Functional characterization of DNA dealkylation repair protein AlkB from Escherichia coli and use of indenone derivatives as inhibitors of AlkB and human AlkB homologue 3 (AlkBH3) Richa Nigam A Dissertation Submitted to Indian Institute of Technology Hyderabad In Partial Fulfillment of the Requirements for The Degree of Doctor of Philosophy Department of Biotechnology November, 2018 Contents Declaration ............................................................................................................................. vi Approval Sheet ...................................................................................................................... vii Acknowledgements .............................................................................................................. viii Abstract ................................................................................................................................... x Synopsis ................................................................................................................................ xii List of abbreviations ............................................................................................................ xxii Chapter 1: Introduction ............................................................................................1 1.1 Alkylation damage to DNA .............................................................................................. 1 1.2 DNA damaging agents ...................................................................................................... 3 1.3 Cellular defence against alkylation damage ...................................................................... 3 1.4 Molecular mechanism of DNA dealkylation repair .......................................................... 5 1.5. Human homologs of AlkB ............................................................................................... 7 1.6 AlkBH3 and cancer ......................................................................................................... 13 1.7 Inhibition of AlkBH3 ...................................................................................................... 16 1.8 Indenone derivatives as AlkBH3 inhibitor ...................................................................... 23 1.9 Significance and specific objectives ............................................................................... 25 1.9.1 Specific objectives ....................................................................................................... 28 1.10 Reference ...................................................................................................................... 29 Chapter 2: Functional characterization of E. coli DNA repair protein AlkB ....53 2.1 Introduction ..................................................................................................................... 53 2.2 Materials and method ...................................................................................................... 55 2.2.1 cloning .......................................................................................................................... 55 2.2.2 Purification of recombinant proteins ............................................................................ 55 2.2.3 N3meC containing oligonucleotide substrate .............................................................. 56 2.2.4 Preparation of methylated DNA substrate ................................................................... 57 2.2.5 Preparation of Acetoacetanillide .................................................................................. 57 2.2.6 Preparation of Ammonium acetate ............................................................................... 57 2.2.7 10X demethylation assay buffer ................................................................................... 57 2.2.8 Demethylation assay by direct detection of formaldehyde .......................................... 58 2.2.9 Preparation of formaldehyde standard curve ............................................................... 59 2.2.10 Demethylation assay by FDH coupled indirect detection of formaldehdye ............... 59 2.2.11 Enzyme kinetic analysis ............................................................................................. 59 ii 2.3 Results and discussion..................................................................................................... 60 2.3.1 SSB facilitated repair of longer ssDNA by AlkB ........................................................ 61 2.3.2 FDH coupled indirect assay to confirm SSB promotes AlkB activity ......................... 66 2.3.3 AlkB enzyme kinetics in presence of SSB ................................................................... 69 2.4 References ....................................................................................................................... 75 Chapter 3: Interaction of AlkB with SSB ..............................................................79 3.1 Introduction ..................................................................................................................... 79 3.2 Materials and method ...................................................................................................... 80 3.2.1 cloning .......................................................................................................................... 80 3.2.2 In vitro GST pull down assay ....................................................................................... 82 3.2.2.1 Purification of recombinant His-AlkB protein .......................................................... 82 3.2.2.2 Preparation of glutathion sepharose bead-bound GST-SSB ..................................... 83 3.2.2.3 Interaction of glutathion bead-bound GST-SSB and His-AlkB ................................ 84 3.2.2.4 SDS-PAGE ................................................................................................................ 84 3.2.2.5 Western blotting ........................................................................................................ 85 3.2.3 Isothermal titration calorimetry(ITC)........................................................................... 86 3.2.3.1 Purification of tagless AlkB ...................................................................................... 86 3.2.3.2 The Peptide ligand for ITC ....................................................................................... 87 3.2.3.3 ITC experiment ......................................................................................................... 87 3.2.4 CD-spectroscopy .......................................................................................................... 87 3.2.4.1 Preparation of protein ................................................................................................ 87 3.2.4.2 CD-experiment .......................................................................................................... 88 3.2.5 Yeast two-hybrid analysis of SSB-AlkB interaction .................................................... 88 3.2.5.1 Yeast strain ................................................................................................................ 88 3.2.5.2 Media preparation ..................................................................................................... 89 3.2.5.3 Yeast transformation ................................................................................................. 89 3.2.5.4 Yeast two-hybrid analysis ......................................................................................... 90 3.2.5.5 HIS3 selection for interaction positive cells ............................................................. 91 3.2.5.6 Colony lift filter assay using lacZ reporter ................................................................ 91 3.3 Results and discussion..................................................................................................... 92 3.3.1 Characterization of direct interaction of AlkB with SSB by yeast-two-hybrid assay 92 3.3.2 Verification of SSB-AlkB interaction in vitro ............................................................. 94 3.3.3 Examination of the strength of SSB-AlkB interaction ................................................. 96 3.4 References ..................................................................................................................... 101 Chapter 4: Screening and evaluation of indenone derivatives as AlkB inhibitors ................................................................................................................104 4.1 Introduction ................................................................................................................... 104 4.2 Materials and method .................................................................................................... 105 iii 4.2.1 cloning ........................................................................................................................ 105 4.2.2 Purification of recombinant AlkB .............................................................................. 105 4.2.3 Preparation of methylated DNA substrate ................................................................. 105 4.2.4 Screening of Indenone analogs for AlkB inhibition ................................................... 106 4.2.5 Determination of IC50 ..............................................................................................