The Tumor Suppressor PML Specifically Accumulates at RPA/Rad51-Containing DNA Damage Repair Foci but Is Nonessential for DNA Damage-Induced Fibroblast Senescence

Abstract

The PML tumor suppressor has been functionally implicated in DNA damage response and cellular senescence. Direct evidence for such a role based on PML knockdown or knockout approaches is still lacking. We have therefore analyzed the irradiation-induced DNA damage response and cellular senescence in human and mouse fibroblasts lacking PML. Our data show that PML nuclear bodies (NBs) nonrandomly associate with persistent DNA damage foci in unperturbed human skin and in high-dose-irradiated cell culture systems. PML bodies do not associate with transient γH2AX foci after low-dose gamma irradiation. Superresolution microscopy reveals that all PML bodies within a nucleus are engaged at Rad51- and RPA-containing repair foci during ongoing DNA repair. The lack of PML (i) does not majorly affect the DNA damage response, (ii) does not alter the efficiency of senescence induction after DNA damage, and (iii) does not affect the proliferative potential of primary mouse embryonic fibroblasts during serial passaging. Thus, while PML NBs specifically accumulate at Rad51/RPA-containing lesions and senescence-derived persistent DNA damage foci, they are not essential for DNA damage-induced and replicative senescence of human and murine fibroblasts.

ACKNOWLEDGMENTS

We thank S. Ohndorf and M. Koch for technical assistance. We are grateful to Hans Will and Hannah Stäge (HPI, Hamburg, Germany) for providing PML knockout mice and help in isolation of MEFs, Nina Reuter and Thomas Stamminger (University of Erlangen, Erlangen, Germany) for providing stable PML knockdown fibroblasts and PML-specific shRNA plasmids, Johannes Norgauer and Mirjana Zimmer (University Hospital Jena, Jena, Germany) for providing skin tissue, and Anja Krüger and Tjard Jörß (animal facility, FLI, Jena, Germany) for patient help and support in work with mice and preparation of primary cells. We also thank Stephan Diekmann and Tobias Ulbricht for enlightening discussions and laboratory support.

This work was supported by grant HE 2484/3-1 from the Deutsche Forschungsgemeinschaft. We also acknowledge JenAge funding by the German Ministry for Education and Research (Bundesministerium für Bildung und Forschung [BMBF]; support code 0315581).