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Defective Mismatch Repair and DNA Damage Sensing in Human Cells Defective Mismatch Repair and DNA Damage Sensing in Human Cells. By Mark John O'Driscoll A thesis submitted for the degree of Ph.D. at The University of London June 1999. Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Hertfordshire, EN6 3LD and Department of Biochemistry and Molecular Biology, University College London, Gower Street, London WCIE 6BT. ProQuest Number: U643118 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest U643118 Published by ProQuest LLC(2015). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Abstract. Base méthylation is a significant contributor to intrinsic DNA damage. Human Mex' cells deficient in the DNA repair enzyme O^-methylguanine-DNA methyltransferase (MGMT), are hypersensitive to killing by methylating agents. DNA O^-meGua-mediated killing requires active mismatch repair (MMR). This property has been exploited to select MMR-defective human cells in the laboratory. Endogenous DNA méthylation is a possible contributor to the emergence of MMR defective colorectal tumours. Using cultured GM0637 human fibroblasts I demonstrated that chronic low dose exposure to a N-methyl-N'- nitrosourea can select for, or induce, MMR defects through defective hMSH6 expression. Since defective MMR is associated with some inflammation- associated tumours, I explored the possibility that nitric oxide produced by activated macrophages might promote the formation of a selective agent for méthylation tolerance. Using azaserine, a potential contribution of DNA damage introduced by carboxymethylating agents to the emergence of MMR defective cells was investigated. Cytotoxic DNA damage introduced by azaserine was repaired by nucleotide excision repair (NER). Azaserine cytotoxicity was not influenced by MMR or MGMT to a detectable extent and DNA carboxymethylation is unlikely to provide a significant selection pressure for loss of the MMR pathway. The effects of MMR on the processing of persistent bulky DNA lesions were examined in MNU B4 a MMR deficient derivative of the NER defective XPA fibroblast cell line XP12RO. MNU B4 cells lacked detectable hMSH2 expression. The combined XPA and hMSH2 defects did not affect sensitivity to persistent DNA damage induced by UV, cross-linking agents (cisplatin, mitomycin C, nitrogen mustard), ionizing radiation, and azaserine. MMR does not appear to act as a general DNA damage sensor. I also isolated several MMR defective derivatives of the XPC cell line GM2249. The MMR defects in these derivatives did not detectably alter their sensitivity to UV irradiation. These variants provide an important resource to investigate the proposed role of MMR in the transcription coupled DNA nucleotide excision repair pathway. Acknowledgements. In the first instance I must express my deepest gratitude to my principle supervisor at ICRF, Dr Peter Karran, for his advice, guidance, debate and encouragement during the drafting of this thesis, and right throughout my time at Clare Hall. I also want to express my gratitude to my principle supervisor. Prof. Peter Swann, for his interest in and encouragement of my work. I would also like to acknowledge everyone in our laboratory, members past and present, for all those great years, Edel O'Reagan, Masami Yamada, Alessandra Bearzatto, Odile Humbert, Peter MacPherson, Shinya Oda, Murielle Masson, Andy Massey (for all the computer help), Richard Hampson and especially Pauline Branch, for keeping me going during those long, long hours of tissue culture. I would also like to thank the people who aided me in completing the work described in this thesis, especially Arron Rae and Derek Davies at the ICRF FACS laboratory, to Lloyd Kelland and Ciaran O'Neill for the atomic absoportion spectroscopy w ork, to S. Martinelli and C. Ciotta for the excellent SCE work on XP12RO and MNU B4, and also to Dr David Shuker for the potassium diazoacetate. I would like to acknowledge the many individuals at Clare Hall, far too many to mention, who combine to make it one of the best environments in which to work. I am especially grateful to all in the Cell Production Unit in Clare Hall. This thesis is for Susan Toft, my words are inadequate. Publications. M. O'Driscoll, 0. Humbert, P Karran. DNA Mismatch Repair. Nucleic Acids and Molecular Biology 12 (1998), 173-197. M. O’Driscoll, S. Martinelli, C. Ciotta, P. Karran. Combined mismatch and nucleotide excision repair defects in a human cell line: mismatch repair processes méthylation but not UV- or ionizing radiation- induced DNA damage. Carcinogenesis 20{5) (1999), 799-804. M. O’Driscoll, P. MacPherson, Yao-Zhong Xu, P. Karran. The cytotoxicity of DNA carboxymethylation and méthylation by the model carboxymethylating agent azaserine in human cells. Carcinogenesis 20(9) (1999), 1855-1862.. Chapter 1 Introduction...................................................................................................................................23 DNA Damage........................................................................................................................24 Spontaneous DNA Damage ................................................................................................ 25 Hydrolytic Base Loss.............................................................................................................25 Deamination..........................................................................................................................25 Oxidative Damage ...............................................................................................................26 Spontaneous Méthylation Damage....................................................................................... 27 Base misincorporation...........................................................................................................28 Induced DNA Damage. ....................................................................................................... 29 Physical DNA Damage. ..................................................................................................... 29 UV light Induced DNA Damage .............................................................................................29 Ionizing Radiation Induced DNA Damage ............................................................................. 30 Chemically Induced DNA damage .......................................................................................32 Simple alkylating agents....................................................................................................... 32 Prevention of DNA Damage ..................................................................................................36 Cellular antioxidants ..............................................................................................................36 Cellular Thiols ..................................................................................................................... 37 Repair of DNA Damage...................................................................................................... 38 Direct Damage Reversal .....................................................................................................39 DNA Photolyase................................................................................................................... 39 Alkyltransferases.................................................................................................................. 40 Alkyltransferase in Bacteria ...................................................................................................42 The adaptive response to alkylation damage in E.coli: the Ada protein .......................................................................................... 42 The Ogt Protein .................................................................................................................... 45 Alkyltransferases in lower eukaryotes ................................................................................... 46 Mammalian Alkyltransferase................................................................................................. 47 Is there an adaptive response to alkylating agents in human cells? ........................................................................................... 48 Regulation of O^-alkylguanine-DNA-alkyltransferase levels in human cells ................................................................................................49 MGMT knockout mice ........................................................................................................... 50 Excision and Resynthesis DNA repair ...................................................................................50 Base Excision Repair............................................................................................................51 3-Methyladenine repair in £ coli...........................................................................................51 Repair of 3-Methyladenine in Eukaryotes ............................................................................
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