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Protein Design of the Mammalian DNA Methyltransferase Dnmt3a

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Protein Design of the Mammalian DNA Methyltransferase Dnmt3a Protein design of the mammalian DNA methyltransferase Dnmt3a by Abu Nasar Siddique A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Molecular Biotechnology Approved, Thesis Committee _______________________ Prof. Dr. Albert Jeltsch _______________________ Prof. Dr. Sebastian Springer _______________________ Prof. Dr. Marianne G. Rots Date of Defense: May 25, 2011 School of Engineering and Science Jacobs University Bremen 1 I. ACKNOWLEDGMENTS I would like to express my deepest gratitude to my supervisor Prof. Dr Albert Jeltsch for his excellent guidance into the Science of Biochemistry, for his encouragement and his enthusiasm, which has always been the source of my inspiration and motivation. I am thankful to Prof. Sebastian Springer for being the co-referee of my PhD thesis, and especially to Prof. Marianne G. Rots, who has traveled a long way to be a part of my thesis committee. I would like to express my appreciation to my Laboratory supervisor Dr. Tomasz Jurkowski for his constructive discussions, excellent commentaries and suggestions. I am grateful to all my lab fellows, especially to Renata, Arumugam, Sanjay, Arun as well as to our neighbor Abhishek for creating a friendly working atmosphere and many useful advices. I would also like to thank my friends: Farhan, Amna, Farhat, Nawab, Nadia, Sadiq, Noorshad, Aasim, Wakeel, Guftaar, Qazi, Imran, Ihteram, Ali, Tariq, Noor Muhammad, Zia, Naveed, Shoaib, Tahir and Amir, whom I will not enumerate due to the lack of space. Thank you for bringing so much fun, support and distraction into my life. I am also thankful to the Higher Education Commission (HEC) in Pakistan and Deutscher Akademischer Austauschdienst (DAAD) in Germany, for providing financial support and help in every aspect during PhD studies. Lastly, but not at all least, I am thankful to my family members especially my wife, brother, sisters and my mother for giving me moral support, when my frustration overbalanced my passion. 2 Declaration The work described in this thesis is my own work, unless otherwise stated or mentioned in the references. The thesis was written by me and nobody else. Abu Nasar Siddique Bremen, May 2011. 3 II. LIST OF PUBLICATIONS Jurkowska RZ*, Siddique AN*, Jurkowski TP, Jeltsch A. Approaches to Enzyme and Substrate Design of the Murine Dnmt3a DNA Methyltransferase. ChembioChem. 2011, in press * Both authors contributed equally to this work (In this paper I performed all the experiment related to the directed evolution of Dnmt3a) Siddique AN, Jurkowska RZ, Jurkowski TP, Jeltsch A. Auto-methylation of the mouse DNA-(cytosine C5)-methyltransferase Dnmt3a at its active site cysteine residue. FEBS J. 2011, in press (In this paper I performed all the experiments) 4 TABLE OF CONTENTS I. ACKNOWLEDGEMENT-------------------------------------------------------------------------II II. LIST OF PUBLICATIONS-----------------------------------------------------------------------4 III. TABLE OF CONTENTS--------------------------------------------------------------------------5 IV. ABSTRACT-----------------------------------------------------------------------------------------6 2. INTRODUCTION-----------------------------------------------------------------------------------7 2.1. Epigenetics--------------------------------------------------------------------------------7 2.1.1 DNA methylation in mammals---------------------------------------------------------------7 2.1.2 Importance of DNA methylation in mammals-------------------------------------------8 2.2. Types of mammalian DNA methyltransferases--------------------------------9 2.2.1 Catalytic mechanism of DNA methylation in mammals---------------------------11 2.2.2 Dnmt3 family-------------------------------------------------------------------------------------13 2.2.3 Structure of Dnmt3a/3a-3L------------------------------------------------------------------14 2.3. Aim of the project - to increase the activity of Dnmt3a-------------------15 2.3.1 Protein Engineering---------------------------------------------------------------------------17 2.3.2 Directed enzyme evolution------------------------------------------------------------------17 3. RESULTS AND DISCUSSION---------------------------------------------------------------19 Directed evolution of the mammalian DNA methyltransferase Dnmt3a towards higher activity------------------------------------------------------------------------22 Auto-methylation of the mouse DNA methyltransferase Dnmt3a-C-----------25 4. REFERENCES------------------------------------------------------------------------------------28 Supplement 1 Supplement 2 Supplement 3 5 IV. Abstract The catalytic domain of the Dnmt3a DNA-(cytosine C5)-methyltransferase (Dnmt3a-C) is active in isolated form but like full-length Dnmt3a it shows only weak DNA methylation activity. To improve this activity by directed evolution, we set up a selection system in which Dnmt3a-C methylate its own expression plasmid in E. coli and protect it from cleavage with methylation specific restriction enzymes. However, despite screening about 400 clones which were selected in 3 rounds from a library of 60000 clones, we were not able to isolate a variant with improved activity, most likely because of a background of uncleaved plasmids and plasmids which have lost the restriction site. We also showed that the catalytic domain of mouse Dnmt3a DNA methyltransferase is able to transfer the methyl group from S-adenosyl-L- methionine (AdoMet) to a cysteine residue in its catalytic centre. This reaction is reversible and slow. The yield of auto-methylation is increased by addition of Dnmt3L, which functions as a stimulator of Dnmt3a AdoMet complexes. In the presence of CpG containing double stranded DNA, the transfer of the methyl group from AdoMet to the flipped target base was preferred and auto-methylation was not detected. This reaction might constitute a regulatory mechanism which could inactivate unused Dnmt3a in the cell. 6 2. Introduction 2.1. Epigenetics In multicellular organism all cells are derived from the zygote and they carry identical genetic information but they follow different developmental pathways. The phenotype and cellular fate is determined by epigenetic regulatory mechanisms that are heritable through cell divisions and function without changing the DNA sequence [Allis et al., 2007; Watson et al., 2008]. Epigenetic mechanisms include DNA methylation, histone protein covalent modifications (such as acetylation, phosphorylation and methylation) and expression of noncoding RNAs that lead to gene silencing [reviewed in Goldberg et al., 2007]. These epigenetic signals control the proper timing of gene expression during cellular development and differentiation. Out of all epigenetic mechanisms, the mechanism and inheritance of DNA methylation is best understood at a molecular level. 2.1.1 DNA methylation in mammals Methylated bases can be found in almost all living organisms, ranging from bacteria and fungi, to plants and mammals. DNA methylation is the best known and the most abundant kind of DNA modifications which adds additional information to the genetic code and thus methylated bases can be considered as additional letters of the genetic alphabet [Jeltsch, 2002]. In mammals, DNA methylation occurs at the C5 position of cytosine residues, predominantly in the CG dinucleotides and only occasionally at non-CG sites. However, only certain 7 CG sites are methylated, resulting in the generation of a tissue and cell-type- specific pattern of methylation [Jurkowska et al., 2011]. Although methylation of the bases in DNA does not influence the Watson- Crick base pairing, the methyl group, which is placed in the major groove of the DNA, can be recognized by DNA interacting proteins and influence the specificity, kinetic properties or the thermodynamics of their interactions with DNA [Auriol et al., 2005]. There are 56 million CG sites in the human genome, approximately 60-80% of them are modified (4-6% of all cytosines) [Lister et al., 2009; Laurent et al., 2010]. 2.1.2 Importance of DNA methylation in mammals DNA methylation plays central regulatory roles in the control of the cellular physiology. It is involved in embryonic development [Li et al., 1992], cellular reprogramming, [Yang et al., 2007], control of gene expression [Lande-Diner et al., 2007] stabilization of X chromosome inactivation, [Heard, 2004; Chang et al., 2006; Yen et al., 2007], brain function and behavior [Sweatt, 2009; Zhang et al., 2010], regulation of parental imprinting [Barlow, 1995; Delaval & Feil, 2004] and maintenance of the genome integrity through protection against endogenous retroviruses and transposons [Lengauer et al., 1997; Howard et al., 2008]. Aberrant methylation causes various human diseases like psychiatric and immune system diseases [Robertson, 2005; Feinberg, 2007], ICF (immunodeficiency, centromeric region instability and facial anomalies) syndrome [Hansen et al., 1999; Xu et al., 1999] and contributes to the development of 8 cancer [Jones & Baylin, 2007; Jones & Baylin, 2002] and ageing [Richardson, 2003]. 2.2. Types of mammalian DNA methyltransferases (MTases) In mammals, 5 different DNA methyltransferases or methyltransferase like proteins have been identified [Jeltsch, 2002]. These include maintenance methyltransferase which is Dnmt1 and de novo methyltransferases that are Dnmt3a and Dnmt3b and an allosteric activator of the de novo methyltransferases – the Dnmt3L protein (Fig. 1). Additionally,
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