The role of homologous recombination repair in the processing of G2-chromosomal breaks and maintenance of G2-checkpoint [Elektronische Ressource] / vorgelegt von Aashish Soni

Ssingh

Sujets

• Biologie

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• Publié par: universitat_duisburg-essen
• Publié le: 01 janvier 2010
• Nombre de lectures: 32
• Langue: Marathi
• Poids de l'ouvrage: 2 Mo

Extrait

The Role of Homologous Recombination Repair in
the processing of G2-chromosomal breaks and
maintenance of G2-checkpoint

Inaugural-Dissertation
zur
Erlangung des Doktorgrades Dr. rer. nat. der Fakultät
Biologie und Geografie
an der Universität Duisburg-Essen
Standort Essen, Germany

vorgelegt von
Aashish Soni
aus
Sagar, Madhya Pradesh, Indien
Juni 2010 Die der vorliegenden Arbeit zugrunde liegenden Experimente wurden
am Institut für Medizinische Strahlenbiologie an der Universität
Duisburg-Essen, Standort Essen, durchgeführt.







1. Gutachter: Prof. Dr. George Iliakis
2. Gutachter: Prof. Dr. Ann Ehrenhofer Murray
Vorsitzender des Prüfungsausschusses: Dr. Juergen Thomale
Tag der mündlichen Prüfung: 27.10. 2010









“An ounce of practice is better than tons of theory” y
(Swami Sivananda Saraswati)




“The thing always happens that you really believe in; and the
belief in thing makes it happen” n
(Frank Lloyd Wright)







Dedicated to
My beloved Shirdi Sai baba

॥ ी सद्रग साईनाथाप णमतर् । शभ भवत ।।
िशरडीस याच लागतील पाय, टळती अपाय सव र् याच
माया समा धी ची पायरी चढ ल, दःख ह हर ल सव र् याच
जरी ह शरीर ग लो मी टाकन, तरी मी धाव न भक्तासाठी
नवसास माझ ी पाव ल समाधी, धरा द्रढ बद्धी माया ठायी
िन य मी िजव ंत जा णा हच ी सय , िन य घ्या प्रचीत अनभव
शरण मज आला आिण वाया ग ला, दाखवा दाखवा ऐसा कोण ी
जो जो मज भज जशा ज शा भाव, तैसा तैसा पाव मीही यासी
तमचा मी भार वाहीन सवथार् , नह ह अयथ ा वचन माझ
जाण ा य थ आह सहाय सवा र्ं स, माग ज ज या स ते ते लाभ
माझा जो जाह ल ा काया वाचा मनी, तयाचा मी ऋणी सवकार् ळ
साई ह ण तोिच, तोिच झाला धय , झाला जो अनय माया पायी

ुेेेेंेुुेेुैेेेुेेेेEेेेूैेेेेेुुंEुेेेTable of content
Table of content
Table of content......................................................................................................... I
List of figures........................................................................................................... IV
List of tables ............................................................................................................. V
List of Abbreviations............................................................................................... VI
1. Abstract................................................................................................................. 1
2. Introduction........................................................................................................... 3
2.1 Ionizing radiation and induction of double strand breaks .......................... 3
2.2 Consequences of DSBs in eukaryotes.......................................................... 5
2.2.1 DSBs and chromosomal aberrations................................................................................. 5
2.2.2 DSBs can also be necessary intermediates in important processes................................. 7
2.3 Mechanisms of DSB repair............................................................................. 7
2.3.1 DDR signalling and cell cycle checkpoints ........................................................................ 8
2.3.1.1 DNA damage sensing .................................................................................................... 8
2.3.1.2 DNA damage signalling.................................................................................................. 9
2.3.1.3 Cell cycle checkpoints ..................................................................................................11
2.3.2 Homologous recombination repair................................................................................... 13
2.3.3 DNA-PK dependent non-homologous end-joining (D-NHEJ).......................................... 16
2.3.4 DNA-PK independent non-homologous end-joining (B-NHEJ) ....................................... 18
2.4 Chromatin remodelling and DSB repair...................................................... 18
2.5 DSB repair pathway selection in different cell cycle phases .................... 20
2.6 Repair deficiency, genomic instability and cancer predisposition........... 22
2.7 Relative roles of repair pathways in DSB repair ........................................ 23
2.8 The goals of the present thesis ................................................................... 25
3. Materials and Methods....................................................................................... 27
3.1 Cell lines ........................................................................................................ 27
3.2 Cell culture and growth conditions ............................................................. 29
3.3 Chemicals and inhibitors ............................................................................. 30
3.4 Irradiation ...................................................................................................... 31

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3.5 Clonogenic survival assay........................................................................... 31
3.6 Cytogenetic assays....................................................................................... 31
3.6.1 Assay to measure the kinetics of G2-chromosome breaks at metaphase: ..................... 32
3.6.2 Premature chromosome condensation (PCCs)............................................................... 32
3.7 Flow cytometry.............................................................................................. 33
3.7.1 Cell cycle analysis by flow cytometry................................................................................... 33
3.7.2 Determination of mitotic index.............................................................................................. 33
4. Results................................................................................................................. 35
4.1 Clonogenic survival assay........................................................................... 35
4.2 The contribution of D-NHEJ and HRR in the repair of IR-induced G2-
chromosome breaks 35
4.2.1 NHEJ deficiency does not affect the repair of chromosome breaks in the G2- phase of
the cell cycle.................................................................................................................................. 37
4.2.2 Is HRR responsible for the repair of G2-chromosomal breaks?...................................... 38
4.2.3 Coupled HRR/NHEJ defects cause increase the initial level of chromatid damage and
inhibit its repair .............................................................................................................................. 38
4.2.4 Is HRR also important for the repair of G2-chromosomal breaks in human cells? ......... 42
4.2.5 Chemical down regulation of Rad51 inhibits the repair of G2-chromosomal breaks in wild
type hamster and human cells ...................................................................................................... 44
4.3 Role of DDR proteins in the repair of G2-chromosomal breaks ............... 46
4.3.1 H2AX and 53BP1 defects do not affect the repair of G2-chormosomal breaks .............. 46
4.3.2 The role of DNA-PK, ATM and ATR in the repair of G2-chromosomal breaks ............... 46
4.3.3 Nbs1 and BRCA1 defects significantly inhibit G2-chromosomal break repair ................ 47
4.3.4 Transitional knock down of CtIP completely inhibits repair of G2-chromosomal breaks in
M059J cells ................................................................................................................................... 49
4.3.5 Chemical inhibition of MRE11 inhibits repair of G2-chromosomal breaks in D-NHEJ
deficient cells................................................................................................................................. 49
4.3.6 Effect of caffeine on the repair of G2-chromosomal breaks ............................................ 49
4.3.7 Effect of a specific ATM inhibitor on the repair of G2-chromosomal breaks ................... 50
4.4 Does the G2-checkpoint play a role in the repair of G2-chromosomal
breaks? 51
4.4.1 The repair of G2-chromosomal breaks is influenced by the G2 checkpoint.................... 53
4.4.2 Study of G2-checkpoint activation and recovery in wild type, D-NHEJ and HRR mutants
56
4.4.2.1 G2-M accumulation after IR in wild type and repair deficient cells .............................. 56

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4.4.2.2 Analysis of the G2-checkpoint in Chinese hamster cells through measurement of
mitotic index (MI) ...................................................................................................................... 57
4.4.2.3 Study of the G2-checkpoint in BRCA1 and BRCA2 deficient human cells by measuring
mitotic index (MI)......... 58
5. Discussion .......................................................................................................... 67
5.1 The role of D-NHEJ in the repair of G2-chromosomal breaks................... 67
5.2 HRR is required for t