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Structure and Function of KH Domain in DDX43 and DDX53 Cancer Antigen Helicases

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Structure and Function of KH Domain in DDX43 and DDX53 Cancer Antigen Helicases Structure and Function of KH Domain in DDX43 and DDX53 Cancer Antigen Helicases A Thesis Submitted to the College of Graduate and Postdoctoral Studies In Fulfillment of the Requirements For the Degree of Master of Science In the Department of Biochemistry, Microbiology and Immunology University of Saskatchewan Saskatoon By Manisha Yadav © Copyright Manisha Yadav, April, 2020. All rights reserved. PERMISSION OF USE STATEMENT I hereby present this thesis in partial fulfilment of the requirements for a postgraduate degree from the University of Saskatchewan and agree that the Libraries of this University may make it freely available for inspection. I further agree that permission for copying of this thesis in any manner, either in whole or in part, for scholarly purposes may be granted by the professor or professors who supervised this thesis or, in their absence, by the Head of the Department or the Dean of the College in which my thesis work was done. It is understood that any copying or publication or use of this thesis or parts of it for any financial gain will not be allowed without my written permission. It is also understood that due recognition shall be given to me and to the University of Saskatchewan in any scholarly use which may be made of any material in my thesis. Requests for permission to copy or to make other use of material in this thesis in whole or part should be addressed to: Head of the Department of Biochemistry, Microbiology and Immunology University of Saskatchewan Saskatoon, Saskatchewan, S7N 5E5 Dean College of Graduate and Postdoctoral Studies University of Saskatchewan 116 Thorvaldson Building, 110 Science Place Saskatoon, Saskatchewan, S7N 5C9 Canada i ABSTRACT DDX43 and DDX53 are two cancer/testis antigens that are overexpressed in various tumors, but absent or expressed at very low levels in normal tissues; therefore, both are proposed to be valid candidates for therapeutic targets. DDX43 and DDX53 belong to the DEAD-box RNA helicase family, and contain a K-homology (KH) domain in their N-terminal regions and a helicase core domain in their C-terminal regions. The KH domain was first characterized in the human heterogeneous nuclear ribonucleoprotein K (hnRNP K). The typical function of KH domain is to bind single-stranded (ss) RNA or ssDNA in a sequence specific manner. The helicase core domain contains two conserved RecA-like domains and unwinds double-strand nucleic acids in a sequence-unspecific manner; therefore, we hypothesize that the KH domain may play a critical role in sequence-specific function of DDX43 and DDX53. In this study, we expressed and purified DDX43-KH and DDX53-KH domains using Ni-NTA column chromatography and size exclusion chromatography. Using electrophoretic mobility shift assay (EMSA), we found that DDX43-KH domain binds ssDNA and ssRNA substrates efficiently but not blunt-end double-stranded (ds) DNA or dsRNA substrates. We applied 1H-15N HSQC NMR spectroscopy to determine the binding affinity of DDX43-KH with dT5, dA5, dC5, dG5 and rU5 oligonucleotides, and found that there are significant chemical shift changes in the HSQC spectra for pyrimidines (dT5, dC5, rU5), but not for purines (dA5 and dG5). Titrations with dT10 and dT5 oligonucleotides revealed that the GRGG loop in DDX43- KH domain is involved in protein-nucleic acids interactions. In addition, we found that the Ala81, adjacent to the GRGG loop, is involved in the binding. A81G and A81S mutations reduced protein stability and binding affinity. To further identify the specific nucleotides bound by the DDX43-KH and DDX53-KH domains, we used SELEX (Systematic evolution of ligands by exponential enrichment) and ChIP-seq (Chromatin Immunoprecipitation Sequencing)/CLIP-seq (Cross-linking immunoprecipitation-sequencing), and found CT-rich sequences are commonly occurring motifs for DDX43-KH domain. However, GTTGA, TG/TTG/TG/T motifs are also common in the SELEX and CLIP samples. Finally, NMR spectroscopy showed that GTTGT has the lowest Kd value (0.0435 mM); suggesting, DDX43- KH binds to TG-rich sequences with high affinity and might partially dictate the substrate specificity for the full length DDX43 protein. ii ACKNOWLEDGEMENTS I wish to express the deepest appreciation to my supervisors Dr. Yuliang Wu and Dr. Miroslaw Cygler for giving me the opportunity to carry out this project in their labs, their endless support on academic studies and their helpful guidance on my career. Their resolute enthusiasm for science kept me constantly engaged with my research. I would also like to express gratitude to my committee member Dr. Jeremy Lee and Dr. Oleg Dmitriev, for their insightful suggestions and guidance throughout my project. Dr. Dmitriev has guided and supported me throughout the NMR spectroscopy experiments and his inputs in developing this project kept me motivated. Dr. Lee has always suggested very important ideas related to the project. Together, my work is a result of constant support and suggestions from this team of professors and I’m highly grateful to work under their supervision. My sincere thanks also go to Dr. Anthony Kusalik and Daniel Hogan for their processing and analyzing the sequence data. I would like to thank Dr. Ivy Yeuk Wah Chung as she has been involved with me in all the protein purification and crystallization experiments. I’m grateful to Dr. Venkatasubramanian Vidhyasagar for his guidance in ChIP/CLIP-seq experiments. I would like to thank Mrs. Eva Ulhemann for her suggestions and support in sharing the protocols for 15N-labelled protein purification. All members of Dr. Wu and Dr. Cygler labs deserve recognition for their constant advice and friendship: Dr. Ravi Shankar Singh, Kingsley Ekumi, Dr. Michal Boniecki, Shizhuo (Sarah) Yang, Aanchal Aggrawal. I would like to extend my thanks to the Saskatchewan Structural Sciences Centre for the 600MHz NMR spectrometer. I would like to thank all members of the Cancer Cluster and faculty of Biochemistry Department. I must express my profound gratitude to my parents, my sister and brother for providing me with continuous encouragement to pursue my dreams and for their patience throughout my years of research. I am also thankful to all my friends for their help and support through my master program. iii TABLE OF CONTENTS PERMISSION OF USE STATEMENT…………………………………………………..……..i ABSTRACT……………………………………………………………………………………..ii ACKNOWLEDGEMENTS…………………………………………………………………….iii TABLE OF CONTENTS………………………………………………………………...……..iv LIST OF FIGURES………………………………………………………………………….....vii LIST OF TABLES……………………………………………………………………………….x LIST OF ABBREVIATIONS…………………………………………………………………..xi 1. INTRODUCTION…………………………………………………………………………...1 1.1 Helicases are classified into six superfamilies…………………………………………...1 1.2 DEAD-box helicases……………………………………………………………………..2 1.3 DDX43 (HAGE) and DDX53 (CAGE) helicases…………………………...………...…4 1.4 The KH domain and KH domain-containing human proteins..………………………....8 1.4.1 Two types of KH domain………………………………………………...……..10 1.4.2 Interaction of Type I KH domains with their specific nucleic-acid sequences……………………………………………………………………….11 1.4.3 Structure of KH domains bound to specific nucleic acid sequences………… .14 2. HYPOTHESIS AND OBJECTIVES……………………………………………………….16 2.1 Hypothesis……………………………………………………………………………...16 2.2 Objectives..……………………………………………………………………………..16 3. MATERIALS AND METHODS…………………………………………………………...17 3.1 Reagent list.……………………………………………………………………...……..17 3.2 Plasmids DNA and mutagenesis………………………………………………………..19 3.3 RNA and DNA substrates………………………………………………………………20 3.4 Expression and purification of recombinant DDX43-KH and DDX53-KH domain proteins……………………………………………………………………………….....26 3.5 Western blot…………………………………………………………………………….26 3.6 Electrophoretic mobility shift assay (EMSA)…………………………………………..27 3.7 Helicase unwinding assays…………………………………………...………………...27 3.8 15N- Isotopic labelling of recombinant DDX43-KH proteins…………………………..28 3.9 Systematic evolution of ligands by exponential enrichment (SELEX) …..……………29 iv 3.10 Cell culture…………………………………………………………………………….30 3.11 Transfection in HEK293T cells and immunoprecipitation…………………………....30 3.12 ChIP-DNA and CLIP-RNA library construction……………………………………...31 3.13 Bioinformatics analysis of SELEX, ChIP-DNA, and CLIP-RNA sequences…..…….32 4. RESULTS…………………………………………………………………………………..33 4.1 Protein overexpression and purification…..……………………………………………33 4.1.1 Purification of DDX43-KH and DDX53-KH domain proteins………………..33 4.1.2 DDX43-KH+RecA1 and DDX53-KH+RecA1 proteins are unstable and difficult to purify…………………………………………………………………………35 4.2 DDX43-KH-74 and DDX43-KH-126 grow into needle like small crystals and DDX53- KH proteins does not crystallize…………………………………………………….….37 4.3 Biochemical characterization of DDX43-KH and DDX53-KH domains using electrophoretic mobility shift assay (EMSA)…………………………………………..40 4.3.1 DDX43-KH and DDX53-KH domains binds ssDNA and RNA but not blunt-end dsDNA and dsRNA substrates………………………………………………….40 4.3.2 DDX43-KH and DDX53-KH domains prefers pyrimidines over purines by EMSA…………………………………………………………………………..42 4.4 DDX43-KH and DDX53-KH domain alone have no helicase unwinding activity…….44 4.5 Nuclear Magnetic Resonance (NMR) spectroscopy analysis of DDX43-KH domain………………………………………………………………………………….44 4.5.1 Expression and purification of 15N-labelled DDX43-KH-89 protein…………..44 4.5.2 Sequential amino acid assignment of DDX43-KH-89 protein and identifying the amino acids interacting with oligonucleotides……………………………….....45 4.5.3 DDX43-KH domain prefers binding pyrimidines than purines
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