The DNA mismatch repair (MMR) system is essential for correcting mispaired nucleotides in double-stranded DNA. Defects in the MMR pathway are evidenced by microsatellite instability, elevated mutation rates, enhanced recombination events, and tolerance to cytotoxic effects of alkylating agents. Loss of DNA MMR in humans is correlated with hereditary nonpolyposis colorectal cancer (HNPCC) and a subset of sporadic tumors. Our overall research program focuses on elucidating the full extent of MMR activity within human cells that contributes towards maintenance of genomic integrity. We have previously determined that fidelity of MMR can be substantially altered at an oncogenic hot spot of mutation, and that this is partially dependent on whether the mismatch-containing plasmid is introduced during cellular quiescence or proliferation. It is not yet known if MMR proficient mammalian cells have decreased MMR activity at phases of the cell cycle other than G2. Most investigations of MMR protein expression within mammalian cells, including ours, have demonstrated constitutive expression of the four key MMR mRNAs and proteins throughout the cell cycle; hMSH2 + hMSH6 (hMutS-alpha) and hMLH1 + hPMS2 (hMutL-alpha). Therefore, if MMR activity in human cells correlates with the E. coil post-replication model of MMR, then post-translational modification of these key MMR proteins may play an important role in the modulation of cellular MMR activities. We hypothesize that alterations in efficiency or fidelity of MMR in the human cell are dependent on phase of the cell cycle and require post-translational MMR protein modification. Our first specific aim is to investigate if the process of DNA replication is required for high efficiency and fidelity of MMR. Specifically, does the frequency of correct MMR differ during G1 (quiescent) versus S (DNA synthesis) or G2 (post synthetic) phase of the human cell cycle? Our second specific aim is to analyze phosphorylation/dephosphorylation events of the key MMR proteins at each different phase of the cell cycle and to determine if this post-translational modification plays a significant role in regulation of specific MMR activities.
