Direct measurement of single-stranded DNA translocation by PcrA helicase using the fluorescent base analogue 2-aminopurine.
Use of the fluorescent base analogue 2-aminopurine has provided a direct demonstration of the translocation of PcrA helicase toward the 5'-end of single-stranded DNA. Single 2-aminopurine bases are introduced into otherwise standard oligonucleotides and produce a fluorescence signal when PcrA reaches their position. We demonstrate that random binding of PcrA to ssDNA is followed by translocation in an ATP-dependent manner toward the 5'-terminus at 80 bases per second at 20 degrees C. The data also provide information on the kinetics of ssDNA binding to the helicase and of the protein dissociation from the 5'-end of ssDNA. A full kinetic model is presented for ATP-dependent DNA translocation by PcrA helicase.
Dillingham
Mark S
MS
Imperial Cancer Research Fund, Clare Hall Laboratories, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3LD, UK.
Wigley
Dale B
DB
Webb
Martin R
MR
D015075:2-Aminopurine / Q000494:pharmacology*; D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D004265:DNA Helicases; D004277:DNA, Single-Stranded; D001411:Geobacillus stearothermophilus / Q000378:metabolism; D007700:Kinetics; D008954:Models, Biological; D009841:Oligonucleotides / Q000737:chemistry; D011485:Protein Binding; D021381:Protein Transport; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013997:Time Factors
D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D009841:Oligonucleotides; C084217:pcrA protein, Bacteria; D015075:2-Aminopurine; D000255:Adenosine Triphosphate; D004265:DNA Helicases