Investigating the roles of high mobility group box proteins in nucleotide excision repair-mediated DNA damage processing




Dangerfield, Jillian Claire

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High mobility group box (HMGB) proteins are non-histone architectural proteins that bind to and bend non-canonical DNA structures. HMGB1, HMGB2, and HMGB3 are structurally similar, sharing ~80% of their sequences, however they have been shown to play biologically different roles. Notably, knockout of HMGB1 in mice is lethal, while knockout of HMGB2 and HMGB3 is not. HMGB1 is a co-factor in nucleotide excision repair (NER)-mediated repair of both DNA intrastrand crosslinks and DNA interstrand crosslinks (ICLs). HMGB1 has been shown to bind with NER proteins on ICLs, increase the mutagenic outcome of intrastrand crosslink and ICL processing, alter the mutation spectra resulting from ICL processing, and recruit the NER protein, Xeroderma pigmentosum complementation group A (XPA), to ICLs in cells. There are still details with respect to the role(s) of HMGB1 that have not yet been determined, such as the potential involvement it may have in a NER sub-pathway or translesion DNA synthesis during ICL processing. Moreover, the role of HMGB3 in DNA damage repair is largely unknown. HMGB3 is overexpressed in many cancers, and is associated with resistance to DNA damaging chemotherapeutic agents, including cisplatin, which induces both intrastrand and interstrand crosslinks. Given the similarity to HMGB1, HMGB3 may also be involved in NER-mediated DNA damage repair. A combination of mutagenesis assays, co-immunoprecipitation experiments, chromatin immunoprecipitation assays, clonogenic assays, slot blot assays, and RNA sequencing were utilized to understand the potential role(s) of HMGB3 in intrastrand crosslink repair, HMGB1 in the two NER sub-pathways and in translesion synthesis, and HMGB2 and HMGB3 in ICL repair. There was no obvious involvement of HMGB1 in the NER subpathways or in translesion synthesis in conjunction with polymerase eta. HMGB3 was not found to play a large role in intrastrand crosslink repair, suggesting that unlike HMGB1, it is not an NER co-factor. Instead, HMGB3 appears to play a role in ICL processing and/or DNA double-strand break (DSB) repair. In addition, HMGB3 knockout in cells resulted in downregulated cell cycle-associated genes and chromatin organization-associated genes, among others. Future directions include continued investigation of the roles of the HMGB proteins in DNA damage repair.


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