SummaryAccurate chromosome segregation requires the assembly of kinetochores, multiprotein complexes that assemble on the centromere of each sister chromatid. A key step in this process involves binding of the constitutive centromere-associated network (CCAN) to CENP-A, the histone H3 variant that constitutes centromeric nucleosomes. This network is proposed to operate as a persistent structural scaffold for assembly of the outer kinetochore during mitosis. Here, we show by fluorescence resonance energy transfer (FRET) that the N-terminus of CENP-N lies in close proximity to the N-terminus of CENP-A in vivo, consistent with in vitro data showing direct binding of CENP-N to CENP-A. Furthermore, we demonstrate in living cells that CENP-N is bound to kinetochores during S phase and G2, but is largely absent from kinetochores during mitosis and G1. By measuring the dynamics of kinetochore binding, we reveal that CENP-N undergoes rapid exchange in G1 until the middle of S phase when it becomes stably associated with kinetochores. The majority of CENP-N is loaded during S phase and dissociates again during G2. We propose a model in which CENP-N functions as a fidelity factor during centromeric replication and reveal that the CCAN network is considerably more dynamic than previously appreciated.

Promyelocytic leukemia (PML) protein binds to and stabilizes CIITA at PML nuclear bodies, which promotes expression of the MHC class II gene locus in response to interferon-γ exposure.

Kinetochores are multi-protein megadalton assemblies that are required for attachment of microtubules to centromeres and, in turn, the segregation of chromosomes in mitosis. Kinetochore assembly is a cell cycle regulated multi-step process. The initial step occurs during interphase and involves loading of the 15-subunit constitutive centromere associated complex (CCAN), which contains a 5-subunit (CENP-P/O/R/Q/U) sub-complex. Here we show using a fluorescent three-hybrid (F3H) assay and fluorescence resonance energy transfer (FRET) in living mammalian cells that CENP-P/O/R/Q/U subunits exist in a tightly packed arrangement that involves multifold protein-protein interactions. This sub-complex is, however, not pre-assembled in the cytoplasm, but rather assembled on kinetochores through the step-wise recruitment of CENP-O/P heterodimers and the CENP-P, -O, -R, -Q and -U single protein units. SNAP-tag experiments and immuno-staining indicate that these loading events occur during S-phase in a manner similar to the nucleosome binding components of the CCAN, CENP-T/W/N. Furthermore, CENP-P/O/R/Q/U binding to the CCAN is largely mediated through interactions with the CENP-N binding protein CENP-L as well as CENP-K. Once assembled, CENP-P/O/R/Q/U exchanges slowly with the free nucleoplasmic pool indicating a low off-rate for individual CENP-P/O/R/Q/U subunits. Surprisingly, we then find that during late S-phase, following the kinetochore-binding step, both CENP-Q and -U but not -R undergo oligomerization. We propose that CENP-P/O/R/Q/U self-assembles on kinetochores with varying stoichiometry and undergoes a pre-mitotic maturation step that could be important for kinetochores switching into the correct conformation necessary for microtubule-attachment.

S100A11 is involved in a variety of intracellular activities such as growth regulation and differentiation. To gain more insight into the physiological role of endogenously expressed S100A11, we used a proteomic approach to detect and identify interacting proteins in vivo. Hereby, we were able to detect a specific interaction between S100A11 and Rad54B, which could be confirmed under in vivo conditions. Rad54B, a DNA-dependent ATPase, is described to be involved in recombinational repair of DNA damage, including DNA double-strand breaks (DSBs). Treatment with bleomycin, which induces DSBs, revealed an increase in the degree of colocalization between S100A11 and Rad54B. Furthermore, S100A11/ Rad54B foci are spatially associated with sites of DNA DSB repair. Furthermore, while the expression of p21 WAF1/CIP1 was increased in parallel with DNA damage, its protein level was drastically down-regulated in damaged cells after S100A11 knockdown. Down-regulation of S100A11 by RNA interference also abolished Rad54B targeting to DSBs. Additionally, S100A11 down-regulated HaCaT cells showed a restricted proliferation capacity and an increase of the apoptotic cell fraction. These observations suggest that S100A11 targets Rad54B to sites of DNA DSB repair sites and identify a novel function for S100A11 in p21-based regulation of cell cycle. INTRODUCTIONProteins may exist in several complexes in a spatial and temporal manner to accomplish distinct functions. Analyses of the interacting partners will provide a strong insight into the physiological role of a particular factor. Therefore, it is essential to identify ideally all interacting partners of proteins in vivo to precisely be able to define their biological function. The investigation of protein complexes of solely endogenously expressed proteins avoid the tendency to detect false positive protein-protein interactions of examinations performed in vitro. A multitude of proteins are involved in both the detection and the repair of DNA damages. It is conceivable that some protein complexes involved in these processes are not yet discovered. A severe form of DNA damage that threaten the integrity of the genome are DNA double-strand breaks (DSBs). DSBs can lead to cell cycle arrest or illegitimate DNA rearrangements that can contribute to cell dysfunction, cell death, or carcinogenesis (Hoeijmakers, 2001). Homologous recombination is a major DNA repair pathway by which DSBs are repaired (Lettier et al., 2006).For the identification of specific interacting proteins of S100A11 (S100C, calgizzarin) we used in the present study a proteomic approach comprising mass spectrometry and immunological techniques. S100A11 belongs to the group of S100 proteins that are considered as multitasking proteins involved in several biological processes such as the Ca 2ϩ signaling network, cell growth and motility, cell cycle progression, transcription, and cell differentiation (Schafer and Heizmann, 1996;Donato, 2001;Eckert et al., 2004). It has been proposed that the S100 proteins are involved in...

Faithful chromatin segregation is mediated and controlled by the kinetochore protein network which assembles at centromeres. In this study, the neighbourhood relations of inner kinetochore and nucleosome-associated complex (NAC) proteins were analysed in living human interphase cells by acceptor photobleaching FRET. The data indicate that CENP-U is in close vicinity to CENP-I as well as to CENP-B and that CENP-M is close to CENP-T.