Biochemical and Structural Studies of RNA and DNA Editing Enzymes
Elias, Youssef
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https://hdl.handle.net/2142/84847
Description
Title
Biochemical and Structural Studies of RNA and DNA Editing Enzymes
Author(s)
Elias, Youssef
Issue Date
2007
Doctoral Committee Chair(s)
Morrissey, James H.
Department of Study
Biochemistry
Discipline
Biochemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Biochemistry
Language
eng
Abstract
RNA and DNA editing, occurring by way of hydrolytic deamination of cytidine (C) and adenosine (A), is a modification event that profoundly effects on an organisms vitality and quality of life. This study focuses on two types of enzymes---ADAT (adenosine deaminase acting on tRNA) and APOBEC3G (apolipoprotein B editing catalytic complex 3G), which deaminate adenosine in the wobble position (position 34) of tRNA and converts cytidine to uridine in single stranded DNA, respectively. Described herein, is the biochemical and structural characterization of prokaryotic and eukaryotic ADAT enzymes, as well as the in vitro reconstitution of the enzymatic activity of the human APOBEC3G cytidine deaminase. We have characterized and confirmed the identity of two putative ADAT enzymes, derived from Aquifex aeolicus and Agrobacterium tumefaciens, by enzymatically assaying for tRNA A34 deamination activity. We have also solved the structure of a stable construct of the A. tumefaciens ADAT enzyme, that has served as the foundation for our structural model of ADAT in complex with its RNA substrate. Our biochemical and structural data have allowed us to identify a novel motif residing in the C-terminus of prokaryotic ADAT enzymes essential for substrate binding and catalysis. We have also extended our studies to encompass the yeast ADAT enzyme, which functions as a heterodimer in vivo, referred to as ADAT2/ADAT3. We successfully obtained highly purified and enzymatically active Saccharomyces cerevisiae ADAT2/ADAT3. Lastly, our studies of nucleic acids editing enzymes have led us to investigate human APOBEC3G (hA3G). Although hA3G is extremely difficult to handle in vitro, we were able to obtain substantial amounts of soluble and enzymatically active hA3G. Our enzymatic data has allowed us to evaluate the turnover efficiency of hA3G in vitro, which is information that had been lacking before now. We have also generated various constructs of hA3G, including a cysteine mutant that can potentially be tethered to a thiol-modified substrate, in efforts to increase the enzymes stability in vitro. Combined together, our studies of ADAT and APOBEC3G enzymes have allowed us to better understand how RNA and DNA editing enzymes recognize and catalyze their different substrates.
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