Instability at Trinucleotide Repeat DNAs A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science By RUJUTA YASHODHAN GADGIL M.Sc. Microbiology, Fergusson College, India, 2013 __________________________________________________________________ 2016 Wright State University i WRIGHT STATE UNIVERSITY GRADUATE SCHOOL August 1, 2016 I HEREBY RECOMMEND THAT THE THESIS PREPARED UNDER MY SUPERVISION BY Rujuta Yashodhan Gadgil ENTITLED Instability at Trinucleotide Repeat DNAs BE ACCEPTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Master of Science. ________________________________ Michael Leffak, Ph.D. Thesis Director ________________________________ Madhavi Kadakia, Ph.D. Committee on Final Examination Chair, Department of Biochemistry and Molecular Biology _____________________________ Michael Leffak, Ph.D. _____________________________ John Paietta, Ph.D. ____________________________ Michael Markey, Ph.D. ____________________________ Robert E. W. Fyffe, Ph.D. Vice President for Research and Dean of the Graduate School ABSTRACT Gadgil, Rujuta Yashodhan. M.S. Department of Biochemistry and Molecular Biology, Wright State University, 2016. Instability at Trinucleotide repeat DNAs. Trinucleotide repeats (TNRs) are sequences prone to formation of non-B DNA structures and mutations; undergo expansions in vivo to cause various inherited neurodegenerative diseases. Hairpin structures formed during DNA replication or repair can cause replication fork stalling and if left unrepaired could cause single or double strand DNA breaks. To test and study this hypothesis we have devised a novel two color marker gene assay to detect DNA breaks at TNRs. By inducing replication stress our results show that TNRs are prone to DNA strand breaks and it is dependent on the repeat tract length. Double strand breaks at structured DNA are repaired differently than ‘clean’ DSBs. The cells which undergo breaks die off, possibly due to inability to repair breaks. Translesion polymerases help tolerate DNA damage at TNR region. iii Table of Contents INTRODUCTION..................................................................................................1 Trinucleotide repeats and related disorders........................................................3 Importance of number of repeats........................................................................5 Expansions or contractions.................................................................................8 Translesion polymerases....................................................................................10 MATERIALS AND METHODS..........................................................................13 Cell culture........................................................................................................13 DF2 Myc (CTG/CAG)100/23.............................................................................. 15 DF2 Myc...........................................................................................................15 DF2...................................................................................................................17 PCR...................................................................................................................17 Hydroxyurea, aphidicolin and telomestatin treatment assays..........................18 I-SCE-I transfection.........................................................................................18 Flow cytometry................................................................................................19 Flow cytometry to detect cell cycle phase.......................................................19 siRNA treatment and flow cytometry..............................................................19 iv siRNA treatment and western blot...................................................................20 RESULTS............................................................................................................21 I. Experimental DF2 Myc CTG cell line repeat length..............................21 II. Analysis patterns for flow cytometry......................................................21 III. CTG100 TNRs are unstable due to hydroxyurea replication stress..........22 IV. Recovery is required for separation of markers......................................27 V. Hydroxyurea shows considerable effect as a replication stress inducer..32 VI. Chk1 is activated at basal levels in CTG100 cells after HU treatment......32 VII. Cells display breaks before mitosis has occurred....................................35 + - VIII. Loss of eGFP , RFP cells after stress induced double strand breaks......37 + - IX. eGFP , RFP cells are unstable.................................................................39 X. Short TNRs are stable under replication stress........................................40 XI. DF2 cells are stable under replication stress............................................40 XII. DF2 Myc cells are stable under hydroxyurea replication stress..............43 XIII. CTG100 TNRs are unstable due to apidicolin replication stress..............46 XIV. CTG100 TNRs are unstable due to oxidative stress.................................46 XV. DSBs at structured DNA are treated differently than ‘clean’ DSBs…..48 XVI. DF2-TTR cells are unstable due to Telomestatin and HU………….…53 XVII. DF2-TTF cells are stable in Telomestatin and HU……………………54 XVIII. CTG 100 TNRs are unstable after Telomestatin treatment……………..57 XIX. DF2 Myc cells are stable under replication stress…………………….59 XX. Loss of translesion polymerases in DF2 Myc CTG100 cells affects DNA damage tolerance....................................................................................61 v XXI. Loss of translesion polymerases in DF2 Myc cells does not affect DNA damage tolerance...................................................................................68 XXII. Knockdown of translesion polymerases in DF2 Myc CTG100 cells......74 DISCUSSION................................................................................................78 Trinucleotide repeats as fragile sites..............................................................78 Trinucleotide repeat length is critical.............................................................80 Unrepaired DNA breaks cause cell death or senescence................................81 Structure forming sequences influence repair mechanisms at broken ends of DNA...................................................................................................82 Translesion polymerases help overcome DNA damage and DNA breaks at trinucleotide repeat .....................................................................................83 Translesion polymerases bypass secondary structures at TNR region..........86 FUTURE DIRECTIONS....................................................................................88 REFERENCES...................................................................................................89 vi List of Figures Figure 1. Microsatellite non-B DNA structures............................................................6 Figure 2. Microsatellite instability related human diseases..........................................7 Figure 3. Model showing replication fork arrest due to formation of hairpin structure .............................................................................................................................................9 Figure 4. Chromosomal integration of c-myc origin construct................................... 14 Figure 5. Cell line constructs......................................................................................16 Figure 6. CTG repeat length in engineered cell lines.................................................24 Figure 7. Analysis pattern for flow cytometry...........................................................25 Figure 8. CTG100 TNRs are unstable due to HU replication stress............................26 Figure 9. Recovery is required for separation of markers.........................................28 Figure 10. Recovery is required for separation of markers.......................................29 Figure 11. Recovery is required for separation of markers.......................................30 Figure 12. Recovery is required for separation of markers.......................................31 Figure 13. Hydroxyurea shows considerable effect as a replication stress inducer...33 Figure 14. Chk1 is activated at basal levels in CTG100 cells after HU treatment.....34 vii Figure 15. Cells undergo DNA breakage before they go through mitosis..............36 Figure 16. Loss of eGFP+, RFP- cells after stress induced double strand breaks....38 Figure 17. GFP positive cells are unstable..............................................................41 Figure 18. Short TNRs are not unstable under replication stress...........................42 Figure 19. DF2 cells are stable under HU replication stress..................................44 Figure 20. DF2 Myc cells are stable under hydroxyurea replication stress...........45 Figure 21. CTG100 TNRs are unstable due to aphidicolin replication stress..........47 Figure 22. CTG100 TNRs are unstable due to oxidative stress...............................50 Figure 23. Cartoon of single-sided and double-sided double strand breaks..........51 Figure 24. DSBs at structured DNA are treated differently than clean DSBs.......52 Figure 25. DF2-TTR cells are unstable