DNA mismatch repair (MMR) recognizes and repairs bases incorrectly incorporated during DNA replication. Germ line mutations in two MMR genes, namely hMSH2 and hMLHl, account for approximately 98% of hereditary non-polyposis colorectal cancers. There is conflicting evidence for the role of hMLHl and hMSH2 in the transcription-coupled repair (TCR) pathway of nucleotide excision repair (NER). Here we have examined the role of these MMR genes in NER using two reporter gene assays. AdHCMVlacZ is a replication-deficient recombinant adenovirus that expresses the B-galactosidase reporter gene under the control of the human cytomegalovirus (HCMV) immediate-early promoter. We have reported a reduced host cell reactivation (HCR) for B-galactosidase expression of UVC-irradiated AdHCMVlacZ in TCRdeficient Cockayne syndrome (CS) fibroblasts compared with normal fibroblasts, indicating that HCR depends, at least in part, on TCR. In addition, we have reported that UVC-enhanced expression of the undamaged reporter gene is induced at lower UVC fluences to cells and at higher levels after low UVC fluences in TCR-deficient compared with normal human fibroblasts, suggesting that persistent damage in active genes triggers increased activity f~om the HCMV-driven reporter construct. We have examined HCR and UV-enhanced expression of the reporter gene in hMLHl-deficient HCT116 human colon adenocarcinoma cells and HCT116-chr3 cells (the MMR-proficient counterpart of HCT116) as well as hMSH2-deficient LoVo human colon adenocarcinoma cells and their hMSH2-proficient counterpart SW 480 cells. We show a greater UV -enhanced expression of the undamaged reporter gene after low UVC exposure in HCT116 compared with HCT 116-chr3 cells ;md in Lo Vo compared with SW 480 cells. We show also a reduced HCR in HCT 116 compared with HCT 116-chr3 cells and in Lo Vo compared with SW 480 cells. However, the reduction in HCR was less or absent when cells were pretreated with UVC. These results suggest that detection of an involvement of hMLHl and hMSH2 in TCR is dependent on UVC (254 nm) fluence to cells.
We have also used these two reporter gene assays to examine the role of hMSH2 and hMLHl in the repair of oxidative DNA damage induced by UV A light (335-400 nm) and H20 2• UV A and H20 2 produce a number of oxidative lesions in DNA (such as 8hydroxyguanines and thymine glycols) that are repaired by the base excision repair (BER) pathway. \ve show a reduced HCR for B-galactosidase expression of UVAtreated AdHCMVlccZ in hMSH2-deficient LoVo human colon adenocarcinoma cells compared to their hMSH2-proficient counterpart SW480 cells, but not in hMLHl-deficient HCT116 human colon adenocarcinoma cells compared to the hMLHl-proficient HCT116-chr3 cells. We also show that pre-treatment of cells with UVA enhances reporter gene expression to higher levels and at lower UV A fluences in Lo Vo compared to SW480 cells but not in HCT116 compared to HCT116-chr3 cells. These results suggest an involvement of hMSH2 but not hMLHl in the repair of UVA-induced oxidative DNA damage. In contrast, no detectable differences were observed between SW480 and LoVo cells, as well as HCT116-chr3 compared to HCT116 cells, in both of the reporter gene assays that used H20 2 as the DNA damaging agent. Based on these results, these findings suggest that neither hMSH2 nor hMLHl play a significant role in the repair of oxidative damage induced by H202. / Thesis / Master of Science (MS)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/23506 |
| Date | 09 1900 |
| Creators | Lee, David F. |
| Contributors | Rainbow, A.J., Biology |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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