Exploring the Relationship of Abasic Sites to Reactive Oxygen Species Generation in a Yeast Model System Público

Abstract

Abasic (AP) sites, the common intermediate in single-base DNA damage/repair, are among the most frequently formed lesions in DNA. DNA damage accumulation may lead to mutations deleterious to health, contributing to carcinogenesis, neurodegeneration, and aging. In Saccharomyces cerevisiae (budding yeast), the amount of intracellular reactive oxygen species (ROS) increases when DNA damage is chemically induced. Base excision repair (BER) and nucleotide excision repair (NER) pathways are crucial in DNA repair (including AP site removal), and cells with deficient BER show an increase in both spontaneous DNA damage accumulation and intracellular ROS level. We hypothesize that, as an abundant common intermediate, abasic site may be responsible for originating genotoxic stress signaling via ROS generation. The yeast DNA glycosylase Ung1 creates abasic sites by uracil excision. Knockout of Ung1 should prevent uracil (incorporated into DNA/spawned from cytosine deamination) from being excised, thereby reducing the level of abasic sites present. If the level of abasic sites positively correlates with ROS generation, Ung1 knockout should lead to a reduction of mutation frequency due to abasic site's mutagenic nature, alongside a decrease in intracellular ROS level. Cells that are completely deficient in BER, as well as those impaired in both BER and NER, showed such correspondence, providing evidence suggesting that abasic sites are responsible for DNA-damage-induced ROS response. To further determine the exact source of ROS generation, we postulate that the ROS response may be compartmentalized, since many enzymes in the BER pathway (including Ung1) are capable of localizing to either the nucleus or mitochondria upon induced DNA damage. Ung1 mutants that selectively localize to either the nucleus or mitochondria were created, and the ROS generation observed was similar to that of Ung1 knockout, indicating that dynamic localization may be required for the ROS response to be maintained.

Table of Contents

BACKGROUND.................................................................................................................. 1

MATERIALS AND METHODS................................................................................................ 5

RESULTS........................................................................................................................ 8

DISCUSSION................................................................................................................... 11

FIGURES

1. Model of DNA damage-induced ROS response.................................................................... 17

2. Putative Ung1 localization sequences and mutations employed............................................. 18

3. Multi-sample overlay of flow cytometry histograms............................................................. 19

4. Distribution fitting of flow cytometry data........................................................................ 20

5. Heating DNA negatively affects the ARP assay.................................................................. 21

6. Ung1 knockout reduces spontaneous mutation frequency.................................................... 22

7. ung1Δ hDNP119 isolates display lowered ROS generation..................................................... 23

8. Correlation between spontaneous mutation frequency and ROS............................................ 24

9. ROS elevation in apn1Δ isolate is independent of Ung1........................................................ 25

10. Similar ROS levels observed in Ung1 localization mutants................................................... 26

11. Partial diagram of yeast BER pathway............................................................................ 27

TABLES

1. Strains and plasmids used............................................................................................. 28

2. ARP assay shows signal elevation in controls.................................................................... 30

3. ANOVA indicates significant effects of ung1Δ.................................................................... 31

FIGURES AND TABLE LEGENDS........................................................................................... 32

REFERENCES.................................................................................................................. 34

About this Honors Thesis

Rights statement

• Permission granted by the author to include this thesis or dissertation in this repository. All rights reserved by the author. Please contact the author for information regarding the reproduction and use of this thesis or dissertation.

School	Emory College
Department	Biology
Degree	BS
Submission	Honors Thesis
Language	English
Research Field	Biology, Genetics Chemistry, Biochemistry Biology, Microbiology
Palabra Clave	Base excision repair Saccharomyces cerevisiae Flow cytometry DNA repair Abasic sites Reactive oxygen species
Committee Chair / Thesis Advisor	Doetsch, Paul, Emory University
Committee Members	Kelly, William G, Emory University Kow, Yoke Wah, Emory University Orloff, Gregg, Emory University

Última modificación

Primary PDF

Thumbnail	Title	Date Uploaded	Actions
	Exploring the Relationship of Abasic Sites to Reactive Oxygen Species Generation in a Yeast Model System ()	2018-08-28 10:38:01 -0400	Press to Select an action Download

Supplemental Files

Thumbnail	Title	Date Uploaded	Actions