The Repair of Topoisomerase DNA-Protein Crosslinks

Our research is dedicated to elucidating molecular mechanisms that repair topoisomerase DNA-protein crosslinks (TOP-DPCs) and PARP-DNA complexes, whose induction is the primary therapeutic mechanism of topoisomerase inhibitors and PARP inhibitors, respectively. We have found that the repair mechanisms are tightly regulated by post-translational modifications such as ubiquitylation, SUMOylation, ADP-ribosylation, and neddylation. Ongoing studies involve the investigation of crosstalk between these modifications in DNA repair. (PMID: 33188014, 34408146, 35013556, 36213144, 38055811)

Targeting the repair of TOP-DPCs and PARP-DNA complexes has been proposed as a strategy to improve tumor response to topoisomerase inhibitors and PARP inhibitors. With unbiased high-throughput genetical and chemical screening, we have identified novel DNA repair pathways involved in TOP-DPC repair as well as small molecule inhibitors (SMIs) targeting these pathways. We are interrogating novel combinations of topoisomerase inhibitors and the SMIs in different preclinical cancer models such as lung cancer and colorectal cancer. (PMID: 35830858, 37353483)

We have developed an imaging-based approach enabling real-time monitoring of drug-induced trapping of TOPccs and PARP1 in live cells at the single-molecule level. Capitalizing on this approach, we are able to measure the fraction of self-fluorescence tag-labeled topoisomerases and PARP single-molecules that are trapped by their respective inhibitors in real-time. Further developing this technique can help us better understand the molecular biology of topoisomerases and facilitate the development of novel topoisomerase inhibitors. (PMID: 37670571)

Non-specific/non-enzymatic DNA-protein crosslinks (DPCs) are among the most detrimental genomic lesions. They are ubiquitously produced by cellular metabolic by-products and by environmental pollutants such as formaldehyde (FA). Failure to repair these DPCs blocks chromatin-based processes, leading to neurodegeneration and cancer. Yet, the types of proteins crosslinked by aldehydes remain largely unknown. We have profiled the proteome of FA-induced DPCs in human cells. We carried out RNAi screening and found that flap endonuclease 1 (FEN1) and several other nucleases are required for cells to survive FA by repairing FA-induced DPCs. Ongoing studies involve the interrogation of their enzymatic roles in DPC repair. (Sun et al., BioRxiv, 2023)

Topoisomerases play essential roles in DNA replication, transcription, chromosome segregation, and recombination. Insufficient activity in the topoisomerases can generate profound effects on DNA and RNA metabolisms and on the accumulation of non-canonical DNA structures, resulting in cancer, immunodeficiency, and neurological disorders. It is conceivable that cells must have evolved mechanisms to compensate for the genetic inactivation of topoisomerases. We are conducting multiple CRISPR-mediated loss-of-function screens to discover synthetically lethal genes

with topoisomerases (TOP1, TOP1MT, TOP2B, TOP3A), which may act as compensatory pathways for topoisomerase-associated cellular processes.