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Mapping Topoisomerase IV Binding and Activity Sites on the E. Coli Genome Hafez El Sayyed

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Mapping Topoisomerase IV Binding and Activity Sites on the E. Coli Genome Hafez El Sayyed Mapping Topoisomerase IV Binding and Activity Sites on the E. coli genome Hafez El Sayyed To cite this version: Hafez El Sayyed. Mapping Topoisomerase IV Binding and Activity Sites on the E. coli genome. Biochemistry, Molecular Biology. Université Paris-Saclay, 2016. English. NNT : 2016SACLS362. tel-01591487 HAL Id: tel-01591487 https://tel.archives-ouvertes.fr/tel-01591487 Submitted on 21 Sep 2017 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. NNT : 2016SACLS362 THESE DE DOCTORAT DE L’UNIVERSITE PARIS-SACLAY PREPAREE A L’UNIVERSITE PARIS-SUD AU SEIN DU CENTRE INTERDISCIPLINAIRE DE RECHERCHE EN BIOLOGIE ECOLE DOCTORALE N° 577 Structure et dynamique des systèmes vivants (SDSV) Spécialité de doctorat : Science de la vie et de la santé Par M. Hafez El Sayyed Mapping Topoisomerase IV Binding and Activity on the E. coli genome Thèse présentée et soutenue au Collège de France, le 26 Octobre 2016 Composition du Jury : M. Confalonieri, Fabrice Pr. I2BC, Orsay, France Président Mme Lamour, Valérie Dr. IGBMC, Illkirch, France Rapporteur M. Nollmann, Marcelo Dr. Centre de Biochimie Structurale, Montpellier Rapporteur M. Koszul, Romain Dr. Institut Pasteur, France Examinateur Mme Sclavi, Bianca DR. ENS, Cachan Examinateur M. Espéli, Olivier Dr. CIRB, Collège de France, Paris Directeur de thèse Acknowledgements I would like to start off by thanking the Espeli Family for really making me feel like I am home. To Olivier, with my utmost sincerity and respect, I would like to thank you for giving me the chance to embark in this PHD adventure. Olivier, I know , I know I am not the easiest to work with and that I am a living hurricane on the bench(es), but it was your patience , understanding , support and guidance that made me a better researcher and I have learned so much thanks to you. Not to flatter you, but you left a milestone in my life and I can’t find the words to express my gratitude. I was blessed to have Ingrid as my first internship student. Thank you for sharing my excitement for Topoisomerases and for all the madness we spread in the lab. You are an amazing and superb student and you will be a great scientist. Elise, we baby-stepped in this lab together, and i t was a pleasure to work with you in the lab. I promise if I am to work in the North Pole, I would come for your thesis defense. Thank you for the help, for being a great friend, for playing “Spot the Cup”, and for having the patience whenever I “accidently” invaded your bench. The same could be extended for Charléne, Victoria and Charlie. Girls, thank you for being you and for really making every second in the lab a living pleasure. Oh Charlie, despite the short time you spent in the lab, you have been an inspiration and the voice of reason whenever needed, your advice and remarks were priceless. Cheers! Not to forget also, are Sylvie and Gaȅlle, who offered valuable discussions and stimulating conversations over the years. Special thanks goes to, Charles Bou Nader for helping me plan, propose, and execute protein purifications and all related biophysics experiments downstream. I would like to state that despite not being a microbiologist, he showed an unequivocal enthusiasm for the project. Cheers Bro! I could not have done it without you. Not to mention, the joyful disposition he has been to me at a personal level. College de France, is the land of sweethearts, I thank each and every person I interacted with on a daily basis for the past three years, especially Fred, Marion, and Mélanie for lighting my days with floods of Kindness and sincerity. With your busy schedules, and coming from faraway places in France I am Thankful and honored to have such esteemed, recognized researchers as my thesis committee. Especially, my thesis examiners, Dr. Valérie Lamour and Dr. Marcelo Nollmann, for taking the time to critically read my manuscript. The reason I am here, the reason I grew up to be who I am today, the love and happiness I possess, are the outcome of my great parents’ sacrifices. Dad you have sacrificed all you have for us to get higher education. You worked 18 hours per day for us to be respectable and successful people. You even sold a piece of land and took many loans to pay for our tuition in schools and universities we could not afford. Mom, you sacrificed your engineering career to give us a good upbringing. You nurtured me and my brothers, you were an ideal mother and you are my role model, and no matter what I say it will never be enough. To my wonderful wife Nadia, if I am to count my blessing, you would be the most precious. Being married to a researcher is not an easy life. Nevertheless, despite practically living in the lab, you never complained and supported me. No matter how far I will get in life, no prizes, nor recognition, tempt me for you are the prize of my life and I will cherish you as long as I live. I had a dream, even before I started my PHD, that my son or daughter would be there the day of my PHD defense. Noé, my sweet lovely son, I could not be happier to have you by my side in this day that I dreamt about for years. Life is not easy, but just by the mere look of you, I find the strength to do the impossible. I hope that you will be proud of your father one day ,and that I can be the father you would like me to be. You are the definition of joy and for that I dedicate my thesis, my life, and my being to serve your innocent smile all my life. I hope I did not forget anyone, and If I did, these are intense times forgive me. Yours sincerely ………….. Table of contents List of figures 5 I-Introduction 7 1.1 The DNA double Helix 12 1.1.1 Aalternative DNA structures 15 1.2 DNA supercoiling 16 1.2.1 Different Types of DNA Supercoils 18 1.3 Regulation of supercoiling homeostasis 20 1.3.1 Modulation of supercoiling during gene expression 20 1.3.2 Impact of NAPs on supercoling homeostasis 23 1.3.3 Regulation of supercoiling during DNA replication 23 1.3.3.1 Positive supercoils and precatenanes 23 1.3.3.2 DNA knots 26 1.3.3.3 Fork regression . 27 1.4 Topoisomerases 29 1.4.1 Toposiomerase IA 30 1.4.1.1 Topoisomerase I 32 1.4.1.2 Topoisomerase III 33 1.4.2 Type IIA topoisomerases 34 1.4.2.1 Topoisomerase IIA inhibitors 37 1.4.2.2 Coumarins and Subunit B 37 1.4.2.3 Quinolones and Subunit A 38 1.4.3 DNA Gyrase 40 1.4.3.1 Roles of DNA gyrase 41 1.4.3.2 Structural insight for substrate specificity 44 1.4.4 Topoisomerase IV 45 1.4.4.1 Strucutral insight in Topo IV chirality sensing 46 1 1.5 The cell cycle 50 1.5.1 B, C, D Periods 50 1.5.1.1 Tools to study the cell cycle 53 1.5.2 DNA Replication 55 1.5.2.1 Initiation of replication 55 1.5.2.2 Fork progression 58 1.5.2.2.1 Fork progression roadblocks 59 1.5.2.2.2 Regulation of fork progression by topisomerases 61 1.5.2.3 Replication termination 62 1.5.3 Insights in chromosome segregation 64 1.5.3.1 Sister chromatids segregation 66 1.5.3.2 Segregation of the terminus 67 1.5.3.2.1 Segregation of fully replicated Chromosomes 68 1.5.3.2.2 Chromosome dimer resolution 68 1.6 Segregation is under the control of many Topo IV partners 71 1.6.1 Topo IV suppressors 71 1.6.2 Topo IV and FtsK 72 1.6.3 Topo IV and SeqA 72 1.6.4 Topo IV and MukB 73 1.7 Genome-wide analysis of genome-architecture organizers 76 II-Thesis objectives 79 III-Results 82 3.1 Publication 83 3.2 Complementary results 84 3.2.1 Validation of ChIP-Seq results 84 3.2.2 Cell cycle analysis on synchronized cell population 86 3.2.3 Peak Identification in ChIP-seq and NorflIP 86 3.2.4 Interpretation of ChIP-seq signals over repetitive elements 89 2 3.2.5 Analysis of ChIP-seq and NorflIP results signal 89 3.2.5.1 Topo IV cleavage is near transcription units 89 3.2.5.2 Topo IV binding and Cleavage sites distribution relative to GC Bias 92 3.2.5.3 Topo IV distribution relative to GATC sites 95 3.2.6 Modeling Topo IV activity relative to cell cycle 97 3.2.7 Characterization of the Topo IV-XerC interaction in vitro 100 3.2.8 Topo IV cleavage at dif is context sensitive 104 3.2.9 Study of the implication of Topo IV, Topo III, and XerC in chromosome segregation 105 3.2.9.1 Role of Topo III in Chromosome segregation 106 3.2.9.2 Topo III is unable to remove chromosome Dimer in the absence of XerC but still shows a role in chromosome segregations 112 3.2.9.3 Extra-genomic expression of XerC or its catalytic mutant (XerCcat) enhances survival of parEts ΔxerC at the semi-permissive temperature but not the non-permissive 116 3.2.9.4 Impact of dif site deletion on cell fitness and morphology 118 IV- Discussion 120 4.1 Genomic profile of Topo IV binding and cleavage 121 4.1.1 Topo IV binding profile 121 4.1.2 Genomic profile of Topo IV cleavage 124 4.2 Does bacterial chromatin influence Topo IV cleavage and binding? 125 4.3 Dif and the control of decatenation 127 4.3.1 Role of Topo IV at dif 127 4.3.2 XerC-Topo IV direct interaction 129 4.4 Role of Topo III in chromosome segregation 130 4.5 Model of Topo IV binding and activity on the E.
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