Coupling dTTP hydrolysis with DNA unwinding by the helicase of bacteriophage T7

Abstract

The functional form of the DNA helicase encoded by bacteriophage T7 is a hexamer of six identical subunits. The helicase couples the hydrolysis of dTTP to unidirectional translocation and unwinding of DNA. The nucleotide binding site of the helicase is located at the subunit interface. In a crystallographic structure of the T7 helicase with bound nucleoside 5′‐triphosphate, Phe523 positioned in a beta‐hairpin loop at the sub‐unit interface is buried within the interface. However, in the unbound state, Phe523 is more exposed towards the outer surface of the helicase. This different orientation suggests that Phe523 and the associated beta‐hairpin loop may undergo a conformational change coupled with nucleotide hydrolysis. Replacement of Phe523 with alanine or valine abolishes the ability of the helicase to unwind DNA and to stimulate T7 polymerase activity on double‐stranded DNA. Genetic and biochemical experiments indicate that a hydrophobic residue with longer side chains is essential at this position. In addition, Phe523 appears to play a critical role in coupling the hydrolysis of dTTP to DNA unwinding. We postulate that upon dTTP hydrolysis, Phe523 moves from within the sub‐unit interface to a more exposed position where it may intercalate into double stranded DNA to facilitate DNA unwinding by the helicase. Results from this study support the strand exclusion mechanism of DNA unwinding by ring helicases.