Date of Award

Spring 5-15-2019

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Molecular Genetics & Genomics)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

DNA alkylation damage is caused by various agents that are present in the environment as well as cellular metabolism and can be induced by certain chemotherapeutic agents. Thus, the repair of damaged DNA is critical for genomic maintenance. The ALKBH family of proteins plays a central role in the repair of specific alkylated lesions, including 1-methyladenine (1meA) and 3-methylcytosine (3meC). A major outstanding question in the field of alkylation repair is the role of associated protein partners in the function of the human AlkB homologues. Here, I demonstrate that the ALKBH3 associated complex ASCC (comprised of ASCC1, ASCC2, and ASCC3) localizes to distinct nuclear foci specifically upon exposure of cells to alkylating agents. These foci associate with alkylated nucleotides and coincide spatially with elongating RNA polymerase II and splicing components. Recruitment of the repair complex to sites of alkylation damage requires recognition of K63-linked polyubiquitin by the CUE (coupling of ubiquitin conjugation to ER degradation) domain of ASCC2. Mutation of ASCC2 to prevent the binding of K63-linked polyubiquitin results in the impediment of alkylation adduct repair kinetics and increased sensitivity to alkylating agents. This work identifies a previously unrecognized ubiquitin-dependent pathway induced specifically to repair alkylation damage. The ASCC1 subunit is also critical for the regulation of this repair complex. ASCC1 is present at nuclear speckles prior to damage but leaves the foci in response to alkylation. ASCC1 loss significantly increases ASCC3 foci formation during alkylation damage, indicating a negative regulatory function. The research presented herein suggest that ASCC1 coordinates the proper recruitment of the ASCC complex during alkylation, a function that appears to depend on a putative RNA-binding motif near the ASCC1 C-terminus. Together, these findings contribute significantly to the identification of regulators of alkylation damage repair in human cells.

Language

English (en)

Chair and Committee

Nima Mosammaparast

Committee Members

Peter Burgers, Shelia Stewart, Luis Batista, Hani Zaher,

Comments

Permanent URL: https://doi.org/10.7936/60vz-vg41

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