Year of Publication
Doctor of Philosophy (PhD)
Dr. Guo-Min Li
DNA mismatch repair (MMR) is a highly conserved pathway that maintains genomic stability primarily by correcting mismatches generated during DNA replication. MMR deficiency leads to microsatellite instability (MSI), which is a hallmark of HNPCC (Hereditary Nonpolyposis Colorectal Cancer). Human mismatch repair is initiated by MutSα, a heterodimer of MSH2 and MSH6 subunits. Mismatch binding by MutSα triggers a series of downstream MMR events including interacting and communicating with other MMR proteins. The ATPase domain of MutSα is situated in the C-termini of its both subunits, and ATP binding is required for dissociation of MutSα from a mismatch. In eukaryotic cells, a strand break, which resides either 3’ or 5’ to the mismatch up to several hundred base pair away, determines the strand specificity of MMR. However, in spite of extensive studies, the mechanism by which MutSα locates and senses a nick from the mismatch, and coordinates the subsequent steps of MMR remains poorly understood. Two controversial models have been proposed to explain how the mismatch and the strand break communicate each other. Sliding model proposes that MutSα slides along the DNA helix from the mismatch to the strand break in an ATP binding-dependent but not ATP hydrolysis-dependent manner. Stationary model postulates that MutSα remains bound at the mismatch, and a protein-mediated DNA loop forms, physically bringing the mismatch and the nick in contact. Here, we tested these models in vitro, using a circular plasmid DNA substrate with a single GT mismatch and two Lac repressor (Lac I) binding sites as conditional physical 'roadblocks', one on either side of the mismatch, which when present, prevent MutSα from sliding bi-directionally along the DNA. The results showed that DNA excision initiates under conditions that block MutSα sliding, suggesting that initiation of excision is independent of whether MutSα slides from the mismatch to the nick. This result implies that the communication between the mismatch and the nick is likely through interactions between the mismatch-bound MutSα and other MMR components at the strand break, supporting the stationary model. Therefore, these studies provide significant insight into the mechanisms of mismatch correction in human cells.
Lee, Sanghee, "MOLECULAR MECHANISM OF HUMAN MISMATCH REPAIR INITIATION" (2014). Theses and Dissertations--Nutritional Sciences. 15.