The recognition of DNA 5-methylcytosine: Studies on Arabidopsis thaliana DNA glycosylase ROS1 and basic leucine-zippertranscription factors in human and Epstein-Barr virus Pubblico

Abstract

Eukaryotic DNA methylation, often a chemical modification of cytosine via methylation of the carbon-5, generates 5-methylcytosine (5mC) in genomes. This modified base serves as a critical epigenetic signal implicated in development, imprinting, immune responses, and various forms of diseases. Characterizing how DNA 5mC is recognized and regulated is critical to effectively understanding the function of DNA methylation. Previous investigations have shown that the base excision repair pathway can regulate active DNA demethylation--the enzyme-driven process of erasing and thus reversing the methyl modification signal. Particularly, Repressor of Silencing 1 (ROS1) and its paralogs in Arabidopsis thaliana can directly excise 5mC to reverse DNA methylation. A major portion of this dissertation describes the molecular mechanism of ROS1 activity. Specifically shown is the interaction between the C-terminal domain and the catalytic domain of ROS1, and the requirement of the C-terminal domain for the 5mC excision activity. This understanding expands the paradigm of DNA repair enzymes from their traditionally understood housekeeping roles to their extended roles in epigenetic regulations. In addition to the discoveries on how DNA 5mC is erased, understanding how proteins specifically recognize this modified base is also critical. It is widely generalized that 5mC is inhibitory for transcription factor binding. However, recent data show that certain transcription factors can preferentially recognize 5mC within specific sequences. As a major extension to this discovery, the other major portion of this dissertation describes the DNA sequence-specific recognition of methylated DNA by human AP-1 and Epstein-Barr virus AP-1-like transcription factors. The study provides the biochemical and structural basis of how DNA methylation can generate novel transcription factor binding sites to dynamically regulate transcription.

Table of Contents

LIST OF ABBREVIATIONS. iii

LIST OF FIGURES. vii

LIST OF TABLES. ix

CHAPTER I. General Introduction. 1

DNA methylation. 1

Oxidative modifications of 5-methylcytosine and active DNA demethylation. 5

Sequence-specific recognition of 5-methylcytosine by transcription factors.11

CHAPTER II. The carboxyl-terminal domain of ROS1 is essential for 5-methylcytosine DNA glycosylase activity. 15

Abstract. 15

Introduction. 17

Results. 19

ROS1 glycosylase domain and the C-terminal domain. 19

ROS1 glycosylase domain and the C-terminal domain associate tightly. 24

Mouse MYH does not possess 5-methylcytosine DNA glycosylase activity. 30

Discussion. 36

Materials and Methods. 40

Protein Expression and Purification. 40

DNA glycosylase activity assay. 41

Acknowledgements. 43

CHAPTER III. Structural basis of methylated DNA recognition by human AP-1 and Epstein-Barr virus Zta transcription factors. 44

Abstract. 44

Introduction. 45

Results. 47

Overall structures. 47

Response elements containing asymmetric half-sites. 50

Position-specific pyrimidine C5-methyl group recognitions: "T-to-5mC switch". 54

Methyl-dependent binding in solution. 58

Effects of oxidative modifications on DNA binding. 62

Resolving the difference of asymmetric half-sites. 65

Discussion. 71

Materials and Methods. 73

Protein Expression and Purification. 73

Crystallography. 74

Fluorescence-based DNA binding Assay. 75

CHAPTER IV. Discussions and Future Directions. 78

Comparison of 5-methylcytosine and thymine. 78

Role of 5-methylcytosine-binding transcription factors. 81

The recognition of oxidative modifications. 82

Future directions for ROS1. 84

APPENDIX.. 87

REFERENCES. 102

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Molecular & Systems Pharmacology
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Chemistry, Biochemistry
Parola chiave	DNA methylation DNA demethylation 5-methylcytosine ROS1 Zta AP-1
Committee Chair / Thesis Advisor	Cheng, Xiaodong, Emory University
Committee Members	Shim, Hyunsuk, Emory University Doetsch, Paul, Emory University Ortlund, Eric, Emory University

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