Pre-steady-state DNA unwinding by bacteriophage T4 Dda helicase reveals a monomeric molecular motor
AUTOR(ES)
Nanduri, Bindu
FONTE
National Academy of Sciences
RESUMO
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 Mg2+ (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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=137486Documentos Relacionados
- Measurement of steady-state kinetic parameters for DNA unwinding by the bacteriophage T4 Dda helicase: use of peptide nucleic acids to trap single-stranded DNA products of helicase reactions
- Long-range intramolecular signaling in a tRNA synthetase complex revealed by pre-steady-state kinetics
- Pre-steady-state kinetics shows differences in processing of various DNA lesions by Escherichia coli formamidopyrimidine-DNA glycosylase
- Substrate Recognition of Anthrax Lethal Factor Examined by Combinatorial and Pre-steady-state Kinetic Approaches*
- Pre-Steady-State Kinetics of Ba-Ca Exchange Reveals a Second Electrogenic Step Involved in Ca2+ Translocation by the Na-Ca Exchanger