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«     A University of Sussex DPhil thesis  Available online via Sussex Research Online:    ...»

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A University of Sussex DPhil thesis 

Available online via Sussex Research Online: 

http://sro.sussex.ac.uk/   

This thesis is protected by copyright which belongs to the author.   

This thesis cannot be reproduced or quoted extensively from without first 

obtaining permission in writing from the Author   

The content must not be changed in any way or sold commercially in any 

format or medium without the formal permission of the Author    When referring to this work, full bibliographic details including the  author, title, awarding institution and date of the thesis must be given  Please visit Sussex Research Online for more information and further details    Cellular and biochemical analyses of TDP1 mediated chromosomal break repair A thesis submitted to the University of Sussex for the degree of Doctor of Philosophy By Owen Spencer Wells 2 Declaration I hereby declare that this thesis has not been and will not be, submitted in whole or in part to another University for the award of any other degree.

Signed ………………………………………………………………………… Owen S. Wells Acknowledgements First of all I would like to express my gratitude to Dr Sherif El-Khamisy for giving me the opportunity to undertake a PhD and for his continued guidance and support throughout. A special thank you to Jessica Hudson for looking after me, spending time listening to my half-baked ideas and sharing her expertise within the laboratory. I would like to thank Aaron, Mariella and Kyle for their encouragement and input in my work, and also to all the members in the El-Khamisy lab, Jude and Conny, my office and the GDSC for putting up with me for the last 3.5 years and making my time here enjoyable.

Finally, a special mention to Diana, Chris, Sophie, Carol and Marcel for helping me through this last year.

Outside of the centre I would like to thank my Mother for whom I am extremely grateful and proud of, for raising my brother and myself and helping us accomplish the goals in our life. My brother, Robert, who I look up too and whose drive and success in anything he puts his mind to has inspired me to do the same, or at least try. The rest of my family especially my Nan, Grandma and Grandad who have all had a pivotal role in my upbringing and enabled me to be the person I am today. Last but not least my uncle Mark whose modest persona has rubbed off on me and has alwayskept me grounded.

Of my friends back home I would like to thank Huw Wilkinson, Jonathan James, Jon Gouran, Gareth Sherrington, James Edwards, Richard Martin and Chris Jones for helping me maintain a social life, for always showing an interest in what I do and listening when I talk about my research. Everyone at Littlemore rugby club and my old house mates Bunk and Tim for helping take my mind off work.

–  –  –

Tyrosyl DNA phosphodiesterase 1 (TDP1) is an end-processing enzyme involved in the repair of abortive topoisomerase I (Top1) complexes. Although not essential for survival, a hypomorphic mutation in TDP1 is linked to the autosomal recessive ataxia, spinocerebellar ataxia with axonal neuropathy 1 (SCAN1).

SCAN1 is a rare human condition linked with neurodegeneration and ataxic gait and patients are usually wheel chair bound by their early teens. TDP1 primarily cleaves lesions at the 3’-end of DNA breaks and its most prominent substrate is stalled Top1 linked to the 3’-terminus of DNA. The enzymatic mechanism by which TDP1 functions are well understood and inhibitors are now being investigated for treatment of cancer. In contrast, the processes involved in TDP1 recruitment, localisation and regulation during the DNA damage response remain unclear. This thesis investigates how the evolutionarily driven N-terminus of TDP1, not conserved in lower Eukaryotes, is required for optimal cellular protection against genotoxic stress. I also characterise how post-translational modifications of TDP1 allow for efficient repair of transcriptionally associated, chromosomal single-strand breaks and uncover new protein interacting partners of TDP1 and their role in TDP1 mediated repair.

–  –  –

Table of Contents Chapter I

Introduction

1.1 General introduction

1.2 Sources of DNA damage

1.2.1 Endogenous sources of DNA damage

1.2.1.1 Reactive oxygen species

1.2.1.2 Methylation

1.2.1.3 Hydrolysis

1.2.1.4 Mismatch of bases

1.2.2 Exogenous sources of DNA damage

1.2.2.1 UV

1.2.2.2 Ionising radiation

1.3 DNA repair

1.3.1 DNA double-strand break repair

1.3.1.1 Homologous recombination

1.3.1.2 Non-homologous end-joining

1.3.2 Nucleotide excision repair

1.3.3 Mismatch repair

1.3.4 Base excision repair

1.3.5 Single-strand break repair

1.3.5.1 Detection: PARP

1.3.5.2 End-processing

1.3.5.3 Gap filling

1.3.5.4 Ligation

1.3.6 Ligase family

1.3.6.1 Ligase I

1.3.6.2 Ligase III

1.3.6.3 Ligase IV

1.3.7 Protein-linked DNA single-strand breaks

1.4 Tyrosyl DNA phosphodiesterase 1

1.5 Diseases associated with defects in single-strand break repair





1.5.1 Neural development

1.5.2 Autosomal recessive ataxias

1.5.3 Spinocerebellar ataxia with axonal neuropathy 1 (SCAN1)

1.5.4 Microcephaly, infantile-onset seizures, and developmental delay (MCSZ).. 31 1.5.5 X-linked mental retardation (XLMR)

1.5.6 Ataxia oculomotor apraxia 1 (AOA1)

1.5.7 Ataxia oculomotor apraxia 2 (AOA2)

1.6 Neurodegeneration: a consequence of repair defect in the nucleus, mitochondria or both?

1.7 Aims and objectives

Chapter II

iv

Materials and Methods

2.1 Materials

2.2 Cloning and Molecular methods

2.2.1 PCR, restriction digests and ligations

2.2.2 Site-directed mutagenesis

2.2.3 Competent cells and transformations

2.2.3.1 E.coli DH5α cells

2.2.3.2 Transformations

2.3 Electrophoresis and western blot analysis

2.3.1 Electrophoresis of DNA

2.3.2 Electrophoresis of proteins

2.3.2.1 Coomassie blue staining

2.3.2.2 Silver staining

2.4 Western blots

2.5 Protein expression and purification

2.5.1 Protein expression

2.5.2 Protein purification

2.5.2.1 Cell lysis

2.5.2.2 Immobilised metal affinity chromatography (IMAC) purification......... 47

2.6 Biochemical assays

2.6.1 SUMOylation assay

2.6.2 SUMO and TDP1 non-covalent binding studies

2.6.2.1 Recombinant TDP1 binding to SUMO immobilised resin

2.6.3 Casein Kinase 2 phosphorylation

2.7 Protein structure and folding

2.7.1 Circular dichroism

2.7.1.1 Quantitative analysis of circular dichroism by Dichroweb

2.7.2 Protein denaturation curves

2.8 Mammalian cell culture

2.8.1 Maintenance of cell lines

2.8.2 Transfection

2.8.2.1 Calcium phosphate transfection

2.8.2.2 Transfection by electroporation

2.9 Cell harvest and lysis

2.10 Co-immunoprecipitation

2.11 Creating stable cell lines in DT40 Tdp1-/- cells

2.12 Viability assays

2.13 FACS analysis

2.14 DNA single-strand break assays

2.14.1 Alkaline comet assay

2.14.1.1 Preparation

2.14.2 Gyrosyl assay

2.14.2.1 Preparation of oligonucleotide substrates:

2.14.2.2 TDP1 fluorescence assay:

2.15 Mass spectroscopy

Chapter III

Results 1: The N-terminal evolutionarily driven domain of TDP1 is required for optimal cellular protection against genotoxic stress

3.1 Introduction

v

3.1.1 TDP1; discovery, substrate and mechanism of action

3.1.2 TDP1’s role in SCAN1

3.1.3 Chapter objectives

3.2 Results

3.2.1 Full-length TDP1 and the catalytic domain show no difference in activity, structure or stability

3.2.1.1 The C-terminal domain does not demonstrate a reduction in enzymatic activity in vitro

3.2.1.2 TDP1 and TDP1151-608 have no difference in thermal stability, or structure

3.2.2 Full-length hTDP1 is required for optimal protection against DNA damaging agents in DT40 cells, whilst hTDP1151-608 gives partial protection

3.2.2.1 Tdp1-/- DT40 cells, complemented with Myc, Myc-hTDP1 or MychTDP1151-608 maintain similar proliferation rates and cell cycle progression..... 77 3.2.2.2 TDP1151-608 does not exhibit reduced activity in cell extracts................. 77 3.2.2.3 Protection of cell lines treated with different genotoxic agents shows that the TDP1151-608 gives partial resistance when compared to the full-length protein

3.2.3 Accumulation of DNA single-strand breaks in hTDP1151-608 compared to hTDP1, is in part transcription dependent

3.2.3.1 DT40 Tdp1-/- cells, and cells complemented with hTDP1151-608 accumulate more single-strand breaks than full-length hTDP1

3.2.3.2 DT40 Tdp1-/- cells complemented with hTDP1151-608 accumulate more single-strand breaks than full-length hTDP1, and these appear to be transcription dependent

3.3 Discussion

Chapter IV

Results 2: SUMOylation facilitates TDP1 driven chromosomal break repair........ 91

4.1 Introduction

4.1.2 Post-translational modification by SUMOylation

4.1.3 SUMO

4.1.4 SUMOylation

4.1.5 SUMO family

4.1.6 Phospho-dependent SUMO motif

4.1.7 SUMO interacting motifs – SIMS

4.1.8 Molecular consequences of SUMOylation and SUMO-SIM interactions..... 96 4.1.9 Chapter objectives

4.2 Results

4.2.1 TDP1 is SUMOylated at lysine 111 by SUMO1 in vitro and vivo.............. 100 4.2.1.1 TDP1 is SUMOylated in vitro

4.2.1.2 TDP1 SUMOylation occurs within the N-terminus in vitro................. 104 4.2.1.3 TDP1 SUMOylation occurs within the N-terminus at K111 in vitro... 106 4.2.1.4 Mutation of lysine to arginine at 111 abrogates SUMOylation in vitro and in vivo

4.2.2 Mutation of lysine 111 to arginine does not alter protein structure, stability or catalytic activity

4.2.2.1 TDP1 and TDP1K111R give similar melting temperatures and unfolding patterns

vi

4.2.2.2 TDP1K111R does not result in a reduced activity compared to wild-type

4.2.2.3 TDP1K111R and TDP1 exhibit no significant structural differences...... 111 4.2.3 SUMOylation-deficient mutant accrues more DNA single-strand breaks that are in part transcription dependent

4.2.3.1 TDP1 K111R mutant shows reduced survival in the presence of CPT 114 4.2.3.2 TDP1 K111R accumulates more DNA single-strand breaks in the presence of CPT

4.2.3.3 Accumulation of DNA single-strand breaks is in part transcription dependant

4.2.4 TDP1 interacts non-covalently with SUMO2/3

4.2.4.1 TDP1 interacts with SUMO2 in vitro

4.2.4.2 TDP1 interacts with SUMO2 via the catalytic domain in vitro............ 119 4.2.4.3 Identification of SIM/s in TDP1

4.2.4.4 Mutation of the first two amino acids in potential SIM sites results in instability and co-expression with GroEL

4.2.4.5 TDP1 fragments within the catalytic domain causes loss of expression in vivo

4.3 Discussion

Chapter V

Results 3: The role of DNA ligases and CK2 during TDP1 mediated repair........ 132

5.1 Introduction

5.1.2 Ligase family and TDP1

5.1.3 Casein kinase II

5.1.4 Chapter objectives

5.2 Results

5.2.1 Ligase I and not ligase IIIα is implicated in TDP1-mediated nuclear repair136 5.2.1.1 Ligase I deficient cells results in hypersensitivity to camptothecin...... 136 5.2.1.2 The conserved catalytic domain of TDP1 is sufficient to maintain interaction with Lig I

5.2.2 TDP1 is a novel substrate for Casein Kinase II

5.2.2.1 TDP1 is phosphorylated in vivo

5.2.2.2 Phosphorylation of TDP1 occurs in the N-terminal domain................. 141 5.2.3 Phosphorylation of TDP1 by CK2 inhibits SUMOylation, in vitro............. 144 5.2.4 TDP1 is phosphorylated by CK2 in whole cell extracts

5.3 Discussion

Chapter VI

Discussion: Cellular and biochemical analyses of TDP1 mediated chromosomal break repair

6.1 Discussion

Publications

References

–  –  –

A-T ataxia-telangiectasia ATM ataxia-telangiectasia mutated ATR ataxia-telangiectasia mutated and Rad3-related ATRIP ATR interacting protein BER base excision repair BSA bovine serum albumin CDK cyclin-dependent kinase CHX cycloheximide CK2 casein kinase 2 CS Chicken serum CPT camptothecin DAPI 4`6-diamino-2-phenylindole DNA-PK DNA-dependent protein kinase DSB double-strand DNA break DRB 5,6-Dichloro-1-β-D-ribofuranosylbenzimidazole FCS foetal calf serum HR homologous recombination hrs hours HU hydroxyurea IF immunofluorescence IR ionising radiation LCL lymphoblastoid cell line MCPH primary microcephaly MEF mouse embryonic fibroblast

viii

MMS methyl methanesulfonate MRN Mre11-Rad50-Nbs1 NER nucleotide excision repair NHEJ non-homologous end-joining NBS Nijmegen Breakage syndrome PARP Poly (ADP-ribose) polymerase PARPi PARP inhibitor PBS phosphate buffered saline PIKK phosphatidylinositol 3-kinase like kinase RNAi RNA interference ROS reactive oxygen species RPA replication protein A SCAN1 spinocerebellar ataxia with axonal neuropathy 1 SD standard deviation S.E.M standard error of the mean SSB single-strand DNA break ssDNA single-stranded DNA TBS tris-buffered saline TBB 4,5,6,7-Tetrabromobenzotriazole TDP1 tyrosyl DNA phosphodiesterase 1 TOP1 topoisomerase I Ub ubiquitin UNT untreated UV ultra-violet WB western blot WCE whole cell extract WT wild-type XRCC1 X-ray repair cross-complementing protein 1

–  –  –

Figure 1.1 DNA strand break repair

8 Figure 1.2 Outcome of DNA strand breaks if left unrepaired.................

13 Figure 1.3 Enzymes involved in the repair of DNA strand breaks........

14 Figure 1.4 DNA structure and damage modifying 3’ and 5’ phosphate backbone of DNA



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