Assembly of the inner kinetochore proteins CENP-A and CENP-B in living human cells.
Chembiochem
; 9(1): 77-92, 2008 Jan 04.
Article
em En
| MEDLINE
| ID: mdl-18072184
ABSTRACT
DNA segregation in mammalian cells during mitosis is an essential cellular process that is mediated by a specific subchromosomal protein complex, the kinetochore. Malfunction of this complex results in aneuploidy and can cause cancer. A subkinetochore complex, the "inner kinetochore", is present at the centromere during the entire cell cycle. Its location seems to be defined by the settlement of CENP-A (CENH3), which replaces histone H3 in centromeric nucleosomes. This suggests that CENP-A can recruit further inner kinetochore proteins by direct binding. Surprisingly, intense in vitro studies could not identify an interaction of CENP-A with any other inner kinetochore protein. Instead, centromere identity seems to be maintained by a unique nucleosome, which might have a modified structure or epigenetic state that serves to distinguish the centromere from the rest of the chromosome. We investigated the association of CENP-A and CENP-B by fluorescence intensity and lifetime-based FRET measurements in living human HEp-2 cells. We observed Förster resonance energy transfer (FRET) between CENP-A and CENP-B at centromere locations; this indicates that these proteins are in the molecular vicinity (<10 nm) of each other. In addition, we analysed protein-protein interactions within the centromeric nucleosome. We could detect energy transfer between CENP-A and histone H4 as well as between CENP-A molecules themselves. On the other hand, no FRET was detected between CENP-A and H2A.1 or H3.1. Our data support the view that two CENP-A molecules are packed with H4, but not with H3, in a single centromeric nucleosome.
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Base de dados:
MEDLINE
Assunto principal:
Autoantígenos
/
Proteínas Cromossômicas não Histona
/
Cinetocoros
/
Hepatócitos
/
Proteína B de Centrômero
Limite:
Humans
Idioma:
En
Ano de publicação:
2008
Tipo de documento:
Article