The orchestration of DNA-protein crosslink recognition and repair in mammalian cells

Abstract

DNA-Protein Crosslinks (DPCs) are large, bulky, and cytotoxic DNA lesions that are subject to repair by Nucleotide Excision Repair (NER) and Homologous Recombination (HR), and post-translationally modified prior to their repair. The orchestration of DPC repair by multiple, redundant repair pathways is not fully understood. A limitation of DPC repair research is that drugs used to form genomic DPCs form other kinds of DNA damage as well. To address this limitation, I have developed a site-specific, chemically homogenous model DPC substrate that can be manipulated to allow for independent study of different kinds of DPCs. DPC substrates were transfected into cell-based or cell-free systems and analyzed utilizing a variety of assays, including qPCR-based and non-qPCR-based assays. I showed that these systems can be used to study DPC repair, removal, and post translational modifications. Genetic manipulation of transfection conditions allows for the study of repair intermediates formed via NER-mediated or HR-mediated DPC repair, independently. Using the tools described above, I found that DPC removal via NER and HR is ubiquitin dependent, however pathway specific removal is modulated by differential polyubiquitination of the crosslinked protein. I also showed that DPC removal by NER, but not HR is proteasome dependent. Finally, I found that DPCs are also modified with SUMO1 and SUMO2/3 proteins, and that DPC ubiquitination and SUMOylation are interdependent, however the mechanisms underlying this interdependence remain to be investigated.