Expression, Purification, Activity, and Crystallization of M.EcoGII, a Sequence Non-specific N6-Adenine Methyltransferase Público

Abstract

M.EcoGII, initially identified as a sequence non-specific N6-adenine DNA methyltransferase in pathogenic Escherichia coli, is purified to >99% homogeneity using an improved method. This improved method is significantly quicker and results in enhanced adenine methyltransferase activity. M.EcoGII activity is greatest at pH 7.0 under conditions of low ionic strength. M.EcoGII is active on various substrates including double stranded DNA (ds-DNA), RNA/DNA hybrid (ds-RNA/DNA), single stranded DNA or RNA (ss-DNA or ss-RNA), as well as tRNA. M.EcoGII exhibits a preference for these substrates in the order of ds-DNA > ss-DNA > ds-RNA/DNA > ss-RNA. M.EcoGII is active on tRNA, irrespective of whether the secondary or tertiary structure is preserved, in agreement with the observation that the enzyme is active on ss-RNA. Computational homology modeling of M.EcoGII suggested conservation of amino acid residues involved in S-adenosyl-L-methionine (AdoMet) binding. Crystallization of M.EcoGII in ternary complexes with ds-DNA or ss-DNA and S-adenosyl-L-homocysteine (AdoHcy), or in a binary complex with the reaction product AdoHcy, was attempted following each purification. Preliminary crystals diffracted X-rays to 4Å with synchrotron radiation.

Table of Contents

Chapter I: Background & Introduction. 1

Chapter II: Materials and Methods. 3

2.1 Cloning and Overexpression of M.EcoGII. 4

2.2 Purification of M.EcoGII. 4

2.3 Preparation of Oligonucleotides. 6

2.4 Radiometric Assay. 6

2.5 Crystallography. 7

2.6 Structural Modeling. 8

Chapter III: Results and Discussion. 8

3.1 Purification of M.EcoGII. 8

3.1.1 Purifications #1-2. 8

3.1.2 Purifications #3-5. 11

3.1.3 Purifications #6-7. 15

3.1.4 Summary of How Original NEB Purification Can Be Optimized. 18

3.2 Enzymatic Activity of M.EcoGII. 20

3.2.1 M.EcoGII Activity is pH and NaCl Dependent. 20

3.2.2 ds-DNA vs. ss-DNA. 22

3.2.3 M.EcoGII Activity on a Wide Variety of Nucleic Acid Substrates. 24

3.2.4 M.EcoGII May Exhibit Sequence Preference. 27

3.2.5 tRNA^Asp Activity. 29

3.2.6 Comparison of Activities of Emory-Purified M.EcoGII to NEB-Purified M.EcoGII. 32

3.3 Computational Structural Modeling of AdoMet Binding Pocket. 34

3.4 Crystallization Trials. 36

Appendix. 40

A.1 Need for Alternative Methyltransferase Assay. 40

A.2 Theoretical Description of Enzyme-coupled SAHH Fluorescence Assay. 40

A.3 Cloning and Overexpression of SAHH. 41

A.4 Purification of SAHH. 41

A.5 Enzyme-coupled SAHH Fluorescence Assay. 42

References. 43

About this Honors Thesis

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School	Emory College
Department	Biology
Degree	BS
Submission	Honors Thesis
Language	English
Research Field	Chemistry, Biochemistry Biology, Molecular Biology, General
Palabra Clave	M.EcoGII N6-Adenine Methyltransferase Sequence Non-specific Methylation Adenine Methylation
Committee Chair / Thesis Advisor	Cheng, Xiaodong, Emory University
Committee Members	Deal, Roger, Emory University Weaver, Jeremy, Emory University

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