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

Allaw, Mohammed Basel (2016)

Permanent URL: https://etd.library.emory.edu/concern/etds/cz30ps78q?locale=en
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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…………………..................................................................................................……........……...…….Page 1

Chapter II: Materials and Methods………………………..........................................................................................…..................….….....…..Page 3

2.1 Cloning and Overexpression of M.EcoGII……………………..…...…............................................................................................................…...…..Page 4

2.2 Purification of M.EcoGII………………………...……................................................................................................……................….…...…....…...…..Page 4

2.3 Preparation of Oligonucleotides……………………..................................................................................................………...................….….......…..Page 6

2.4 Radiometric Assay…………………………….……………...........................................................................................................................….…......…..Page 6

2.5 Crystallography……………………………………..………...........................….…...….….............................................................................................…...Page 7

2.6 Structural Modeling……………………………………...................................................................................................................................…...…....Page 8

Chapter III: Results and Discussion………..………….......................................................................……………………….….......................…..Page 8

3.1 Purification of M.EcoGII……………………………….............................................................................................................................………......…..Page 8

3.1.1 Purifications #1-2……………………………………..................................…...……..........................................................................................…......Page 8

3.1.2 Purifications #3-5…………………………………….............................................................................................................................................Page 11

3.1.3 Purifications #6-7……………..…………………..................................…….....................................................................................................…..Page 15

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

3.2 Enzymatic Activity of M.EcoGII…………………………………..............................................................................................................................…Page 20

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

3.2.2 ds-DNA vs. ss-DNA………………….........................................................................................................................................................…Page 22

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

3.2.4 M.EcoGII May Exhibit Sequence Preference…………...........................……….......................................................................................…..…..Page 27

3.2.5 tRNAAsp Activity………………………………………………...............................................................................................................................…..….Page 29

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

3.3 Computational Structural Modeling of AdoMet Binding Pocket………….…........................................................................................................Page 34

3.4 Crystallization Trials………………………………………………................................................................................................................................…..Page 36

Appendix……………………………………………..…….…...………........…….....................................................................…....................….…...….Page 40

A.1 Need for Alternative Methyltransferase Assay………………………....................………...........................................................................................Page 40

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

A.3 Cloning and Overexpression of SAHH……………………………..................................................................................................................…………..Page 41

A.4 Purification of SAHH……………………………………………….........................................................................................................................……...…..Page 41

A.5 Enzyme-coupled SAHH Fluorescence Assay………….....................…………............................................................................................…..……..Page 42

References……………………………………………………........…....……………...….........................................................................................….....Page 43

List of Figures

Figure 1: Schematic detailing the enzyme-catalyzed addition of a methyl group to the N6-position of adenine………………………………………………….……………..Page 3

Figure 2: Stained SDS-PAGE gels and chromatogram peaks corresponding to successive purification stages of preparation #1………………………..….………..Page 9

Figure 3: Stained SDS-PAGE gels and chromatogram peaks corresponding to successive purification stages of preparation #2……………………………………..Page 10

Figure 4: Stained SDS-PAGE gels and chromatogram peaks corresponding to successive purification stages of preparation #3………………………………..…..Page 12

Figure 5: Stained SDS-PAGE gels and chromatogram peaks corresponding to successive purification stages of preparation #4…………………………...….…..Page 13

Figure 6: Stained SDS-PAGE gels and chromatogram peaks corresponding to successive purification stages of preparation #5…………………………….….…..Page 14

Figure 7: Stained SDS-PAGE gels and chromatogram peaks corresponding to successive purification stages of preparation #6………………………………..…..Page 17

Figure 8: Stained SDS-PAGE gels and chromatogram peaks corresponding to successive purification stages of preparation #7………………………...…....…..Page 18

Figure 9: pH and NaCl Dependence of M.EcoGII…………………......................................................................................................................…......Page 22

Figure 10: M.EcoGII Activity on ds-DNA vs. ss-DNA……………….......................................................................................................................…..Page 23

Figure 11: M.EcoGII Activity on Wide Variety of Nucleic Acid Substrates.......................................................................................................Pages 26-27

Figure 12: M.EcoGII Activity on Oligonucleotides with Different Number of Adenines………………………………………………………..................................……..…..Page 29

Figure 13: M.EcoGII Activity on tRNAAsp-GTC……………………………...……..................................................................................................................Page 32

Figure 14: Comparison of Emory-Purified M.EcoGII to NEB-Purified M.EcoGII…………………………..………………………………………......................................Pages 33-34

Figure 15: Structural Modeling of AdoMet/AdoHcy Binding Pocket of M.EcoGII…………………………………………………………….………...............................…..Pages 35-36

Figure 16: Crystal Images and Diffraction Pattern……….........................................................................................................................…...Pages 37-38

Figure 17: Matthews Probability Plots……………….…………………........................................................................................................................……..Page 39

List of Tables

Table 1: M.EcoGII Yield from Each Purification…………………………....................................................................................................................……..Page 20

Table 2: X-ray Diffraction Data Set Collected from SER-CAT 22-ID beamline at the Advanced Photon Source, Argonne National Laboratory and processed using HKL2000……………………………………………………………………….…............................................................................................................................………..Page 38

Table 3: Matthews Probabilities Based on Fraction of Crystal Volume Occupied By Solvent….……………………………………………........................………...…..Pages 39-40


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