O'Driscoll, Mark John; (1999) Defective mismatch repair and DNA damage sensing in human cells. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Base methylation is a significant contributor to intrinsic DNA damage. Human Mex- cells deficient in the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT), are hypersensitive to killing by methylating agents. DNA O6-meGua-mediated killing requires active mismatch repair (MMR). This property has been exploited to select MMR-defective human cells in the laboratory. Endogenous DNA methylation 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 methylation 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 raciiation, 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.

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