Investigating DNA Mismatch Repair Through Single-Molecule Approaches

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Intellectual Merit:During DNA synthesis in living cells, potential for erroneous incorporation of nucleobases (e.g. introduction of a mismatch such as a thymine instead of a cytosine across from a guanine) is as high as 10% per nucleotide. These pro-mutagenic DNA alterations need to be removed through post-replication mismatch repair (MMR) systems. Malfunction of MMR has disastrous consequences. Even though tremendous progress has been made in elucidating DNA mismatch repair, mainly through biochemical studies, many critically important details of DNA repair mechanisms remain obscure and controversial, even for bacterial (E. coli) MMR system, which is the simplest and best characterized. This is because MMR involves an orchestrated activity of many proteins, which form transient complexes that are difficult to monitor in real time. In the previous project, atomic force microscopy (AFM) was used to directly visualize and examine the behavior of MutS, the key E.coli MMR protein that detects mismatched bases. Those AFM results, which reconciled the main features of previous, mutually exclusive models, provided new insights into the mechanism by which E.coli MMR is initiated. The main goal of the current research is to further unravel the mechanism of E. coli mismatch repair through the application of a suite of single-molecule techniques such as AFM,Optical Tweezers (OT) and single-molecule fluorescence resonance energy transfer (SMFRET). Specifically, AFM imaging and volumetric measurements will be used to examine the structure of MutS tetramers and MutS complexes with MutL to determine the oligomeric status of both proteins in ternary complexes with heteroduplex DNA in the absence and presence of adenine nucleotides. AFM force spectroscopy will be used to measure the interactions within MutS dimers as well as the effect of adenine nucleotides on these interactions, while optical tweezers will be used to measure similar interactions within MutS tetramers as well as between MutS tetramers and DNA. MutS-controlled DNA looping mechanisms and pathways will be thoroughly examined using Optical Tweezers. SM-FRET will be used to follow, in real time, the position of the MMR proteins relative to key DNA sites (the mismatch site and strand signal site) and the distance and contact between these key DNA sites will be probed during the MMR reaction by all three techniques through mechanical and optical (fluorescence) detection. These single-molecule approaches will test a new MMR signaling model, in which the mismatch site and the site of DNA incision are brought into contact by the concerted work of two MutS dimers through a DNA looping-sliding mechanism. These approaches promise to unravel the details of the MMR mechanism that so far have proved very difficult to elucidate by traditional methods. The new results will significantly expand the knowledge base about one of the key mechanisms that is responsible for the maintenance of genomic stability and will lay the groundwork for future studies of eukaryotic MMR including human MMR. Methodologies developed for this model system may prove useful for studying other DNA repair systems and more generally to investigate protein-protein and DNA-protein interactions.Broader impacts. This project will provide an exciting education and research opportunity for two graduate students and one undergraduate student. Because the project crosses several disciplines (biophysics, biochemistry, molecular biology, mechanics, single-molecule instrumentation) it will provide a rich learning experience for all people involved, junior and senior researchers alike. It will expose engineering students to important problems at the interface between modern biology and nanoscience. One of the graduate students involved will visit South Korea to receive training in sophisticated single-molecule optical methods to be used in this research. This international exchange will be beneficial to expanding international collaborations between leading US and foreign research and educational institutions. The potential benefit to society is in the increase in fundamental knowledge of mechanisms by which pre-mutagenic lesions are repaired in DNA. Outreach activities will involve K-12 students, their teachers and the general public.