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Biochemical Characterization of Homing Endonucleases Encoded By Biochemical characterization of homing endonucleases encoded by fungal mitochondrial genomes By Tuhin Kumar Guha A thesis submitted to the Faculty of Graduate Studies of the University of Manitoba in partial fulfilment of the requirements of the degree of: DOCTOR OF PHILOSOPHY Department of Microbiology University of Manitoba Winnipeg Copyright © 2016 by Tuhin Kumar Guha I Abstract The small ribosomal subunit gene of the Chaetomium thermophilum DSM 1495 is invaded by a nested intron at position mS1247, which is composed of a group I intron encoding a LAGLIDADG open reading frame interrupted by an internal group II intron. The first objective was to examine if splicing of the internal intron could reconstitute the coding regions and facilitate the expression of an active homing endonuclease. Using in vitro transcription assays, the group II intron was shown to self-splice only under high salt concentration. Both in vitro endonuclease and cleavage mapping assays suggested that the nested intron encodes an active homing endonuclease which cleaves near the intron insertion site. This composite arrangement hinted that the group II intron could be regulatory with regards to the expression of the homing endonuclease. Constructs were generated where the codon-optimized open reading frame was interrupted with group IIA1 or IIB introns. The concentration of the magnesium in the media sufficient for splicing was determined by the Reverse Transcriptase-Polymerase Chain Reaction analyses from the bacterial cells grown under various magnesium concentrations. Further, the in vivo endonuclease assay showed that magnesium chloride stimulated the expression of a functional protein but the addition of cobalt chloride to the growth media antagonized the expression. This study showed that the homing endonuclease expression in Escherichia coli can be regulated by manipulating the splicing efficiency of the group II introns which may have implications in genome engineering as potential ‘on/off switch’ for temporal regulation of homing endonuclease expression . Another objective was to characterize native homing endonucleases, cytb.i3ORF and I- OmiI encoded within fungal mitochondrial DNAs, which were difficult to express and purify. For these, an alternative approach was used where two compatible plasmids, HEase.pET28b (+)- II kanamycin and substrate.pUC57-chloramphenicol, based on the antibiotic markers were maintained in Escherichia coli BL21 (DE3). The in vivo endonuclease assays demonstrated that these homing endonucleases were able to cleave the substrate plasmids when expressed, leading to the loss of the antibiotic markers and thereby providing an indirect approach to screen for potential active homing endonucleases before one invests effort into optimizing protein overexpression and purification strategies. III Acknowledgements The journey during the last five years has been incredible. I am thankful to many people for their contribution in my research, either directly involved in my research or through friendly discussions. I am very thankful to my supervisor, Dr. Georg Hausner for giving me the opportunity to work in his laboratory, for believing in me, for the immense guidance and continuous support. The patience, detailed explanation on the research subject whenever needed, friendly approach, your understanding and concern towards the students, have been some of the added benefits working in your lab and I will greatly cherish them forever. My sincere gratitude to Dr. Peter C Loewen for his mentorship and constant support over the years. This thesis would not have been possible without his support. To my committee members, Drs. Jörg Stetefeld (Department of Chemistry), Brian Mark, Ivan Oresnik (Department of Microbiology) for their valuable suggestions and comments that helped me to shape the research project better. Moreover, I want to thank the committee members for reading my thesis and for your constructive suggestions. I am thankful to Dr. Joe O’Neil (Department of Chemistry) for generously allowing access to his laboratory and assisting the circular dichroism spectropolarimetry work. To Dr. Steven Zimmerly (Biological Sciences, University of Calgary, Canada) for his suggestions on group II introns relating to the in vitro splicing assay and also for agreeing to be my external examiner. The necessary corrections/suggestions have been really helpful. I also want to thank Dr. Barry Stoddard (Fred Hutchinson Cancer Research Centre, Seattle, USA) for undertaking the DNA-protein cocrystallographic work. IV I am thankful to my past lab mates Dr. Mohamed Hafez, Chen Shen, and Megan Hay for their help, support and friendship and Dr. Mohamed Hafez, again for his suggestions and sharing ideas about mS1247 twintron. Also I am thankful to my present lab mates, Alvan Wai, Iman Bilto, Zubaer Abdullah, Talal Abboud for their suggestions and above all, friendship. My sincere gratitude to Alvan, who has been always beside me, helping me in my research in every possible way and for his constant encouragement. I am thankful to my departmental colleagues who have helped me, and taught me in handling necessary instruments. My sincere thanks to my fellow colleagues in the department, who have allowed me to use their instruments and chemicals whenever needed. I greatly appreciate yours’ generosity. I would like to extend my gratitude to all the staff members of the Microbiology department. Moreover, I acknowledge the financial support from the Faculty of Graduate Studies, the Faculty of Science and the Graduate Students’ Association at the University of Manitoba. The travel grants did help me to attend and present my research findings in various scientific conferences. The funding from the Faculty of Graduate Studies GETS (Graduate Enhancement of Tri-Council Stipends) program is also gratefully acknowledged. Last but not least, I am very thankful to my family members who have always believed in me. I am ever grateful to them for their immense support, suggestion and encouragement during my entire Ph.D. program. They have definitely helped me to stay focus in my research work and effectively complete this saga. V This thesis is dedicated to Ma Manasha Ma and Dadu, whose blessings have been a significant part of my life. VI Table of contents Abstract .......................................................................................................................................... II Acknowledgements ..................................................................................................................... IV Dedication……………………………………………………………………………………….VI Table of contents ........................................................................................................................ VII List of tables.............................................................................................................................. XIV List of figures .............................................................................................................................. XV List of abbreviations .............................................................................................................. XVIII General introduction .................................................................................................................... 1 Chapter 1. Literature review ....................................................................................................... 4 1.0. Introduction .......................................................................................................................... 5 1.1. Discovery ............................................................................................................................. 6 1.2. Homing ................................................................................................................................. 7 1.3 Evolutionary speculations ..................................................................................................... 8 1.4. Distribution of homing ......................................................................................................... 9 1.5. Homing mechanisms .......................................................................................................... 12 1.5.1. Group I intron homing ................................................................................................. 12 1.5.2. Group II intron ‘retrohoming’ ..................................................................................... 13 1.6. The homing cycle ............................................................................................................... 15 1.7. Homing endonucleases ....................................................................................................... 16 1.7.1. Distinguishing characteristics of HEases .................................................................... 17 1.7.2. Nomenclature conventions .......................................................................................... 18 1.8. Homing endonuclease families .......................................................................................... 19 1.8.1. LAGLIDADG family .................................................................................................. 20 1.8.1.1. Structure ................................................................................................................ 21 VII 1.8.1.2. DNA recognition ..................................................................................................
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