Characterization of O6-Alkylguanine-DNA Alkyltransferase Degradation and Labeling with Fluorophores
Open Access
- Author:
- Lewis, Candice
- Graduate Program:
- Genetics
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- September 24, 2010
- Committee Members:
- Kristin Eckert, Dissertation Advisor/Co-Advisor
Kristin Eckert, Committee Chair/Co-Chair
Dr Sreenivas Kanugula, Committee Chair/Co-Chair
Hui Ling Chiang, Committee Member
Ira Joseph Ropson, Committee Member
Robert G Levenson, Committee Member - Keywords:
- DNA repair
AGT
O6-Benzylguanine - Abstract:
- O6-Alkylguanine-DNA alkyltransferase (AGT) is a highly conserved DNA repair protein, that removes alkyl adducts at the O6-position of guanine in a single irreversible, stoichiometric reaction. AGT is unique among all the other DNA repair pathways as it requires no co-factors or chaperones to carry out DNA repair. O6-Alkylguanine is a highly cytotoxic lesion, and when left un-repaired, leads to G:C to A:T transition mutations. Upon alkyl transfer, AGT continues to bind to DNA and could impede other AGT molecules from repairing DNA. Therefore, degradation might be the only mechanism to remove the alkylated AGT. To study human AGT (hAGT) degradation and intracellular dynamics, we created a Green Fluorescent Protein (GFP)-hAGT fusion and expressed this tagged AGT in Chinese Hamster Ovary (CHO) cells. GFP-hAGT expressing CHO cells enabled Fluorescent Recovery After Photobleaching (FRAP) experiments showing for the first time, the rapid mobility of AGT within the nucleus. When the entire nucleus was bleached, recovery of fluorescence occurred only after 30 minutes, indicating that AGT accumulates in the nucleus over long periods of time, probably soon after synthesis. The addition of GFP at the N-terminus of hAGT changed some physiological features of hAGT. The half life of the alkylated protein changed from 9h for the native form to 26h for the GFP-tagged form. However, the formation of putative Ub-GFP-AGT species was similar to the ubiquitination pattern and time frame, seen in wt hAGT expressed in CHO cells. This finding suggests that the N-end rule pathway for ubiquitin-mediated degradation may not be the only proteolytic pathway regulating degradation of hAGT. Fluorophores were used to characterize a novel 16kDa AGT truncation species detected in both CHO and HeLa cells. The cleavage site of this species was determined to be at codon 50, causing the elimination of three of the four ligands required to bind a zinc atom and maintain structural stability. Despite the truncation of the N-terminus, this 16kDa hAGT species showed significant protection from N-Methyl-N’-Nitro-N-Nitrosoguanidine (MNNG) induced cytotoxicity in Escherichia coli (E.coli). Together with purified protein AGT activity assays, these data indicate that this truncated AGT species retains the ability to repair DNA. This intriguing finding suggests that this truncated species is either part of a previously unknown degradation cycle, or it has a unique role in the cytosol. The biological role of AGT is to protect cells from alkylation damage generated from endogenous as well as environmental sources. However, as a consequence of its biological role, hAGT plays an important part in tumor cells by causing resistance to some chemotherapeutic drugs such as temozolomide or BCNU, by neutralizing the drug induced alkylation damage. Therefore, in order to combat this effect, a series of AGT inhibitors were created previously to inactivate hAGT and increase the efficacy of chemotherapy. O6-Benzylguanine (O6-BG) is one such, potent inhibitor of AGT, which is currently in clinical trials. In this dissertation, a series of mutant hAGT proteins were created to test for increased sensitivity to O6-BG. Three codons at sites 157, 159 and 160 were targeted because of their location within the O6-BG binding pocket. Our studies confirm that these sites, particularly, a tryptophan at position 160, contribute to a ten-fold lowering of O6-BG ED50. These mutants are unique and rare because a majority of previously characterized AGT mutations within the O6-BG binding pocket cause resistance to O6-BG.