The Regulation of Base Excision Repair: The Effects of Sumoylation on Ntg1 Function Público
Mehdi, Syed Kaunain (2013)
Abstract
Unrepaired oxidative damage to DNA leads to the development of cancer and other degenerative diseases. The cellular mechanism base excision repair (BER) ensures proper repair of damaged DNA in both the nucleus and mitochondria. Unfortunately, not a great deal of information is known about the regulation of this crucially important mechanism. The BER protein of Saccharomyces cerevisiae, Ntg1, which is post-translationally modified by sumoylation, dynamically localizes to either the nucleus or mitochondria upon cellular increases in oxidative DNA damage. While the phenomenon of dynamic localization is understood, the mechanism of Ntg1 regulation in repairing a diverse range of lesions in the nucleus and mitochondria is unknown. I propose that sumoylation of Ntg1, by a small ubquitin like modifying protein (SUMO), will regulate Ntg1 function by driving dynamic localization to areas of oxidative DNA damage, and/or altering its enzymatic activity. In order to observe whether sumoylation of Ntg1 altered its dynamic localization and/or its enzymatic activity, it was first necessary to produce artificial SUMO fusion proteins. Localization of one fusion protein was observed under standard growth conditions and upon introduction of oxidative DNA damage using fluorescence microscopy. Enzymatic activity of the other fusion protein, when introduced to a dihydrouracil substrate, was observed using an enzyme assay. Our results indicate sumoylated Ntg1 shares the same localization pattern as wild type Ntg1 under standard growth conditions and upon introduction to oxidative DNA damage. Similarly, the Ntg1 SUMO fusion possesses comparable enzymatic activity with wild type Ntg1 towards a dihydrouracil containing substrate. Overall, these results are significant in the understanding of the role of sumoylation in the regulation of a vital DNA repair mechanism in base excision repair. Further study of this sumoylation regulation pathway is necessary if we are to understand how base excision repair is not only regulated in Saccharomyces cerevisiae, but ultimately in humans as well. By studying and understanding the regulation of BER in yeast, we will be able to further understand how DNA repair is regulated in humans, and possibly discover new methods of fighting cancer.
Table of Contents
Table of Contents
BACKGROUND.......................................................................................................................1
MATERIALS AND METHODS..............................................................................................4
RESULTS..................................................................................................................................8
DISCUSSION...........................................................................................................................12
REFERENCES.........................................................................................................................17
FIGURES AND TABLES LEGENDS...................................................................................19
TABLES
1. Strains and Plasmids..............................................................................................................24
2. Averaged Results of Localization Scoring by Visualization..................................................24
FIGURES
1. Model of the base excision repair
pathway............................................................................25
2. Model of Sumoylation...........................................................................................................25
3. Locations of Putative Consensus SUMO Modification Sites on Ntg1...................................26
4. Hypothetical Model of Sumoylated Ntg1 Localization and Enzymatic Activity....................26
5. Localization of Ntg1-GFP and Ntg1-Smt3-GFP Under Standard Growth Conditions..........27
6. Averaged Results for Localization Scoring by Visualization and Localization
Scoring by Q-SCAn program....................................................................................................27
7. Western Blot of Ntg1-Smt3-His and Controls with and without IPTG Induction.................28
8. SDS Gel of Nickel and FPLC Purifications of Ntg1-Smt3-His6 and Controls......................29
9. Western Blot of Nickel and FPLC Purifications of Ntg1-Smt3-His6.....................................30
10. SDS Gel of Ntg1-Smt3-His6 After Adjusting Purification...................................................30
11. Western Blot of Ntg1-Smt3-His6 After Adjusting Purification ............................................31
12. Western Blot of Modified Purifications of Ntg1-Smt3-His6 and Controls............................32
13. Typhoon Image of Elution 1 and Elution 1 Concentrate of Ntg1-Smt3-His6
and Controls................................................................................................................................33
14. Typhoon Image of Ntg1-Smt3-His6 Enzymatic Activity Assay...........................................33
15. Quantification Results of Ntg1-Smt3-His6 Enzymatic Activity Assay..................................34
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