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.
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.
Nanduri
B
B
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, 4301 West Markham, Little Rock, AR 72205, USA.
Eoff
R L
RL
Tackett
A J
AJ
Raney
K D
KD
D000255:Adenosine Triphosphate / Q000378:metabolism; D017122:Bacteriophage T4 / Q000201:enzymology*; D020029:Base Pairing; D001483:Base Sequence; D002384:Catalysis; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004265:DNA Helicases / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005454:Fluorescence Polarization; D007700:Kinetics; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D009693:Nucleic Acid Hybridization; D020135:Peptide Nucleic Acids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D014764:Viral Proteins
D004277:DNA, Single-Stranded; D020135:Peptide Nucleic Acids; D014764:Viral Proteins; D000255:Adenosine Triphosphate; D004247:DNA; D004265:DNA Helicases; C077401:dda DNA helicase protein, Bacteriophage T4