Date of Completion
mismatch repair, colon cancer
Field of Study
Doctor of Philosophy
Lynch syndrome is the most common form of hereditary colorectal cancer. It is caused by genetic mutations in the DNA mismatch repair (MMR) pathway. The MMR pathway is a cellular mechanism for repairing nucleotide mismatches that arise during DNA replication. Generally, MSH2-MSH6 heterodimers bind mismatches then are themselves bound by a second mismatch repair heterodimer, MLH1-PMS2. This complex regulates an exonuclease, Exo1, to remove the mismatch-containing DNA that is re-synthesized and sealed by DNA polymerase δ and DNA ligase. There are multiple models to explain the specific MMR molecular mechanism including the molecular switch model. In this model, mismatch binding acts as an exchange factor that reduces ADP affinity and increases ATP affinity in MSH2-MSH6. In the molecular switch model, the form of MSH2-MSH6 that is not bound to DNA and ATP is incapable of initiating downstream repair, thus MMR is “off”. The DNA and ATP-bound form of MSH2-MSH6 is capable of initiating repair, thus MMR is “on”. The MMR pathway also acts as a sensor for some kinds of DNA damage and can activate a cell cycle arrest and apoptosis response. The DNA methylating agent, N-methyl-N’-nitro-N-nitrosoguanidine (MNNG), causes O6-methylguanine (O6-me-G) lesions that strongly activate the MMR-dependent DNA damage response by causing O6-me-G:T mismatches. Most work on the MMR pathway has been in vitro biochemistry in lower organisms. Because of this, there is a substantial knowledge gap about the molecular mechanism of MMR in vivo in humans. Understanding the cellular MMR mechanism in repair and the damage response will have the greatest impact clinically for Lynch syndrome patients. Using three MSH2 mutants in the ATPase domain of the protein that we hypothesized would impair the molecular switch model of MMR at different steps, we restored expression of MSH2 in an MSH2-null cell line. Using in vivo chromatin localization, MMR and cell cycle arrest assays, we generated data that support the molecular switch model of MMR in vivo in humans for both repair and activation of the MMR-dependent DNA damage response to MNNG lesions.
Brown, Graham D., "Biochemical and Cellular Studies of Human MSH2 ATPase Domain Mutants Support the Molecular Switch Model of Mismatch Repair In Vivo" (2014). Doctoral Dissertations. 302.