Dynamic Fluorescence Studies of DNA-Protein Complexes

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DESCRIPTION (provided by applicant): The goal of this project is to understand the mechanisms by which DNA polymerases achieve high fidelity DNA replication, using the Kienow fragment of E. coli (pol I family) and RB69 bacteriophage DNA polymerase (p01alphaI family) as model systems. Single-molecule fluorescence methods will be developed to monitor large scale motions of protein domains and of the DNA substrate that occur during nucleotide selection and exonucleolytic proofreading. A detailed description of the conformational dynamics of DNA polymerases will lead to a deeper understanding of how these enzymes regulate and coordinate their different activities to achieve high fidelity DNA replication. We will investigate how the different domain arrangements in Kienow fragment and RB69 polymerase determine the pathway by which DNA is transferred between polymerase and exonuclease active sites during proofreading. Mutations will be introduced into the tip and base of the thumb subdomain to determine whether this domain helps to guide the DNA substrate between the two sites. The role of duplex melting in active-site switching will be investigated for both polymerases. Conformational dynamics of the fingers subdomain will be observed in the presence and absence of nucleotide substrates to elucidate the role of the fingers in nucleotide selection. The rate of template base flipping will be measured to determine whether these transitions are triggered by closure of the fingers. We will also test the hypothesis that finger domain movements control the rate of exonucleolytic proofreading by modulating the site-switching kinetics.