? DNA helicases are required for virtually every aspect of DNA metabolism, including replication, repair, recombination and transcription. A comprehensive understanding of these essential biochemical processes requires detailed understanding of the mechanism of helicases. We are studying the Dda helicase, from bacteriophage T4, as a representative of super family (SF) 1 helicases, which is the largest class of these enzymes. In the previous grant cycle, we tested the hypothesis that unwinding of double-stranded (ds) DNA by Dda is largely a consequence of unidirectional translocation on single-stranded (ss) DNA. Our results have supported our hypothesis and the data are consistent with an 'inchworm model' for helicase activity. There are several discrepancies in the details for exactly how the 'inchworm' functions, and it is within this context that the current specific aims have been designed. Our current model for Dda function may explain some of the discrepancies. We suggest that Dda can function as a monomer, however, multiple monomers can cooperate to enhance translocation and unwinding. We term this new model the cooperative inchworm model. The role of cooperativity in the mechanism may be to reduce slippage that occurs when the helicase encounters a challenge to translocation such as duplex DNA or a DNA-binding protein. In the current cycle, we propose to test this new hypothesis, as well as expand the goals of the project as we continue to focus on Dda. We will determine the kinetic step size for DNA unwinding for monomeric and multimeric forms of Dda. We will measure the quantity of ATP hydrolyzed under pre-steady state conditions and in the presence of excess enzyme. Processivity of DNA unwinding will be studied as a function of the number of Dda molecules bound to the substrate. The interaction of DNA with Dda will be investigated by crosslinking coupled with mass spectrometry. Crystallographic and structural modeling studies will be pursued to relate the structure of the helicase to the biochemical function. Lastly, new methods will be developed to observe helicase translocation and unwinding directly in single molecule experiments. ? ?
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