Crystal structure of the CENP-B protein-DNA complex: the DNA-binding domains of CENP-B induce kinks in the CENP-B box DNA.
(9/99)
The human centromere protein B (CENP-B), one of the centromere components, specifically binds a 17 bp sequence (the CENP-B box), which appears in every other alpha-satellite repeat. In the present study, the crystal structure of the complex of the DNA-binding region (129 residues) of CENP-B and the CENP-B box DNA has been determined at 2.5 A resolution. The DNA-binding region forms two helix-turn-helix domains, which are bound to adjacent major grooves of the DNA. The DNA is kinked at the two recognition helix contact sites, and the DNA region between the kinks is straight. Among the major groove protein-bound DNAs, this 'kink-straight-kink' bend contrasts with ordinary 'round bends' (gradual bending between two protein contact sites). The larger kink (43 degrees ) is induced by a novel mechanism, 'phosphate bridging by an arginine-rich helix': the recognition helix with an arginine cluster is inserted perpendicularly into the major groove and bridges the groove through direct interactions with the phosphate groups. The overall bending angle is 59 degrees, which may be important for the centromere-specific chromatin structure. (+info)
Kinetochore localisation of the DNA damage response component 53BP1 during mitosis.
(10/99)
53BP1 is a vertebrate BRCT motif protein, originally described as a direct interactor of p53, which has recently been shown to be implicated in the early response to DNA damage. Upon DNA damage, 53BP1 re-localises to discrete nuclear foci that are thought to represent sites of DNA lesions and becomes hyperphosphorylated. Several observations suggest that 53BP1 is a direct substrate for the ataxia telangiectasia mutated (ATM) kinase. So far, 53BP1 behaviour during mitosis has not been reported in detail. We have examined 53BP1 subcellular distribution in mitotic cells using several antibodies against 53BP1, and ectopic expression of GFP-tagged 53BP1. We found that 53BP1 significantly colocalised with CENP-E to kinetochores. 53BP1 is loaded to kinetochores in prophase, before CENP-E, and is released by mid-anaphase. By expressing various GFP-tagged 53BP1 truncations, the kinetochore binding domain has been mapped to a 380 residue portion of the protein that excludes the nuclear localisation signal and the BRCT motifs. Like many kinetochore-associated proteins involved in mitotic checkpoint signalling, more 53BP1 appears to accumulate on the kinetochores of chromosomes not aligned on the metaphase plate. Finally, we show that 53BP1 is hyperphosphorylated in mitotic cells, and undergoes an even higher level of phosphorylation in response to spindle disruption with colcemid. Our data suggest that 53BP1 may have a role in checkpoint signalling during mitosis and provide the evidence that DNA damage response machinery and mitotic checkpoint may share common molecular components. (+info)
CENP-A, -B, and -C chromatin complex that contains the I-type alpha-satellite array constitutes the prekinetochore in HeLa cells.
(11/99)
CENP-A is a component of centromeric chromatin and defines active centromere regions by forming centromere-specific nucleosomes. We have isolated centromeric chromatin containing the CENP-A nucleosome, CENP-B, and CENP-C from HeLa cells using anti-CENP-A and/or anti-CENP-C antibodies and shown that the CENP-A/B/C complex is predominantly formed on alpha-satellite DNA that contains the CENP-B box (alphaI-type array). Mapping of hypersensitive sites for micrococcal nuclease (MNase) digestion indicated that CENP-A nucleosomes were phased on the alphaI-type array as a result of interactions between CENP-B and CENP-B boxes, implying a repetitive configuration for the CENP-B/CENP-A nucleosome complex. Molecular mass analysis by glycerol gradient sedimentation showed that MNase digestion released a CENP-A/B/C chromatin complex of three to four nucleosomes into the soluble fraction, suggesting that CENP-C is a component of the repetitive CENP-B/CENP-A nucleosome complex. Quantitative analysis by immunodepletion of CENP-A nucleosomes showed that most of the CENP-C and approximately half the CENP-B took part in formation of the CENP-A/B/C chromatin complex. A kinetic study of the solubilization of CENPs showed that MNase digestion first released the CENP-A/B/C chromatin complex into the soluble fraction, and later removed CENP-B and CENP-C from the complex. This result suggests that CENP-A nucleosomes form a complex with CENP-B and CENP-C through interaction with DNA. On the basis of these results, we propose that the CENP-A/B/C chromatin complex is selectively formed on the I-type alpha-satellite array and constitutes the prekinetochore in HeLa cells. (+info)
CENP-C binds the alpha-satellite DNA in vivo at specific centromere domains.
(12/99)
CENP-C is a fundamental component of the centromere, highly conserved among species and necessary for the proper assembly of the kinetochore structure and for the metaphase-anaphase transition. Although CENP-C can bind DNA in vitro, the identification of the DNA sequences associated with it in vivo and the significance of such an interaction have been, until now, elusive. To address this problem we took advantage of a chromatin-immunoprecipitation procedure and applied this technique to human HeLa cells. Through this approach we could establish that: (1) CENP-C binds the alpha-satellite DNA selectively; (2) the CENP-C region between amino acids 410 and 537, previously supposed to contain a DNA-binding domain, is indeed required to perform such a function in vivo; and (3) the profile of the alpha-satellite DNA associated with CENP-C is essentially identical to that recognized by CENP-B. However, further biochemical and ultrastructural characterization of CENP-B/DNA and CENP-C/DNA complexes, relative to their DNA components and specific spatial distribution in interphase nuclei, surprisingly reveals that CENP-C and CENP-B associate with the same types of alpha-satellite arrays but in distinct non-overlapping centromere domains. Our results, besides extending previous observations on the role of CENP-C in the formation of active centromeres, show, for the first time, that CENP-C can associate with the centromeric DNA sequences in vivo and, together with CENP-B, defines a highly structured organization of the alpha-satellite DNA within the human centromere. (+info)
Centromere proteins Cenpa, Cenpb, and Bub3 interact with poly(ADP-ribose) polymerase-1 protein and are poly(ADP-ribosyl)ated.
(13/99)
Poly(ADP-ribose) polymerase-1 (PARP-1) is activated by DNA strand breaks during cellular genotoxic stress response and catalyzes poly(ADP-ribosyl)ation of acceptor proteins. These acceptor proteins include those involved in modulation of chromatin structure, DNA synthesis, DNA repair, transcription, and cell cycle control. Thus, PARP-1 is believed to play a pivotal role in maintaining genome integrity through modulation of protein-protein and protein-DNA interactions. We previously described the association of PARP-1 with normal mammalian centromeres and human neocentromeres by affinity purification and immunofluorescence. Here we investigated the interaction of this protein with, and poly(ADP-ribosyl)ation of, three constitutive centromere proteins, Cenpa, Cenpb, and Cenpc, and a spindle checkpoint protein, Bub3. Immunoprecipitation and Western blot analyses demonstrate that Cenpa, Cenpb, and Bub3, but not Cenpc, interacted with PARP-1, and are poly(ADP-ribosyl)ated following induction of DNA damage. The results suggest a role of PARP-1 in centromere assembly/disassembly and checkpoint control. Demonstration of PARP-1-binding and poly(ADP-ribosyl)ation in three of the four proteins tested further suggests that many more centromere proteins may behave similarly and implicates PARP-1 as an important regulator of diverse centromere function. (+info)
Development of a CENP-A/CENP-B-specific immune response in a patient with systemic sclerosis.
(14/99)
Antibodies directed against an epitope motif on CENP-A have been shown to cross-react with mimotopes on other autoantigens and on Epstein-Barr nuclear antigen 1 (EBNA-1), suggesting a molecular mimicry. We describe here the gradual development of an anticentromere immune response in a patient with systemic sclerosis, which started from an antihistone response and was not mediated by molecular mimicry. Via an epitope on histone H3, the antibody response spread to a homologous epitope in the H3 homology domain of CENP-A. This was followed by an intramolecular epitope spreading to N-terminal peptides of CENP-A containing the known epitope motif G-P-X(1)-R-X(2). From there it spread to corresponding epitopes on CENP-B and to mimotopes of the major CENP-A epitope motif on other autoantigens including EBNA-1. Whether the D-penicillamine treatment received by this patient was involved in the triggering of this cascade remains a matter of speculation. (+info)
Recognition of Granzyme B-generated autoantigen fragments in scleroderma patients with ischemic digital loss.
(15/99)
OBJECTIVE: To examine whether autoantibodies recognizing granzyme B (GB)-cleaved autoantigens are associated with ischemic digital loss (IDL) in limited systemic sclerosis (SSc). METHODS: Fifteen of 19 patients with limited SSc and IDL were matched by age, sex, race, and duration of disease to controls with limited SSc but without IDL. The sera were used to immunoblot HeLa cell lysates and chromosome preparations that had been incubated in vitro in the absence or presence of GB. Anticentromere antibodies (ACAs) were assayed by immunofluorescence and immunoprecipitation of in vitro-translated centromere proteins (CENP-B and CENP-C). Immunoprecipitation of GB-cleaved CENPs was also performed. RESULTS: GB-cleaved autoantigens were immunoblotted by 16 of 19 IDL sera (84.2%) compared with 6 of 15 non-IDL sera (40.0%) (odds ratio 8.0, 95% confidence interval 1.6-40.0). This association persisted after adjustment for ACA status. Furthermore, the presence of antibodies to centromere proteins as well as to GB-cleaved antigens was highly specific for IDL, occurring in 12 of 19 IDL patients (63.2%) and in none of 15 controls (P < 0.0001). An identical 60-kd GB-generated fragment was recognized by 5 of 16 IDL sera (31.3%) and was demonstrated to arise through GB-mediated cleavage of CENP-C. GB-cleaved CENP-C fragments were recognized preferentially over the intact CENP-C molecule by antibodies from patients with IDL. CONCLUSION: The striking recognition of GB-generated autoantigen fragments by sera from patients with limited SSc and IDL constitutes the first in vivo evidence that antibodies against GB-generated centromeric peptide fragments identify a distinct clinical subset. (+info)
Heterochromatin is a functionally important chromosomal component, especially at centromeres. In fission yeast, conserved heterochromatin-specific modifications of the histone H3 tail, involving deacetylation of Lys 9 and Lys 14 and subsequent methylation of Lys 9, promote the recruitment of a heterochromatin protein, Swi6, a homolog of the Drosophila heterochromatin protein 1. However, the primary determinants of the positioning of heterochromatin are still unclear. The fission yeast proteins Abp1, Cbh1, and Cbh2 are homologs of the human protein CENP-B that bind to centromeric alpha-satellite DNA and associate with centromeric heterochromatin. We show that the CENP-B homologs are functionally redundant at centromeres, and that Abp1 binds specifically to centromeric heterochromatin. In the absence of Abp1 or Cbh1, the centromeric association of Swi6 is diminished, resulting in a decrease in silencing of the region. CENP-B-homolog double disruptants show a synergistic reduction of Swi6 at centromeric heterochromatin, indicating that the three proteins are functionally redundant in the recruitment of Swi6. Furthermore, using chromatin immunoprecipitation assays, we show that disruption of CENP-B homologs causes a decrease in heterochromatin-specific modifications of histone H3. These results indicate that the CENP-B homologs act as site-specific nucleation factors for the formation of centromeric heterochromatin by heterochromatin-specific modifications of histone tails. (+info)