| Abstract: | Measurement of steady-state rates of unwinding of double-stranded oligonucleotides by helicases is hampered due to rapid reannealing of the single-stranded DNA products. Including an oligonucleotide in the reaction mixture which can hybridize with one of the single strands can prevent reannealing. However, helicases bind to single-stranded DNA, therefore the additional oligonucleotide can sequester the enzyme, leading to slower observed rates for unwinding. To circumvent this problem, the oligonucleotide that serves as a trap was replaced with a strand of peptide nucleic acid (PNA). Fluorescence polarization was used to determine that a 15mer PNA strand does not bind to the bacteriophage T4 Dda helicase. Steady-state kinetic parameters of unwinding catalyzed by Dda were determined by using PNA as a trapping strand. The substrate consisted of a partial duplex with 15 nt of single-stranded DNA and 15 bp. In the presence of 250 nM substrate and 1 nM Dda, the rate of unwinding in the presence of the DNA trapping strand was 0.30 nM s–1 whereas the rate was 1.34 nM s–1 in the presence of the PNA trapping strand. PNA prevents reannealing of single-stranded DNA products, but does not sequester the helicase. This assay will prove useful in defining the complete kinetic mechanism for unwinding of oligonucleotide substrates by this helicase. |
