The histone H3 variant CENP-A marks centromeres epigenetically and is essential for mitotic fidelity. Previous crystallographic studies of the CENP-A nucleosome core particle (NCP) reconstituted with a human α-satellite DNA derivative revealed both DNA ends to be highly flexible, a feature important for CENP-A mitotic functions. However, recent cryo-EM studies of CENP-A NCP complexes comprising primarily Widom 601 DNA reported well-ordered DNA ends. Here, we report the cryo-EM structure of the CENP-A 601 NCP determined by Volta phase-plate imaging. The data reveal that one ('left') 601 DNA end is well ordered whereas the other ('right') end is flexible and partly detached from the histone core, suggesting sequence-dependent dynamics of the DNA termini. Indeed, a molecular dynamics simulation of the CENP-A 601 NCP confirmed the distinct dynamics of the two DNA extremities. Reprocessing the image data using two-fold symmetry yielded a cryo-EM map in which both DNA ends appeared well ordered, indicating that such an artefact may inadvertently arise if NCP asymmetry is lost during image processing. These findings enhance our understanding of the dynamic features that discriminate CENP-A from H3 nucleosomes by revealing that DNA end flexibility can be fine-tuned in a sequence-dependent manner.

Computational Biology Branch National Center for Biotechnology Information National Library of Medicine National Institutes of Health Bethesda MD 20894 USA

Département de Génomique Fonctionnelle et Cancer Institut de Génétique et Biologie Moléculaire et Cellulaire Université de Strasbourg CNRS INSERM 67404 Illkirch Cedex France

Department of Molecular Structural Biology Max Planck Institute of Biochemistry 82152 Martinsried Germany

Institute for Advanced Biosciences Inserm U 1209 CNRS UMR 5309 Université Grenoble Alpes 38000 Grenoble France

Izmir Biomedicine and Genome Center Dokuz Eylul University Health Campus Balcova Izmir 35330 Turkey

Laboratoire de Biologie et de Modélisation de la Cellule CNRS ENSL UCBL Ecole Normale Supérieure de Lyon 69007 Lyon France

Laboratory of the Biology and Pathology of the Eye Institute of Biology and Medical Genetics 1st Faculty of Medicine Charles University and General Teaching Hospital 128 00 Prague Czech Republic

Université Grenoble Alpes CEA CNRS Institut de Biologie Structurale 38000 Grenoble France

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