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Date of Award
Doctor of Philosophy (PhD)
DNA repair is essential to prevent the cytotoxic or mutagenic effects of various types of DNA lesions. These lesions are sensed by distinct pathways to recruit repair factors specific to type of damage. In particular, the ALKBH family of proteins recognizes and repairs specific alkylated lesions, including 1-methyladenine (m1A) and 3-methylcytosine (m3C). A major outstanding question in the field is how the AlkB homologue ALKBH3 and its associated protein partners are recruited to sites of alkylation damage and how this repair activity is regulated. Understanding the upstream signaling events that mediate recognition and repair of DNA alkylation damage is particularly important as alkylation chemotherapy is one of the most widely used systemic modalities for cancer treatment and environmental chemicals may trigger DNA alkylation. Here, I demonstrate that human cells have a previously unrecognized signaling mechanism for sensing DNA damage induced by alkylation. The ALKBH3-ASCC alkylation repair complex (consisting of the dealkylase ALKBH3 and the ASCC complex subunits ASCC1, ASCC2, and ASCC3) relocalizes to distinct nuclear foci specifically upon exposure of cells to alkylation agents. These foci associate with alkylated nucleotides and are coincident with elongating RNA Polymerase II and other splicing components. Proper recruitment of the complex requires K63-polyubiquitin recognition via the CUE (coupling ubiquitination to ER degradation) domain of the subunit ASCC2. The E3 ligase RNF113A is responsible for the upstream ubiquitin signaling necessary to recruit the repair complex. Conversely to ASCC2 and ASCC3, the subunit ASCC1 is present at nuclear speckles prior to alkylation but leaves in response to damage. Upon loss of ASCC1, ASCC3 foci significantly increase upon alkylation damage, suggesting a negative regulatory function for ASCC1. Indeed, ASCC1 appears to coordinate the proper recruitment of the ASCC complex in response to alkylation in a manner dependent on its putative RNA-binding motif. Interestingly, expression of an AlkB homologue from the blueberry scorch RNA virus containing an NLS (nuclear localization signal) fusion was sufficient to significantly reduce HA-ASCC2 foci during alkylation damage. Overexpression of the RNA-specific methyltransferase METTL8, which produces m3C on mRNA, with an NLS fusion was sufficient to induce recruitment of ASCC3 to the nucleus, and primarily the nucleolus, even in the absence of alkylating agents, suggesting that RNA alkylation is both necessary and sufficient to recruit the ALKBH3-ASCC repair complex. Together, these findings significantly contribute to the notion that human cells have specialized DNA repair mechanisms and that these mechanisms can also repair damaged RNA.
Chair and Committee
Susana Gonzalo, Albert Kim, Alessandro Vindigni, Zhongsheng You,
Brickner, Josh, "Activation and Regulation of the ALKBH3-ASCC Alkylation Repair Pathway" (2019). Arts & Sciences Electronic Theses and Dissertations. 1991.
Available for download on Sunday, November 08, 2020