Coupling of DNA unwinding to nucleotide hydrolysis in a ring-shaped helicase.
The ring-shaped T7 helicase uses the energy of dTTP hydrolysis to perform the mechanical work of translocation and base pair (bp) separation. We have shown that the unwinding rate of T7 helicase decreases with increasing DNA stability. Here, we show that the dTTPase rate also decreases with increasing DNA stability, which indicates close linkage between chemical transition steps and translocation steps of unwinding. We find that the force-producing step during unwinding is not associated with dTTP binding, but dTTP hydrolysis or P(i) release. We determine that T7 helicase extracts approximately 3.7 kcal/mol energy from dTTPase to carry out the work of strand separation. Using this energy, T7 helicase unwinds approximately 4 bp of AT-rich DNA or 1-2 bp of GC-rich DNA. T7 helicase therefore adjusts both its speed and coupling ratio (bp/dTTP) to match the work of DNA unwinding. We discuss the mechanistic implications of the variable bp/dTTP that indicates T7 helicase either undergoes backward movements/futile hydrolysis or unwinds DNA with a variable bp-step size; 'long and fast' steps on AT-rich and 'short and slow' steps on GC-rich DNA.
Donmez
Ilker
I
Department of Biochemistry, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ, USA.
Patel
Smita S
SS
D017123:Bacteriophage T7 / Q000201:enzymology*; D020029:Base Pairing; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004265:DNA Helicases / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008954:Models, Biological; D009711:Nucleotides / Q000378:metabolism*; D013379:Substrate Specificity; D013816:Thermodynamics; D013997:Time Factors
D009711:Nucleotides; D004247:DNA; D004265:DNA Helicases