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Our group has done tons of work looking at single DNA-protein complexes and protein-protein complexes using AFM. However, there are some limitations to AFM, including the inability to directly measure conformational dynamics or to decipher large multi-protein complexes.
We are currently developing single-molecule FRET methods to study conformational dynamics of DNA-protein complexes as well as to analyze large multi-protein complexes related to DNA repair. |
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| Single-molecule FRET to measure conformational dynamics of DNA induced by mismatch repair factor MutS |
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| Combined AFM-FRET development to study large multi-protein complexes related to DNA repair |
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| Background and Motivation for Single-molecule FRET Work |
| Our group focuses primarily on structure-functions studies of the DNA mismatch repair pathway. |
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| DNA Mismatch Repair Background |
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AFM studies and crystallographic data have been used to study interactions of mismatch repair factor MutS with mismatched DNA. We want to better understand how MutS may recognize a single base mismatch when hundreds upon thousands of correctly-paired bases are present. The crystal structure of MutS bound to mismatched DNA showed that MutS induced a kink in the DNA at the mismatch, which may play a role in how MutS recognizes mismatches over homoduplex DNA. This structure does not explain why some mismatches are repaired more efficiently than others.
Our group sought-out to relate DNA structure with repair efficiency. AFM results showed that when MutS was bound at a DNA mismatch, two DNA conformations ensued: bent and unbent. Because only mismatched DNA displayed the unbent conformation, we developed a model for mismatch repair initiation that suggested that MutS first bent the DNA at the mismatch, and this bent conformation drove the formation of the unbent conformation, which was the conformation that went on to be repaired in the cell. Our model, shown in Figure 1, suggests that it is totally essential for the unbent complex to form for the DNA to be repaired. |
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| Figure 1. Our DNA mismatch repair initiation model. We hypothesize that when MutS finds a mismatch (ie. heteroduplex DNA) it first forms the kinked-specific complex (B), and that conformation drives the formation of the unbent-specific complex (C) which goes on to be repaired. |
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| Because AFM does not give us dynamic changes in the DNA conformation, we are pursuing other techniques to understand the dynamic equilibrium between bent and unbent DNA conformations in the presence of MutS and how this equilibrium is affected by other repair proteins and cofactors in the cell. |
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| We have also used AFM to study how MutS and MutL (the first two repair factors and the most highly conserved of all the repair factors) interact at a mismatch to invoke repair. We found that multi-MutL-MutS complexes form large tracks along the DNA. We are not able to differentiate between MutS and MutL molecules in these large complexes, so we would like to combine AFM with fluorescence to add a new dimension of information to these images. |
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| We are interested in combining AFM with FRET to get a topographical image of complex multi-protein systems as well as a fluorescence image to tell us which protein is which, and how many are involved in the complex. |
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| Read on to see more about these projects. |
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| Single-molecule FRET to measure conformational dynamics of DNA induced by mismatch repair factor MutS |
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| Combined AFM-FRET development to study large multi-protein complexes related to DNA repair |
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