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«by Zhuobin Liang A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Molecular, Cellular, and ...»

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REJOINING OF DNA DOUBLE-STRAND BREAKS AND GENOME STABILITY:

FROM HOST-PATHOGEN INTERACTIONS TO BREAK-INDUCED MUTAGENESIS

by

Zhuobin Liang

A dissertation submitted in partial fulfillment

of the requirements for the degree of

Doctor of Philosophy

(Molecular, Cellular, and Developmental Biology)

in the University of Michigan

2015

Doctoral Committee:

Associate Professor Thomas E. Wilson, Co-Chair Associate Professor Lyle Simmons, Co-Chair Associate Professor Anuj Kumar Professor Jianming Li Associate Professor JoAnn Sekiguchi © Zhuobin Liang All Right Revered 2015

DEDICATION

To my beloved wife, parents, parents-in-law and brother ii

ACKNOWLEDGMENTS

The completion of my dissertation research would not be possible without help and support from a number of people. First, I would very much like to thank my graduate mentors Dr. Thomas Wilson and Dr. Tzvi Tzfira for the opportunities and patient guidance they provided.

They instilled in me the passion and spirit of science and taught me valuable techniques for becoming an independent investigator. My sincere thanks also go to great lab colleagues whom I closely collaborated with, especially Dr. Kishore Chiruvella, Siva Nallasivam, Dr. Yoel Shiboleth and Vardit Zeevi.

Next, I am extremely grateful to the members of my thesis committee, Dr. Lyle Simmons, Dr. Anuj Kumar, Dr. Jianming Li and Dr. JoAnn Sekiguchi, for their constant support and valuable suggestions. I also greatly benefited from discussions with a number of labs including but not limited to the Mats Ljungman, Xiaochun Yu, Thomas Glover, David Ferguson, Christine Canman and Patrick O'Brien labs.

I also want to express my thanks to the departments of MCDB, Pathology, Human Genetics, Rackham Graduate School and International Center in the University of Michigan for their assistance throughout my graduate study.

Finally I want to offer my deepest gratitude to my many friends and family members for their companion and encouragement.

iii

PREFACE

This dissertation consists of the research projects I conducted in the former lab of Dr.

Tzvi Tzfira in the University of Michigan from Sept 2008 to April 2011 and in Dr. Thomas E Wilson lab from May 2011 to present. The overall objective is to investigate the complex contribution of rejoining of DNA double-strand breaks to genome stability Parts of Chapter 1 were modified from an invited book chapter of Repair, Mutagenesis and Other Responses to DNA Damage published in Cold Spring Harb Perspect Biol 2013 May;5(5): a012757 (co-first author), and from another invited review in preparation (first author).

All the contents in Chapter 2 have been published in Nat Commun 4:2253 doi:

10.1038/ncomms3253 (first author).

All the contents in Chapter 3 have been published in Plant Physiol 2012 Jan;158(1): 132co-first author), and in Nat Biotechnol 2011 Dec;29(12): 1072-4 (middle author).

All the contents in Chapter 4 have been published in PLoS Genet 2013 June;9(6):

e1003599 (second author).

Chapter 5 is currently in preparation and will submit shortly (first author).

–  –  –

DEDICATION…………………………………………………………………………………...ii ACKNOWLEDGEMENTS…………………………………………………………………….iii PREFACE……………………………………………………………………………….……….iv LIST OF FIGURES…………………………………………………………………….……….ix LIST OF TABLES……………………………………………………………………………...xii LIST OF ABBREVIATIONS…………………………………………………………………xiii ABSTRACT………………………………………………………………………………….....xiv CHAPTER

1. Introduction…………………………………………………………………………………..1 Repair of DNA double-strand breaks by end-joining……………………….……...…….........1

1.1 Canonical NHEJ core proteins…………………………………………………..…….2

1.2 Alternative NHEJ……………………………………………………………...………6

1.3 Disposition of DSBs between c-NHEJ and HR……………………....………………..8 T-DNA integration into genomic double-strand breaks…………………….……...….……..10

1.4 Gene targeting in plants………………...…………………………….……..………..10

1.5 Voyage of Agrobacterium T-DNA in the host cell………………….………………..11

1.6 DNA repair and T-DNA integration………………………………….……..………..12 Outline of subsequent chapters…………………………………………………....………….15 v Figures…………………………………………………………………………….…….……16 Tables………………………………………………………………………..……………….25 References……………………………………………………………………………………27





2. In Vivo Formation of Double-Stranded T-DNA Molecules by T-Strand Priming…....…41 Abstract……………………………………………………………………………………....41 Introduction………………………………………………………………………………..…42 Materials and Methods…………………………………………………………………….....44 Results………………………………………………………………………………………..48 Discussion…………………………………………………………………………………....57 Acknowledgements………………………………………………………………………..…59 Figures………………………………………………………………………………….….…60 References………………………………………………………………………………..…..65

3. Assembly of Multigene Vectors for Agrobacterium-Mediated Plant Transformation and Generation of the Potent Anti-Malarial Drug Artemisinin in Tobacco…………………68 Abstract…………………………………………………………………………..…………..68 Introduction………………………………………………………………………..…………70 Materials and Methods…………………………………………………………..…………...76 Results……………………………………………………………………………..…………83 Discussion…………………………………………………………………………..………..92 Acknowledgements…………………………………………………………………………100 Figures………………………………………………………………………………………101 vi Tables……………………………………………………………………………………….109 References…………………………………………………………………………………..111

4. Saccharomyces Cerevisiae DNA Ligase IV Supports Imprecise End Joining Independently of Its Catalytic Activity…………………………………………….…….120 Abstract……………………………………………………………………………………..120 Introduction…………………………………………………………………………………122 Materials and Methods……………………………………………………………………...125 Results………………………………………………………………………………………131 Discussion…………………………………………………………………………………..142 Acknowledgements…………………………………………………………………………147 Figures………………………………………………………………………………………148 Tables……………………………………………………………………………………….161 References…………………………………………………………………………………..164

5. Overhang Polarity of Chromosomal Double-Strand Breaks Impacts Kinetics and Fidelity of Yeast Nonhomologous End Joining…………………………….……..…..…169 Abstract……………………………………………………………………………………..169 Introduction…………………………………………………………………………………171 Materials and Methods……………………………………………………………………...173 Results………………………………………………………………………………………179 Discussion…………………………………………………………………………………..197 Acknowledgements…………………………………………………………………………203 vii Figures………………………………………………………………………………………204 Tables……………………………………………………………………………………….216 References…………………………………………………………………………………..217

6. Concluding Remarks……………………………………………………………………...223 Summary of results………………………………………………………...……….……….223 Study the genetic requirements for T-strand conversion and its kinetics in yeast…….…….224 Study the molecular mechanisms of break-end structure recognition in yeast……….……..227 Figures……………………………………………………………………………………....229 References…………………………………………………………………………………..231

–  –  –

Figure 1-1. Ku and DNA-PKcs…………………………………………………………….……..16 Figure 1-2. DNA ligase IV assembly………………………………...……………….….……….17 Figure 1-3. Mre11-Rad50-Nbs1 (MRN) complex…………………………...……….….……….18 Figure 1-4. PolX polymerases…………………………………………………………….......….19 Figure 1-5. Disposition of DSBs between repair pathways………………………………..……...20 Figure 1-6. Transportation of the T-DNA from Agrobacterium to the host nucleus……….…….21 Figure 1-7. Hypothetical models of T-DNA integration to the host genome………………...……22 Figure 1-8. The NHEJ-mediated integration machinery for T-DNA integration…..…….…...…..23 Figure 1-9. The HR-mediated integration machinery for T-DNA integration…………..…....…..24 Figure 2-1. Detection of T-strand conversion to dsT-DNA by in-vivo annealing of complementary T-strands…………………………..……………………..………….60 Figure 2-2. Detection of T-strand conversion to dsT-DNA by site-specific mutagenesis…...……61 Figure 2-3. Confocal fluorescence microscopy analysis of in vivo formation of dsT-DNAs.........62 Figure 2-4. In vivo priming of T-strand molecules by synthetic primers………………….….…..63 Figure 2-5. Hypothetical model of dsT-DNA formation in plant cells……………….……….…..64 Figure 3-1. General features of ZFN- and homing-endonuclease-mediated multigene binary vector assembly system……………………….……………………….….……...…101 Figure 3-2. Multiple gene expression in protoplasts from triple- and quadruple-gene long binary vectors……………………………………………………………………..………..102

–  –  –

Figure 3-4. Phenotypic analysis seven-transgene-long transgenic Arabidopsis plants…......…….103 Figure 3-5. Phenotypic analysis of nine-transgene-long transgenic Arabidopsis plants……..…...104 Figure 3-6. PCR analysis of T-DNA inserts in offspring of transgenic Arabidopsis plants….…...105 Figure 3-7. The pathway and constructs for engineering artemisinin production in tobacco........106 Figure 3-8. Characterization of transgenic tobacco and subcellular localization of ADS and mtADS…………………………………………………………………..…………..107 Figure 3-9. Production of artemisinin in engineered tobacco plants……………….……….…...108 Figure 4-1. Dnl4 mutations under study…………………………………………………….…..148 Figure 4-2. Dnl4 catalytic mutations severely impair auto-adenylation and c-NHEJ efficiency…………………………………………………………..………........…..149 Figure 4-3. Extensive recruitment of catalytically inactive Dnl4 and associated c-NHEJ factors to a chromosomal DSB………………………………………………….………...…..150 Figure 4-4. Catalytically inactive Dnl4 protein does not impede DSB resection……….….……151 Figure 4-5. Residual NHEJ in the chromosomal suicide deletion assay with catalytically defective Dnl4………………………………………………………………..……..152 Figure 4-6. End joining profiles after extensive HO recleavage depend on Dnl4 status…..…......153 Figure 4-7. Cdc9 recruitment to a chromosomal DSB during the NHEJ repair phase……......….154 Figure 4-8. DSB resection is inefficient in the absence of a fermentable carbon source….…......155 Figure 4-9. DSB repair by homologous recombination in Dnl4 catalytic mutant strains….….…156 Figure 4-10. Imprecise joints in the suicide deletion assay…………………………...….……...157

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