Abstract
The prime function of nucleoli is ribogenesis, however, several other, non-canonical functions have recently been identified, including a role in genotoxic stress response. Upon DNA damage, numerous proteins shuttle dynamically between the nucleolus and the nucleoplasm, yet the underlying molecular mechanisms are incompletely understood. Here, we demonstrate that PARP1 and PARylation contribute to genotoxic stress-induced nucleolar-nucleoplasmic shuttling of key genome maintenance factors in HeLa cells. Our work revealed that the RECQ helicase, WRN, translocates from nucleoli to the nucleoplasm upon treatment with the oxidizing agent H2O2, the alkylating agent 2-chloroethyl ethyl sulfide (CEES), and the topoisomerase inhibitor camptothecin (CPT). We show that after treatment with H2O2 and CEES, but not CPT, WRN translocation was dependent on PARP1 protein, yet independent of its enzymatic activity. In contrast, nucleolar-nucleoplasmic translocation of the base excision repair protein, XRCC1, was dependent on both PARP1 protein and its enzymatic activity. Furthermore, gossypol, which inhibits PARP1 activity by disruption of PARP1-protein interactions, abolishes nucleolar-nucleoplasmic shuttling of WRN, XRCC1 and PARP1, indicating the involvement of further upstream factors. In conclusion, this study highlights a prominent role of PARP1 in the DNA damage-induced nucleolar-nucleoplasmic shuttling of genome maintenance factors in HeLa cells in a toxicant and protein-specific manner.
Highlights
The nucleolus is a dynamic, non-membrane bound compartment of the nucleus[1,2]
(6) XRCC1 requires DNA damage-bound and PARylated PARP1 as a loading platform, which leads to XRCC1 retention in the nucleoplasm until its tasks in base excision repair (BER) are completed
(7) The latter hypothesis is supported by results revealing that in cells without PARP1 activity, XRCC1 relocates quickly to nucleoli
Summary
The nucleolus is a dynamic, non-membrane bound compartment of the nucleus[1,2]. Its prime function is ribosome biogenesis, comprising rRNA synthesis and ribosome assembly. In total >150 DNA repair proteins localize to nucleoli under non-stress conditions[16] and at least some of them undergo DNA damage-specific nucleolar-nucleoplasmic shuttling upon induction of genotoxic stress[17] It is a matter of active investigations if the nucleolus acts as a mere reservoir and storage site for genome maintenance factors that operate in the nucleoplasm upon their nucleolar release, or if such factors play specific roles in nucleolar processes, e.g. in the repair of damaged rDNA sequences or in DNA repair-unrelated functions such as RNA metabolism - or both. PARP inhibitors have attracted much attention in the last years for their use in cancer chemotherapy, either by their use as chemosensitizers in combination with classical DNA-damaging chemotherapeutics or as stand-alone drugs in homologous-deficient tumors following the concept of synthetic lethality[24,25]
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