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Biochemical Characterization of DDX43 (HAGE) Helicase

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Biochemical Characterization of DDX43 (HAGE) Helicase Biochemical Characterization of DDX43 (HAGE) Helicase A Thesis Submitted to the College of Graduate Studies and Research In Fulfillment of the Requirements For the Degree of Master of Science In the Department of Biochemistry University of Saskatchewan Saskatoon By Tanu Talwar © Copyright Tanu Talwar, March, 2017. 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 University of Saskatchewan 107 Wiggins Road Saskatoon, Saskatchewan, Canada S7N 5E5 i ABSTRACT DDX43, DEAD-box polypeptide 43, also known as HAGE (helicase antigen gene), is a member of the DEAD-box family of RNA helicases. It is highly expressed in many tumor types compared with normal tissues, and is therefore considered as a potential target for immunotherapy of cancers. Despite its unique expression and potential as a therapy target, little is known about its biochemical and physiological functions. In this study, we purified recombinant DDX43 protein to near homogeneity and our gel filtration results showed that DDX43 exists as a monomer in solution. Biochemical assays using monomer fractions of DDX43 demonstrated that it could unwind both RNA and DNA substrates in an ATP- dependent manner, and most efficiently in the presence of Mg2+; no significant unwinding activity was detected with other nucleoside triphosphates or divalent cations. Replacing the conserved lysine in motif I (K292A) or aspartic acid in motif II (D396A) abolished the unwinding activity. Intriguingly, DDX43 could unwind RNA substrates without strict polarity, but it showed higher unwinding activity on a 5’ tail RNA substrate compared to a 3’ tail or blunt end RNA substrates. However, for DNA substrates, it exhibited unidirectional translocation and unwound DNA in a 3’to 5’ direction only. A K-homology (KH) domain in the N-terminal region of DDX43 was found to possess strong nucleic acid binding ability and the N-terminal domain showed novel strand exchange and required for the unwinding activity. Compared to the full-length protein, the C-terminal helicase domain had weaker unwinding activity, but an increase was observed in the presence of the N-terminal domain. Using Co- immunoprecipitation and mass spectrometry, we found that DDX43 associates with pICln and MEP50, two methylosome subunits that are involved in assembly of the spliceosome complex. Collectively, our results suggest that DDX43 is a KH domain containing ATP- dependent dual helicase, where unwinding activity is mediated through cooperation between its N-terminal domain and helicase domain, and potentially involved in pre-mRNA splicing. ii ACKNOWLEDGEMENTS I wish to express the deepest appreciation to my supervisor Dr. Yuliang Wu for his endless support on academic studies and his helpful guidance on my life. His 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, Dr. Stanley Moore and Dr. Erique K. Lukong for their insightful suggestions and guidance throughout my project. My sincere thanks also go to everyone in Dr. Wu’s lab, including Dr. Manhong Guo, Dr. Venkatasubramanian Vidhyasagar, Hao ding and Ahmad Kareim for their stimulating discussions. 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 brother and my In-laws for providing me with continuous encouragement and for their patience throughout my years of research. And finally, I acknowledge the love of my life, my husband, Vishal Taimni, who blessed me with a life of joy and always encouraged me to pursue my dreams. I am also thankful to all my friends for their help and support through my master program. iii TABLE OF CONTENTS Contents PERMISSION OF USE STATEMENT .................................................................................... i ABSTRACT ............................................................................................................................ii ACKNOWLEDGEMENTS .................................................................................................. iii TABLE OF CONTENTS ........................................................................................................ iv LIST OF FIGURES ............................................................................................................. viii LIST OF TABLES ................................................................................................................... x LIST OF ABBREVIATIONS ................................................................................................. xi 1. INTRODUCTION ............................................................................................................... 1 1.1 Helicases and their Functions ........................................................................................ 1 1.2 The Mechanisms of Helicases ........................................................................................ 2 1.3 Superfamily 2 Helicases ................................................................................................. 3 1.4 DEAD-box Helicases ..................................................................................................... 7 1.5 The KH domain .............................................................................................................. 9 1.6 DDX43 (HAGE) Helicase ........................................................................................... 12 2. HYPOTHESIS AND OBJECTIVES ................................................................................. 17 2.1 Hypothesis:................................................................................................................... 17 2.2 Objectives: ................................................................................................................... 17 3. MATERIALS AND METHODS ....................................................................................... 18 3.1 Reagents ....................................................................................................................... 18 3.2 Plasmids DNA and Mutagenesis .................................................................................. 20 iv 3.3 RNA and DNA Substrates ............................................................................................ 20 3.4 Expression and Purification of Recombinant DDX43 Protein .................................... 23 3.5 Western Blot ................................................................................................................. 23 3.6 Helicase Unwinding Assays ......................................................................................... 24 3.7 Translocase Assays ....................................................................................................... 24 3.8 Annealing Assay ........................................................................................................... 25 3.9 Strand Exchange Assay ................................................................................................ 25 3.10 Electrophoretic Mobility Shift Assay (EMSA) .......................................................... 26 3.11 ATP Hydrolysis Assays .............................................................................................. 26 3.12 Cell Culture ................................................................................................................ 27 3.13 Immunoprecipitation .................................................................................................. 27 4. RESULTS .......................................................................................................................... 29 4.1 Protein Overexpression and Purification ..................................................................... 29 4.3 DDX43 Protein Unwinds DNA Substrates .................................................................. 33 4.3.1 DDX43 Protein Unwinds DNA in only 3’ to 5’ Direction..................................... 33 4.3.2 DDX43 Protein Exhibits High Processivity on DNA Substrates .......................... 34 4.4 DDX43 has Weak 3’-5’ Translocase Activity .............................................................. 34 4.5 DDX43-K292A and D396A Mutants Abolish Helicase Unwinding Activity.............. 35 4.6 DDX43 Protein Unwinds DNA:RNA hybrid Substrates ............................................. 37 4.7 ATP Hydrolysis and Mg2+ is Essential for the Unwinding Activity
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