Studying the replication mechanism of the yeast retrotransposon Ty5 by molecular and computational approaches

vi CHAPTER I. GENERAL INTRODUCTION 1 Taxonomy of transposable elements 1 Retroviruses and LTR retrotransposons 2 Overview of reverse transcription 7 Retroelement proteins involved in reverse transcription 10 Diversity in priming LTR retroelements reverse transcription 12 Integration and recombination of cDNA 15 Evolution of retroelements 19 The Ty5 system 19 Dissertation organization 23 References 24 CHAPTER II. Ty5 gag MUTATIONS INCREASE Ty5 cDNA SYNTHESIS AND SUGGESTS A ROLE FOR HYDROGEN BONDING IN THE FUNCTION OF THE NUCLEIC ACID ZINC FINGER 35 Abstract 35 Introduction 36 Materials and Methods 38 Strain and plasmid construction 38 Mutagenesis and library screening 39 Assays for integration, recombination and target specificity 40 Protein preparation and immunoblot analysis 40 Assaying Ty5 cDNA 41 Calculating hydrogen bonding potential 41 Results 42 Two mutations in gag increase Ty5 transposition frequency 42 Effects of gag mutations on integration and recombination 43 Effects of gag mutations on cDNA level 44 Effects of gag mutations on protein processing and solubility 45 Effects of gag mutations on target bias 46 Features of the zinc finger important for transposition 47 Discussion 48 gag mutations increase cDNA synthesis 49 Mutations in the zinc finger implicate a role for hydrogen bonding in NCp function 52