Investigation into the mechanism of DNA unwinding by the bacteriophage T4 Dda helicase: Characterization of the transition from a monomeric to a multimeric molecular motor

Helicases couple the energy released during nucleotide tri-phosphate hydrolysis with translocation along the nucleic acid lattice and the thermodynamically unfavorable separation of complementary strands of nucleic acid. The ubiquitous nature of these enzymes across the spectrum of known life forms, and the absolute requirement for the function they provide make them excellent targets for mechanistic studies. This study utilized the bacteriophage T4 Dda helicase to investigate several aspects of the helicase catalyzed mechanism of DNA unwinding, focusing primarily upon determination of the kinetic step-size for Dda. One limitation in performing even simple steady-state kinetic analysis of helicase mechanism involves the ability of helicase catalyzed single-stranded nucleic acid product to spontaneously re-anneal. This obstacle has been overcome by using peptide nucleic acid oligonucleotides to 'trap' product and prevent re-annealing.; The complex and dynamic interaction of helicases with their nucleic acid substrate has been observed in a static manner through several structural studies. Many of these functional interactions have been elucidated through studies that used either site directed mutagenesis of the enzyme, or as described herein, chemically modified nucleic acid substrates to carefully dissect out the elements that play an important role in helicase action.; Several oligomeric states have been described for helicase function. Dda can function as a monomer, but several Dda molecules can separate longer regions of duplex DNA, indicating that multiple engines may serve to prevent enzyme dissociation from the substrate. The kinetic transition from a monomeric to a multiple motor species has been illustrated through use of transient state kinetic approaches, leading to a model describing the ability of Dda to transition from a relatively non-processive monomeric helicase to a processive species comprised of multiple helicase molecules. The accuracy of the determined kinetic parameters was increased substantially through use of non-linear least squares global analysis of the data sets and by the development of novel experimental designs, which eliminated sources of uncertainty from the analysis of the data.; Finally, Dda was shown to convert roughly 74% of the energy associated with ATP hydrolysis into the mechanical separation of double-stranded DNA, illustrating the highly efficient nature of helicase evolution.