? 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. ? ?

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Biochemistry Study Section (BIO)
Program Officer
Ikeda, Richard A
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of Arkansas for Medical Sciences
Schools of Medicine
Little Rock
United States
Zip Code
Byrd, Alicia K; Matlock, Dennis L; Bagchi, Debjani et al. (2012) Dda helicase tightly couples translocation on single-stranded DNA to unwinding of duplex DNA: Dda is an optimally active helicase. J Mol Biol 420:141-54
Eoff, Robert L; Raney, Kevin D (2010) Kinetic mechanism for DNA unwinding by multiple molecules of Dda helicase aligned on DNA. Biochemistry 49:4543-53
Perumal, Senthil K; Raney, Kevin D; Benkovic, Stephen J (2010) Analysis of the DNA translocation and unwinding activities of T4 phage helicases. Methods 51:277-88
Blair, Lauren P; Tackett, Alan J; Raney, Kevin D (2009) Development and evaluation of a structural model for SF1B helicase Dda. Biochemistry 48:2321-9
Sikora, Bartek; Eoff, Robert L; Matson, Steven W et al. (2006) DNA unwinding by Escherichia coli DNA helicase I (TraI) provides evidence for a processive monomeric molecular motor. J Biol Chem 281:36110-6
Eoff, Robert L; Raney, Kevin D (2006) Intermediates revealed in the kinetic mechanism for DNA unwinding by a monomeric helicase. Nat Struct Mol Biol 13:242-9
Byrd, Alicia K; Raney, Kevin D (2006) Displacement of a DNA binding protein by Dda helicase. Nucleic Acids Res 34:3020-9
Spurling, Travis L; Eoff, Robert L; Raney, Kevin D (2006) Dda helicase unwinds a DNA-PNA chimeric substrate: evidence for an inchworm mechanism. Bioorg Med Chem Lett 16:1816-20
Mackintosh, Samuel G; Raney, Kevin D (2006) DNA unwinding and protein displacement by superfamily 1 and superfamily 2 helicases. Nucleic Acids Res 34:4154-9
Eoff, Robert L; Spurling, Travis L; Raney, Kevin D (2005) Chemically modified DNA substrates implicate the importance of electrostatic interactions for DNA unwinding by Dda helicase. Biochemistry 44:666-74

Showing the most recent 10 out of 17 publications