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Reconstitution and characterization of kinetochore units assembled on centromeric nucleosome templates
Kinetochores, large multi-protein assemblies on centromeric DNA, interact with spindle microtubules to mediate faithful chromosome segregation during mitosis. The inner part of the kinetochore, the 16-subunit constitutive centromere associated network (CCAN), flanks the centromere throughout the cell cycle and provides the structural foundation for the peripheral kinetochore subunits. Centromere protein (CENP)-C is a fundamental CCAN subunit that provides a foundation, either directly or indirectly, to all other kinetochore components onto the specialized centromeric chromatin. Recent biochemical reconstitution studies provided insights into the complex human kinetochore architecture but still lacked the full-length version of CENP-C. My PhD work aimed to characterize the interactions of recombinant full-length CENP-C with nucleosomes and the other kinetochore members in vitro. Furthermore, my work aimed to define the determinants for the centromere recruitment of CENP-C. Lastly, in my studies, I gained new insights into the propagation of centromeric chromatin. Combining biochemical reconstitution and biophysical methods, I revealed that the full-length CENP-C protein binds two nucleosomes and that this interaction strictly depends on two conserved nucleosome binding motifs. Using dinucleosomes bound to full-length CENP-C as a template, a 30-subunit kinetochore particle was reconstituted containing all inner and outer kinetochore subcomplexes. Electroporation of CENP-C into living human cells showed that CENP-C depends on the interactions with nucleosomes and with the CCAN members CENP-HIKM/-LN for its proper recruitment to the centromere. Furthermore, homo-dimerization mediated by the Cupin-like domain was identified as a mechanism promoting affinity towards the centromere. Immunoprecipitation experiments from cell lysates combined with mass spectrometry analysis identified Polo-like kinase 1 (PLK1) as a crucial factor that promotes centromere propagation. In conclusion, this work demonstrates that CENP-C can ...
Reconstitution and characterization of kinetochore units assembled on centromeric nucleosome templates
Kinetochores, large multi-protein assemblies on centromeric DNA, interact with spindle microtubules to mediate faithful chromosome segregation during mitosis. The inner part of the kinetochore, the 16-subunit constitutive centromere associated network (CCAN), flanks the centromere throughout the cell cycle and provides the structural foundation for the peripheral kinetochore subunits. Centromere protein (CENP)-C is a fundamental CCAN subunit that provides a foundation, either directly or indirectly, to all other kinetochore components onto the specialized centromeric chromatin. Recent biochemical reconstitution studies provided insights into the complex human kinetochore architecture but still lacked the full-length version of CENP-C. My PhD work aimed to characterize the interactions of recombinant full-length CENP-C with nucleosomes and the other kinetochore members in vitro. Furthermore, my work aimed to define the determinants for the centromere recruitment of CENP-C. Lastly, in my studies, I gained new insights into the propagation of centromeric chromatin. Combining biochemical reconstitution and biophysical methods, I revealed that the full-length CENP-C protein binds two nucleosomes and that this interaction strictly depends on two conserved nucleosome binding motifs. Using dinucleosomes bound to full-length CENP-C as a template, a 30-subunit kinetochore particle was reconstituted containing all inner and outer kinetochore subcomplexes. Electroporation of CENP-C into living human cells showed that CENP-C depends on the interactions with nucleosomes and with the CCAN members CENP-HIKM/-LN for its proper recruitment to the centromere. Furthermore, homo-dimerization mediated by the Cupin-like domain was identified as a mechanism promoting affinity towards the centromere. Immunoprecipitation experiments from cell lysates combined with mass spectrometry analysis identified Polo-like kinase 1 (PLK1) as a crucial factor that promotes centromere propagation. In conclusion, this work demonstrates that CENP-C can ...
Reconstitution and characterization of kinetochore units assembled on centromeric nucleosome templates
Walstein, Kai (author) / Musacchio, Andrea
2021-02-18
Theses
Electronic Resource
English
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