A CAF-1–PCNA-Mediated Chromatin Assembly Pathway Triggered by Sensing DNA Damage

Abstract

Sensing DNA damage is crucial for the maintenance of genomic integrity and cell cycle progression. The participation of chromatin in these events is becoming of increasing interest. We show that the presence of single-strand breaks and gaps, formed either directly or during DNA damage processing, can trigger the propagation of nucleosomal arrays. This nucleosome assembly pathway involves the histone chaperone chromatin assembly factor 1 (CAF-1). The largest subunit (p150) of this factor interacts directly with proliferating cell nuclear antigen (PCNA), and critical regions for this interaction on both proteins have been mapped. To isolate proteins specifically recruited during DNA repair, damaged DNA linked to magnetic beads was used. The binding of both PCNA and CAF-1 to this damaged DNA was dependent on the number of DNA lesions and required ATP. Chromatin assembly linked to the repair of single-strand breaks was disrupted by depletion of PCNA from a cell-free system. This defect was rescued by complementation with recombinant PCNA, arguing for role of PCNA in mediating chromatin assembly linked to DNA repair. We discuss the importance of the PCNA–CAF-1 interaction in the context of DNA damage processing and checkpoint control.

ACKNOWLEDGMENTS

We are most grateful to B. Stillman (Cold Spring Harbor Laboratory) and his laboratory for their generous collaboration in providing antibodies as well as recombinant human CAF-1. We gratefully acknowledge F. Bunz (Cold Spring Harbor Laboratory) for making and characterizing the RF-C p140 monoclonal antibody and J. Moreau (Institut Jacques Monod) for providing the Xenopus laeviscDNA library used in the two-hybrid screen. We thank D. M. J. Roche for sharing her expertise and unpublished data on the analysis of chromatin assembly in the human cell-free system. We thank all members of our laboratory for help and advice and E. Moustacchi and E. Bailly for critical reading of the manuscript.

J.G.M. was supported first by an EMBO long-term fellowship and then by a European Union Training, Mobility and Research (TMR) fellowship. P.G. was supported by an EMBO long-term fellowship. Z.O.J. and U.H. were supported by the Swiss National Science Foundation (grant 31-43138.35/2) and by the Canton of Zürich. This work was supported by the Association pour la Recherche sur le Cancer, La Ligue Nationale contre le Cancer, Fondation de la Recherche Medicale, and a TMR Network grant from the European Union (G.A.).