title: Poly(ADP-ribose) polymers regulate DNA topoisomerase I (Top1) nuclear dynamics and camptothecin sensitivity in living cells.
abstract: Topoisomerase 1 (Top1) is essential for removing the DNA supercoiling generated during replication and transcription. Anticancer drugs like camptothecin (CPT) and its clinical derivatives exert their cytotoxicity by reversibly trapping Top1 in covalent complexes on the DNA (Top1cc). Poly(ADP-ribose) polymerase (PARP) catalyses the addition of ADP-ribose polymers (PAR) onto itself and Top1. PARP inhibitors enhance the cytotoxicity of CPT in the clinical trials. However, the molecular mechanism by which PARylation regulates Top1 nuclear dynamics is not fully understood. Using live-cell imaging of enhanced green fluorescence tagged-human Top1, we show that PARP inhibitors (Veliparib, ABT-888) delocalize Top1 from the nucleolus to the nucleoplasm, which is independent of Top1-PARP1 interaction. Using fluorescence recovery after photobleaching and subsequent fitting of the data employing kinetic modelling we demonstrate that ABT-888 markedly increase CPT-induced bound/immobile fraction of Top1 (Top1cc) across the nuclear genome, suggesting Top1-PARylation counteracts CPT-induced stabilization of Top1cc. We further show Trp205 and Asn722 of Top1 are critical for subnuclear dynamics. Top1 mutant (N722S) was restricted to the nucleolus in the presence of CPT due to its deficiency in the accumulation of CPT-induced Top1-PARylation and Top1cc formation. This work identifies ADP-ribose polymers as key determinant for regulating Top1 subnuclear dynamics.
authors.affiliation: Laboratory of Molecular Biology, Department of Physical Chemistry, Indian Association for the Cultivation of Science, 2A & B, Raja S. C. Mullick Road, Jadavpur, Kolkata-700032, India.
authors.forename: Subhendu K
authors.initials: SK
authors.lastname: Das
authors.affiliation: Laboratory of Molecular Biology, Department of Physical Chemistry, Indian Association for the Cultivation of Science, 2A & B, Raja S. C. Mullick Road, Jadavpur, Kolkata-700032, India.
authors.forename: Ishita
authors.initials: I
authors.lastname: Rehman
authors.affiliation: Laboratory of Molecular Biology, Department of Physical Chemistry, Indian Association for the Cultivation of Science, 2A & B, Raja S. C. Mullick Road, Jadavpur, Kolkata-700032, India.
authors.forename: Arijit
authors.initials: A
authors.lastname: Ghosh
authors.affiliation: Laboratory of Molecular Biology, Department of Physical Chemistry, Indian Association for the Cultivation of Science, 2A & B, Raja S. C. Mullick Road, Jadavpur, Kolkata-700032, India.
authors.forename: Souvik
authors.initials: S
authors.lastname: Sengupta
authors.affiliation: Laboratory of Molecular Biology, Department of Physical Chemistry, Indian Association for the Cultivation of Science, 2A & B, Raja S. C. Mullick Road, Jadavpur, Kolkata-700032, India.
authors.forename: Papiya
authors.initials: P
authors.lastname: Majumdar
authors.affiliation: Physical Chemistry Department, Indian Association for the Cultivation of Science, 2A & B, Raja S. C. Mullick Road, Jadavpur, Kolkata-700032, India.
authors.forename: Biman
authors.initials: B
authors.lastname: Jana
authors.affiliation: Laboratory of Molecular Biology, Department of Physical Chemistry, Indian Association for the Cultivation of Science, 2A & B, Raja S. C. Mullick Road, Jadavpur, Kolkata-700032, India pcbbd@iacs.res.in.
authors.forename: Benu Brata
authors.initials: BB
authors.lastname: Das
mesh_terms: D001562:Benzimidazoles / Q000494:pharmacology; D002166:Camptothecin / Q000494:pharmacology*; D002466:Cell Nucleolus / Q000187:drug effects / Q000378:metabolism; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects; D004247:DNA / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D004058:Diffusion; D019008:Drug Resistance, Neoplasm / Q000187:drug effects; D036681:Fluorescence Recovery After Photobleaching; D049452:Green Fluorescent Proteins / Q000378:metabolism; D045325:HCT116 Cells; D006801:Humans; D007700:Kinetics; D050505:Mutant Proteins / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011064:Poly Adenosine Diphosphate Ribose / Q000378:metabolism*; D000067856:Poly(ADP-ribose) Polymerase Inhibitors / Q000494:pharmacology
chemical_list: D001562:Benzimidazoles; D050505:Mutant Proteins; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; C485184:enhanced green fluorescent protein; C521013:veliparib; D049452:Green Fluorescent Proteins; D011064:Poly Adenosine Diphosphate Ribose; D004247:DNA; D004264:DNA Topoisomerases, Type I; C528909:TOP1 protein, human; D002166:Camptothecin