Research Projects

Topoisomerase 1 is an enzyme that relaxes DNA torsional stress and plays an essential role during transcription, replication, and recombination. TOP1 cuts one strand of DNA forms a transient covalent bond with DNA and then re-ligates the cleaved ends. Although TOP1 introduces transient DNA breaks, their re-ligation is aborted by topoisomerase-poisons, forming covalently-trapped topoisomerase 1-DNA protein crosslinks (TOP1-DPCs), a severe form of DNA lesions. If not repaired, the accumulated TOP1-DPCs can threaten genome integrity. Interestingly, the cellular toxicity of TOP1-DPCs also made TOP1 an attractive target for anti-cancer chemotherapeutic agents that abort re-ligation step of TOP1 reaction and thus promote the formation of TOP1-DPCs. Despite significant progress in our understanding of TOP1-DPC repair, the exact mechanism of TOP1-DPCs is not fully understood. First, it is unclear how different TOP1-DPC repair pathways are regulated and how the choice between overlapping repair pathways is made. Second, it also not studied how different TOP1-DPC excision proteins are coupled to downstream DNA repair pathways that seal the protein-free breaks. Third, the role of local chromatin structure and dynamics in TOP1-DPC repair is yet to be understood. Fourth, it is also conceivable that additional not yet identified repair proteins or pathways are involved in the regulation, pathway choice, and repair of TOP1-DPCs. Thus, decoding TOP1-DPC interactome is essential for a better understanding of repair mechanisms of TOP1-DPCs, and their exploitation to for cancer chemotherapies. Here, we propose to decode on-chromatin interactome of TOP1-DPCs using proximity labeling-based proteomic approaches. Proximity proteomics relies on using a biotin ligase to biotinylate proteins proximal to a target protein. We will express the Turbo-ID, an engineered biotin ligase, tagged TOP1 in a human cell line, induce the formation of TOP1-DPCs by TOP1 poisons, and biotinylate TOP1-DPCs proximal proteins, followed by immunoprecipitation and mass spectrometry to identify proteins proximal to TOP1-DPCs. Identifying proteins that interact with TOP1-DPCs is essential for a better understanding of the TOP1-DPC repair process, which is critical for improving TOP1 poisoning-based chemotherapies. Proposed studies may also help in understanding the role of TOP1-DPCs in neurodegenerative diseases as well as the normal functioning of TOP1. The functions of identified proteins will then be studied in TOP1-DPC repair. In addition to nuclear TOP1, other topoisomerases, DNA methyltransferases, apurinic/apyrimidinic lyases, Poly (ADP-ribose) polymerase-1, DNA polymerases, and DNA glycosylases, and a variety of non-enzymatic reactions also lead to the formation of DPCs. In fact, DPCs are one of the most common and severe DNA lesions generated spontaneously in a cell. Therefore, the proposed study will also help understand the repair of other enzymatic and non-enzymatic DPCs.