Every day each cell in the body is under threat from DNA damaging agents that have the potential to disrupt the passing of intact genetic information from one generation to the next. The presence of a wide variety of threats has led to the evolution of numerous DNA damage response pathways in order to deal with the damage that they may cause. The mismatch repair system is primarily responsible for the removal of incorrect base insertions and deletions occurring during replication and its importance is highlighted by its conservation from bacteria to humans. MLHl is one of the main components of this repair pathway and loss of this protein has been associated with a number of cancers, particularly hereditary colon cancer. Defects in the mismatch repair machinery have also been associated with resistance to a number of chemotherapeutic drugs and instability of the genome at repeat sequences. The present data initially examines the role of MLHl in DNA damage responses following induction of damage by a number of different treatments in telomerase- immortalised human fibroblasts stably depleted of MLHl using an integrated shRNA plasmid. The importance of a number of specific pathways, namely ATM/ATR, caspase, p53 and PARP, involved in the damage response is assessed through the use of inhibitors. A number of studies have shown that loss of the DNA methyltransferase maintenance protein DNMTl initiates p53-dependent cell death in differentiated cells. While the absence of DNMTl is tolerated in undifferentiated embryonic stem cells in terms of viability, these cells exhibit the hallmarks of mismatch repair deficiency, i.e. drug resistance and genomic instability, although transcription levels of the mismatch genes remains unaffected. Depletion of DNMTl in the colon cancer cell line RT29 resulted in induction of rapid cell death which could be ablated through the inhibition of not just p53 but also P ARP. Analysis of the mismatch repair components showed that although transcription levels were not affected, a reduction in DNMTl resulted in the depletion of a number of DNA repair proteins, suggesting that DNMTl is a key protein involved not only in DNA methylation, but also in the stability of the mismatch repair system.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:551590 |
| Date | January 2010 |
| Creators | Dunne, Philip D. |
| Publisher | University of Ulster |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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