Elucidating Mechanisms of Base Excision Repair and Genetic Instability in Saccharomyces cerevisiae Pubblico

Morris, Lydia Patrice (2012)

Permanent URL: https://etd.library.emory.edu/concern/etds/np1939585?locale=it
Published

Abstract

A large subset of DNA damage acquired by cells is repaired by the base excision repair
(BER) pathway. Though defects in many BER genes have been associated with
neurodegenerative diseases and cancer, the molecular basis for such associations is not well
understood. Further, when cells cannot repair oxidative DNA lesions normally targeted
goals of the studies presented
here are to better understand BER mechanisms at the level of individual proteins and on the
genome-wide level. We employed Saccharomyces cerevisiae because the biochemical steps
of BER are highly conserved, and S. cerevisiae is a well developed model for DNA repair
studies. AP endonucleases play a central role in the repair of DNA damage through the BER
pathway, thus our studies focus on the major yeast AP endonuclease, Apn1, to better
understand how BER protects cells against genomic instability, an important characteristic of
cancer.
In an unbiased, forward genetic screen to identify mutations in APN1 that impair cellular
DNA repair capacity we identified and characterized variant Apn1 V156E, which was
predicted to decrease catalytic function based on homology modeling. We found that, unlike
wild type Apn1, the V156E is targeted for degradation by a proteasome-independent
mechanism, leading to decreased steady-state levels. Inducing transcription of APN1-V156E
using a regulatable promoter restored protein to levels comparable to wild type Apn1 and
functionally restored DNA repair capacity. Thus, the V156 residue plays a critical role in
maintaining Apn1 protein levels and normal levels of repair independent of catalytic
function.
In genome-wide chromatin immunoprecipitation studies aimed at exploring the relationship
between DNA damage repair and genomic instability using Apn1 as the target protein, we
found that the level of oxidative stress dictates the distribution of Apn1 across the genome.
Regardless of oxidative stress level, Apn1 binding sites are enriched for C and G nucleotides,
suggesting that Apn1 targets particular regions in a base content-specific manner. These
results have implications for understanding how the genomic distribution of DNA repair
activities preserves genome integrity and for understanding how defects in the major human
AP endonuclease may contribute to disease.

Table of Contents


TABLE OF CONTENTS

Chapter 1

General Introduction 1


References 22


Chapter 2
Saccharomyces cerevisiae Apn1 Mutation Affecting Stable Protein Expression Mimics
Catalytic Activity Impairment: Implicationsfor Assessing DNA Repair Capacity in Humans 55

Abstract 56


Introduction 57

Materials and Methods 60

Results 69

Discussion 79

References 83


Chapter 3
Apn1 Localizes to Sites for Prioritized Repair of Oxidative DNA Damage in
Saccharomyces cerevisiae 112

Abstract 113

Introduction 114

Materials and Methods 118

Results 122

Discussion 126

References 130


Chapter 4

Discussion and Future Directions 143

References 164



FIGURES AND TABLES

Chapter 1

General Introduction 1

Table 1
Base excision repair genes from bacteria, yeast and humans 47

Figure 1
Target sites for intracellular DNA decay 48

Figure 2
Examples of base lesions caused by DNA damaging agents 50

Figure 3
Schematic representation of the base excision repair pathway 52

Figure 4
Processing of oxidative and spontaneous damage in Saccharomyces cerevisiae 54


Chapter 2

Saccharomyces cerevisiae Apn1 Mutation Affecting Stable Protein Expression

Mimics Catalytic Activity Impairment: Implicationsfor Assessing

DNA Repair Capacity in Humans 55

Table 1
Genotypes of strains used in this study 92

Figure 1
Amino acid alignment of E. coli endo IV and S. cerevisiae Apn1 93

Figure 2
MMS sensitivity of apn1 mutant strains 94

Figure 3
Homology modeling of Apn1 96

Figure 4
Measurement of AP site incision activity in cell lysates containing Apn1 variant proteins 98

Figure 5
Quantification of endogenous Apn1 protein and APN1 mRNA levels 100

Figure 6
Thermostability and degradation of Apn1 variant proteins 102

Figure 7
Apn1 V156E overexpression functionally restores cellular DNA repair activity 106

Table S1
Plasmids used in this study 109



Table S2
Mutations in APN1 identified in random mutagenesis screen 110

Figure S1
MMS sensitivities of strains containing C-terminal TAP-tagged versions of APN1

wild type and mutants 111


Chapter 3
Apn1 Localizes to Sites of Prioritized Repair
of Oxidative DNA Damage in Saccharomyces cerevisiae 112

Table 1
GC content in Apn1 binding peaks 135

Table 2
Intragenic and intergenic content in Apn1 binding peaks 136

Table 3
Apn1 binding peaks overlapping oxidative stress-related fragile sites 137

Figure 1
H2O2-induced cytotoxicity analysis 138

Figure 2
Characteristics of Apn1 binding peaks 139

Figure 3
Model: Apn1 genomic occupancy 141


Chapter 4
Discussion and Future Directions 143

Figure 1
Apn1 protein structure 172

Figure 2
Model: Apn1 genomic occupancy 174










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