DNA damage control in the nucleus and mitochondria: exploring new pathways and novel post-translational regulations
DNA topoisomerase I (Top1) regulate DNA supercoiling both in the nucleus and mitochondria to enable faithful transmission of our genetic information to the offspring. However, Top1 are toxic when trapped on the DNA (Top1 cleavage complexes; Top1cc) in the presence of anticancer drug camptothecin or endogenous DNA damage generated by reactive oxygen species (ROS). A key repair enzyme for Top1cc is tyrosyl-DNA phosphodiesterase (TDP1), which hydrolyzes the phosphodiester bond between the DNA 3'-end and the Top1 tyrosyl moiety. Active site mutation of TDP1 causes the severe neurodegenerative syndrome spinocerebellar ataxia with axonal neuropathy (SCAN1). TDP1 repairs Top1cc by forming complexes with the DNA single stranded break repair enzymes like PARP1-XRCC1-PNKP-Polymerase beta-ligaseIII. Top1cc also generates DNA double-strand breaks, and induces phosphorylated TDP1, H2AX and Chk2 nuclear foci by activation of ATM. Therefore, Top1cc-induced DNA damage response is a complex signaling network and is not fully understood. The goal of this application is to unravel the complexity of this signaling network in mechanistic detail and to identify new regulators and novel post-translational modifications of proteins essential for genome maintenance. We will also explore the potential role of mitochondrial DNA (mtDNA) damage, dynamics and metabolism in the pathological-etiology of SCAN1 and related genetic disorders.
Anti-cancer drug camptothecin-induced gH2AX focal accumulations (green) and reactive oxygen species induced mitochondrial hyperfusion (red).
PARP1–TDP1 coupling for the repair of Top1–DNA covalent complexes. (1) The C-terminus domain of PARP1 binds the N-terminus regulatory domain of TDP1 (double-headed arrow). The PARP1–TDP1 molecular complex is shown as a black node. (2) PARP coupling with TDP1 stimulates (open arrow) the excision of Top1-DNA covalent complexes by the phosphodiesterase activity of TDP1 (jigsaw line) (3). The parallel pathway for Top1–DNA complex removal involves various endonucleases (4) including XPF-ERCC1, CtIP and Mre11. (Das et al., Nucleic Acids Research, 2014) https://nar.oxfordjournals.org/content/early/2014/01/31/nar.gku088.long
Top1 dynamics in the live cell nucleus. (A) Cartoon representing live cells Top1 dynamics in the presence and absence of CPT. Fluorescence tagged-human Top1 was evaluated with fluorescence recovery after photobleaching (FRAP) technology. Cells exhibit mainly two types of fluorescent molecule: mobile (unbound) and immobile (bound) (as indicated in the cartoon). After photobleaching at a region of interest (ROI) cells exhibit bleached molecules (grey). Exchanges occur between the mobile parts of two compartments. Top1 cleaves one strand of duplex DNA via the nucleophilic attack of its active site tyrosine (Y723) on the DNA phosphodiester backbone to yield a 3'-phosphotyrosyl bond. The short-lived covalent Top1-DNA cleavage complex (reversible Top1cc) is readily reversed by a second transesterification reaction in which the 5'-hydroxyl end acts as a nucleophile to religate the DNA and to free Top1 (No CPT). Top1 poisons i.e. CPT binds in the interface of Top1-DNA complex, stabilizes Top1cc (Top1 bound state/immobile) and inhibits the Top1-religation reaction. Bold arrow indicates the shift in the cleavage/religation equilibrium, with increasing population of bound/immobile fraction, fluorescence exchange rate is reduced in FRAP recoveries. (B) Representative images showing the fluorescence recovery after photobleaching (FRAP) of wild type Top1 (EGFP-Top1WT) transiently expressed in HCT116 cells and their response to indicated CPT concentrations (1-10 µM). A sub?nuclear spot (ROI) indicated by a circle was bleached (BLH) and photographed at regular intervals of 3 ms thereafter. Successive images taken for ~90s after bleaching illustrate fluorescence return into the bleached areas. (C) Quantification of FRAP data showing mean curves of Top1 variants in the presence and absence of CPT. (Das et al., Nucleic Acids Research, 2016) Adapted from: https://www.ncbi.nlm.nih.gov/pubmed/27466387