.