Generation of Mismatch Repair Fusion Proteins for Single-Molecule Analysis
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Date
2014-05
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The Ohio State University
Abstract
Single molecule visualization studies require proteins that are labeled with known fluorophores at known locations on the molecule. This project examines the viability of one method that is capable of specifically and efficiently labeling proteins with fluorophores. The labeling method uses the Formylglycine Generating Enzyme (FGE) that recognizes a hexa-amino acid sequence and catalyzes the conversion of a central cysteine into a formyl-glycine containing a reactive aldehyde group. The converted aldehyde may be used in conjunction with Hydrazino-Pictet-Spengler (HIPS) ligation to attach a high quantum yield fluorophore such as Cy3 or Cy5 to a specific site within any protein. We used MutS as an initial test of this method. The fusion cloning method was then extended to the eukaryote PCNA trimer. We then cloned and modified the RecJ protein to more fully test the labeling methodology. RecJ is a 5'-3' exonuclease that functions in conjunction with MutS and MutL to execute bacterial MMR. To date, no exonuclease has been labeled and visualized for single molecule analysis. Finally, in order to test whether fluorophore-labeling RecJ affected its activity, we developed a novel fluorophore-based exonuclease assay.
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Keywords
Mismatch Repair, Formylglycine Generating Enzyme (FGE), Single-Molecule Analysis, MutS, PCNA, RecJ