Pre-steady-state DNA unwinding by bacteriophage T4 Dda helicase reveals a monomeric molecular motor.
Helicases are molecular motor enzymes that unwind and translocate nucleic acids. One of the central questions regarding helicase activity is whether the process of coupling ATP hydrolysis to DNA unwinding requires an oligomeric form of the enzyme. We have applied a pre-steady-state kinetics approach to address this question with the bacteriophage T4 Dda helicase. If a helicase can function as a monomer, then the burst amplitude in the pre-steady state might be similar to the concentration of enzyme, whereas if the helicase required oligomerization, then the amplitude would be significantly less than the enzyme concentration. DNA unwinding of an oligonucleotide substrate was conducted by using a Kintek rapid quench-flow instrument. The substrate consisted of 12 bp adjacent to 12 nucleotides of single-stranded DNA. Dda (4 nM) was incubated with substrate (16 nM) in buffer, and the unwinding reaction was initiated by the addition of ATP (5 mM) and Mg(2+) (10 mM). The reaction was stopped by the addition of 400 mM EDTA. Product formation exhibited biphasic kinetics, and the data were fit to the equation for a single exponential followed by a steady state. The amplitude of the first phase was 3.5 +/- 0.2 nM, consistent with a monomeric helicase. The burst amplitude of product formation was measured over a range of enzyme and substrate concentrations and remained consistent with a functional monomer. Thus, Dda can rapidly unwind oligonucleotide substrates as a monomer, indicating that the functional molecular motor component of a helicase can reside within a single polypeptide.
Nanduri
Bindu
B
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock 72205, USA.
Byrd
Alicia K
AK
Eoff
Robert L
RL
Tackett
Alan J
AJ
Raney
Kevin D
KD
D017122:Bacteriophage T4 / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D004265:DNA Helicases / Q000378:metabolism*; D004279:DNA, Viral / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D014764:Viral Proteins
D004279:DNA, Viral; D009838:Oligodeoxyribonucleotides; D014764:Viral Proteins; D004265:DNA Helicases; C077401:dda DNA helicase protein, Bacteriophage T4