EWSR1 maintains centromere identity.

The centromere is essential for ensuring high-fidelity transmission of chromosomes. CENP-A, the centromeric histone H3 variant, is thought to be the epigenetic mark of centromere identity. CENP-A deposition at the centromere is crucial for proper centromere function and inheritance.

Localization of SNARE proteins in the brain and corpus allatum of Bombyx mori.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) make up the core machinery that mediates membrane fusion. SNAREs, syntaxin, synaptosome-associated protein (SNAP), and synaptobrevin form a tight SNARE complex that brings the vesicle and plasma membranes together and is essential for membrane fusion. The cDNAs of SNAP-25, VAMP2, and Syntaxin 1A from Bombyx mori were inserted into a plasmid, transformed into Escherichia coli, and purified.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A).

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e.

CENP-A Ubiquitylation Is Indispensable to Cell Viability.

CENP-A is a centromere-specific histone H3 variant that epigenetically determines centromere identity, but how CENP-A is deposited at the centromere remains obscure. We previously reported that CENP-A K124 ubiquitylation, mediated by the CUL4A-RBX1-COPS8 complex, is essential for CENP-A deposition at the centromere. However, a recent report stated that CENP-A K124R mutants show no defects in centromere localization and cell viability.

CENP-A Ubiquitylation Contributes to Maintaining the Chromosomal Location of the Centromere.

The centromere plays an essential role in accurate chromosome segregation, and the chromosomal location of the centromere is determined by the presence of a histone H3 variant, centromere protein A (CENP-A), in centromeric nucleosomes. However, the precise mechanisms of deposition, maintenance, and inheritance of CENP-A at centromeres are unclear. We have reported that CENP-A deposition requires ubiquitylation of CENP-A lysine 124 mediated by the E3 ligase activity of Cullin 4A (CUL4A)-RING-box protein 1 (RBX1)-COP9 signalsome complex subunit 8 (COPS8).

Metabolism of steroids by cytochrome P450 2C9 variants.

CYP2C9 is a human microsomal cytochrome P450c (CYP). Much variation in CYP2C9 levels and activity can be attributed to polymorphisms of this gene. Wild-type CYP2C9 and ten mutants were co-expressed with NADPH-cytochrome P450 reductase in Escherichia coli.

SGT1-HSP90 complex is required for CENP-A deposition at centromeres.

The centromere plays an essential role in accurate chromosome segregation, and defects in its function lead to aneuploidy and thus cancer. The centromere-specific histone H3 variant CENP-A is proposed to be the epigenetic mark of the centromere, as active centromeres require CENP-A-containing nucleosomes to direct the recruitment of multiple kinetochore proteins. CENP-A K124 ubiquitylation, mediated by CUL4A-RBX1-COPS8 E3 ligase activity, is required for CENP-A deposition at the centromere.

The inheritance of centromere identity.

CENP-A (Centromere protein A) is a histone H3 variant that epigenetically determines the centromere position, but the mechanism of its centromere inheritance is obscure. We propose that CENP-A ubiquitylation, which is inherited through dimerization between rounds of cell division, is a candidate for the epigenetic mark of centromere identity.

CENP-A Ubiquitylation Is Inherited through Dimerization between Cell Divisions.

The presence of chromatin containing the histone H3 variant CENP-A dictates the location of the centromere in a DNA sequence-independent manner. But the mechanism by which centromere inheritance occurs is largely unknown. We previously reported that CENP-A K124 ubiquitylation, mediated by CUL4A-RBX1-COPS8 E3 ligase activity, is required for CENP-A deposition at the centromere.

CENP-A K124 Ubiquitylation Is Required for CENP-A Deposition at the Centromere.

CENP-A is a centromere-specific histone H3 variant that epigenetically determines centromere identity to ensure kinetochore assembly and proper chromosome segregation, but the precise mechanism of its specific localization within centromeric heterochromatin remains obscure. We have discovered that CUL4A-RBX1-COPS8 E3 ligase activity is required for CENP-A ubiquitylation on lysine 124 (K124) and CENP-A centromere localization. A mutation of CENP-A, K124R, reduces interaction with HJURP (a CENP-A-specific histone chaperone) and abrogates localization of CENP-A to the centromere.

Synthetic genetic array screen identifies PP2A as a therapeutic target in Mad2-overexpressing tumors.

The spindle checkpoint is essential to ensure proper chromosome segregation and thereby maintain genomic stability. Mitotic arrest deficiency 2 (Mad2), a critical component of the spindle checkpoint, is overexpressed in many cancer cells. Thus, we hypothesized that Mad2 overexpression could specifically make cancer cells susceptible to death by inducing a synthetic dosage lethality defect.

HECT-E3 ligase ETC-1 regulates securin and cyclin B1 cytoplasmic abundance to promote timely anaphase during meiosis in C. elegans.

The anaphase inhibitor securin plays a crucial role in regulating the timing of sister chromatid separation during mitosis. When sister chromatid pairs become bioriented, the E3 ligase anaphase promoting complex/cyclosome (APC/C) ubiquitylates securin for proteolysis, triggering sister chromatid separation. Securin is also implicated in regulating meiotic progression.

Key players in chromosome segregation in Caenorhabditis elegans.

In contrast to many eukaryotic organisms in which kinetochores are assembled on localized centromeres of monocentric chromosomes, Caenorhabditis elegans has diffuse kinetochores, termed holo-kinetochores, which are assembled along the entire length of the mitotic chromosome. Despite this cytologically distinct chromosomal architecture, holo-kinetochores of C. elegans and kinetochores of other eukaryotes share structurally and functionally conserved properties.

The spindle assembly checkpoint in Caenorhabditis elegans: one who lacks Mad1 becomes mad one.

The spindle assembly checkpoint (SAC) monitors the microtubule attachment status of the kinetochore and arrests cells before anaphase until all pairs of sister kinetochores achieve bipolar attachment of microtubules, thereby ensuring faithful chromosome transmission. The evolutionarily conserved coiled-coil protein MAD1 has been implicated in the SAC signaling pathway. MAD1 forms a complex with another SAC component MAD2 and specifically localizes to unattached kinetochores to facilitate efficient binding of MAD2 to its target, CDC20, the mitotic substrate-specific activator of the anaphase promoting complex or cyclosome (APC/C).

SPDL-1 functions as a kinetochore receptor for MDF-1 in Caenorhabditis elegans.

The spindle assembly checkpoint (SAC) ensures faithful chromosome segregation by delaying anaphase onset until all sister kinetochores are attached to bipolar spindles. An RNA interference screen for synthetic genetic interactors with a conserved SAC gene, san-1/MAD3, identified spdl-1, a Caenorhabditis elegans homologue of Spindly. SPDL-1 protein localizes to the kinetochore from prometaphase to metaphase, and this depends on KNL-1, a highly conserved kinetochore protein, and CZW-1/ZW10, a component of the ROD-ZW10-ZWILCH complex.

Spindle assembly checkpoint gene mdf-1 regulates germ cell proliferation in response to nutrition signals in C. elegans.

When newly hatched Caenorhabditis elegans larvae are starved, their primordial germ cells (PGCs) arrest in the post-S phase. This starvation-induced PGC arrest is mediated by the DAF-18/PTEN-AKT-1/PKB nutrient-sensing pathway. Here, we report that the conserved spindle assembly checkpoint (SAC) component MDF-1/MAD1 is required for the PGC arrest.

Suppressors of spindle checkpoint defect (such) mutants identify new mdf-1/MAD1 interactors in Caenorhabditis elegans.

The spindle assembly checkpoint (SAC) governs the timing of metaphase-to-anaphase transition and is essential for genome stability. The Caenorhabditis elegans mutant strain gk2 carries a deletion within the mdf-1/MAD1 gene that results in death of the homozygous strain after two or three generations. Here we describe 11 suppressors of the mdf-1(gk2) lethality, 10 identified in an ethyl methanesulfonate (EMS) mutagenesis screen and 1 isolated using the dog-1(gk10) (deletions of guanine-rich DNA) mutator strain.

Spatial organization of the mammalian genome surveillance machinery in response to DNA strand breaks.

We show that DNA double-strand breaks (DSBs) induce complex subcompartmentalization of genome surveillance regulators. Chromatin marked by gamma-H2AX is occupied by ataxia telangiectasia-mutated (ATM) kinase, Mdc1, and 53BP1. In contrast, repair factors (Rad51, Rad52, BRCA2, and FANCD2), ATM and Rad-3-related (ATR) cascade (ATR, ATR interacting protein, and replication protein A), and the DNA clamp (Rad17 and -9) accumulate in subchromatin microcompartments delineated by single-stranded DNA (ssDNA).

The Rad50S allele promotes ATM-dependent DNA damage responses and suppresses ATM deficiency: implications for the Mre11 complex as a DNA damage sensor.

Genetic and cytologic data from Saccharomyces cerevisiae and mammals implicate the Mre11 complex, consisting of Mre11, Rad50, and Nbs1, as a sensor of DNA damage, and indicate that the complex influences the activity of ataxia-telangiectasia mutated (ATM) in the DNA damage response. Rad50(S/S) mice exhibit precipitous apoptotic attrition of hematopoietic cells. We generated ATM- and Chk2-deficient Rad50(S/S) mice and found that Rad50(S/S) cellular attrition was strongly ATM and Chk2 dependent.

Phosphorylation of SMC1 is a critical downstream event in the ATM-NBS1-BRCA1 pathway.

The ATM protein kinase is activated by intermolecular autophosphorylation in response to DNA damage and initiates cellular signaling pathways that facilitate cell survival and reduce chromosomal breakage. Here, we show that NBS1 and BRCA1 are required for the recruitment of previously activated ATM to the sites of DNA breaks after ionizing irradiation, and that this recruitment is required for the phosphorylation of SMC1 by ATM. To explore the functional importance of SMC1 phosphorylation, murine cells were generated, in which the two damage-induced phosphorylation sites in SMC1 are mutated.

The Cdc20 homolog, FZY-1, and its interacting protein, IFY-1, are required for proper chromosome segregation in Caenorhabditis elegans.

Accurate chromosome segregation is achieved by a series of highly regulated processes that culminate in the metaphase-to-anaphase transition of the cell cycle. In the budding yeast Saccharomyces cerevisiae, the degradation of the securin protein Pds1 reverses the binding and inhibition of the separase protein Esp1. Esp1 cleaves Scc1.