The function of backup pathways of non-homologous end joining in relation to growth state and lesion quality in cells of higher eukaryotes [Elektronische Ressource] / vorgelegt von Satyendra Kumar Singh

Ssingh

Sujets

• Biologie

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• Publié par: universitat_duisburg-essen
• Publié le: 01 janvier 2010
• Nombre de lectures: 65
• Langue: English
• Poids de l'ouvrage: 4 Mo

Extrait

The Function of Backup Pathways of Non-Homologous End Joining
in Relation to Growth State and Lesion Quality in Cells of Higher
Eukaryotes


Inaugural-Dissertation
zur
Erlangung des Doktorgrades Dr. rer. nat. der Fakultät
Biologie und Geografie
an der Universität Duisburg-Essen
Standort Essen, Germany
vorgelegt von
Satyendra Kumar Singh
aus
Mirzapur,Uttar Predesh, 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. Ralf Küppers
3. Gutachter: Prof. Dr. Peter Bayer
Vorsitzender des Prüfungsausschusses: Prof. Dr. Ralf Küppers
thTag der mündlichen Prüfung: August 5 2010










“Try not to become a man of success, but rather try to become a man of
value.” Albert Einstein


“There are two kinds of people; those who do the work and those who take the
credit. Try to be in the first group; there is less competition there.”
Indira Gandhi







Dedicated to
My dearest Brother Sumit Kumar Singh
(10 Nov 1981 – 25 July 2008)

“The saddest summary of a life contains three descriptions: could have, might have,
and should have.” Louis E. Boone
Table of content
Table of content
Table of content............................................................................................................................... I
Figures and Tables....................................................................................................................... VI
Abbreviations. IX
Abstract.........XII
1. Introduction..1
1.1. Preamble ........................................................................................................................................... 1
1.2. Homologous Recombination Repair (HRR) .................................................................................. 2
1.3. Non-homologous end joining (NHEJ) ............................................................................................ 5
1.3.1. Detection of DSBs and tethering of DNA ends.......................................................................................... 6
1.3.1.1. The Ku70/Ku80 heterodimer .............................................................................................................. 6
1.3.1.2. DNA-PKcs........... 6
1.3.2. Processing of DNA Ends............................................................................................................................ 9
1.3.2.1. Artemis ............................................................................................................................................... 9
1.3.2.2. DNA polymerase μ and λ ................................................................................................................... 9
1.3.2.3. Polynucleotide Kinase (PNK)........................................................................................................... 10
1.3.2.4. Other DNA end processing enzymes................................................................................................ 10
1.3.3. Ligation of the DNA ends ........................................................................................................................ 11
1.3.3.1. The Ligase IV-XRCC 4 complex ..................................................................................................... 11
1.4. Back up pathways of NHEJ (B-NHEJ) ........................................................................................ 11
1.4.1. The Mre11-Rad50-Nbs1 (MRN) complex and CtIP ................................................................................ 13
1.4.2. Poly (ADP-ribose) polymerase 1, Xrcc1 and DNA ligase III................................................................... 14
1.4.3. Histone H1................................................................................................................................................ 15
1.5. Regulation of DSB repair pathway choice by DNA-PKcs.......................................................... 15
1.6. Growth factor signaling and DNA double strand break repair................................................. 17
1.7. Defining Characteristics of IR-induced DNA damage................................................................ 20
1.7.1. Linear energy transfer (LET).................................................................................................................... 20
1.7.2. Complex lesions induced by IR................................................................................................................ 22
1.7.3. Principles of measurement of clustered DNA lesions .............................................................................. 23

I Table of content
1.8. IR induced heat labile sites and their contribution to DSB induction....................................... 24
1.8.1. Measurement of prompt DSBs in mammalian cells ................................................................................. 24
1.9. Heavy ion irradiation facility at the GSI in Darmstadt.............................................................. 26
1.10. Aim and Scope of the work ......................................................................................................... 27
1.10.1. Cell cycle dependent fluctuations of B-NHEJ activity ........................................................................... 28
1.10.2. The Growth state dependence of B-NHEJ.............................................................................................. 28
1.10.3. Determination of B-NHEJ activity by PFGE after exclusion of HLSs................................................... 28
2. Materials and Methods..............................................................................................................30
2.1. Material........................................................................................................................................... 30
2.1.1. Laboratory Apparatus 30
2.1.2. Disposable Elements.. 31
2.1.3. Chemical Reagents.... 32
2.1.4. Antibodies.................. 34
2.1.5. Buffers and solutions.34
2.2. Methods............ 37
2.2.1. Cell lines and cell culture ......................................................................................................................... 37
2.2.2. Radiation................................................................................................................................................... 38
2.2.2.1. X-ray irradiation (low LET).............................................................................................................. 38
2.2.2.2. Heavy ion irradiation (high LET) ..................................................................................................... 38
2.2.3. The GSI Accelerator Facilities ................................................................................................................. 39
2.2.4. Cell sorting methods.. 40
2.2.4.1. Cell cycle analysis by flow cytometry .............................................................................................. 40
2.2.4.2. Cell freezing...................................................................................................................................... 41
2.2.4.3. Cell staining for sorting .................................................................................................................... 41
2.2.4.4. Cell sorting......... 42
2.2.5. Centrifugal elutriation 42
2.2.6. Asymmetric field inversion gel electrophoresis (AFIGE) ........................................................................ 42
2.2.7. Methods to obtain plateau phase culture................................................................................................... 44
2.2.7.1. Normal growth into a plateau-phase ................................................................................................. 44
2.2.7.2. Artificially generated non-growing cells .......................................................................................... 44
2.2.8. Clonogenic survival assay ........................................................................................................................ 45
2.2.9. Methods to monitor Heat Labile Sites (HLSs) ......................................................................................... 45
2.2.9.1. Low temperature lysis....................................................................................................................... 45
2.2.9.2. Measurement of induction of HLSs induction into DSBs at different temperature .......................... 46

II Table of content
2.2.10. Electrophoresis and Immunobl