Mismatch repair (MMR) is a highly conserved genome maintenance process that primarily resolves polymerase misincorporation errors. Mutation of the MMR genes dramatically increase spontaneous mutation rates and have been linked to adaptation as well as associated multi-drug resistance in several pathogenic bacteria. MMR mutations in humans are the cause of the common human cancer predisposition Lynch syndrome as well as numerous sporadic cancers. MMR is an excision-resynthesis process that is initiated at a DNA strand break, which may be hundreds to thousands of nucleotides away from the mismatch. The fidelity of MMR depends on establishing the ssDNA break on the error-containing DNA strand. A subset of ?-proteobacteria, including E.coli and the ESKAPE pathogens Klebsiella and Enterobacter, recently evolved DNA adenine methylation (Dam) and MutH to introduce a ssDNA break onto the newly replicated strand containing a misincorporation error. An important distinction between bacteria that utilize the Dam/MutH MMR Pathway and all other organisms is that dam mutations are lethal in combination with mutation of several homologous recombination genes (synthetic lethality). Similarly, mutations of MMR excision components are lethal in combination with replication editing gene mutations. The mechanisms that lead to MMR-dependent synthetic lethality are largely hypothetical. Deterministic models have historically underpinned MMR mechanisms, where definite complexes and progressions are proposed to complete the biochemical events. Using newly developed single molecule- imaging techniques we showed that the initial steps of MMR rely on random DNA diffusion mechanics that are modulated by the two most highly conserved MMR proteins in terrestrial biology, MutS and MutL. This new application will test the hypothesis that the entire multi-component MMR excision process is Stochastic (fully governed by random processes; the complete opposite of Deterministic). Understanding such biochemical randomness should impact future research and therapeutic strategies targeting MMR. We propose to use real-time single molecule imaging in vitro and in vivo to resolve the mechanics of E.coli MMR and its role in synthetic lethality.
The Specific Aims are: 1.) quantitative biophysical imaging of individual MMR component activities, 2.) visualization of the complete MMR excision reaction on single mismatched DNA molecules, and 3.) analysis of MMR component interactions in live cells.
DNA mismatch repair (MMR) mutations have been linked to adaptation and associated multi-drug resistance in pathogenic bacteria. Many of these clinically relevant bacteria including E.coli have recently evolved DNA adenine methylation (Dam) and MutH as integral components of their MMR process. We propose to utilize innovative real-time imaging tools to visualize the molecular details of E.coli MMR to ascertain the detailed mechanics and identify potentially useful therapeutic patterns.
