Abstract
A continuous fluorescence-based assay is described for measuring helicase- mediated unwinding of duplex DNA. The assay utilizes an oligonucleotide substrate containing the fluorescent adenine analog, 2-aminopurine, at regular intervals. 2-Aminopurine forms a Watson-Crick-type base pair with thymine and does not distort normal B-form DNA. Fluorescence of the 2- aminopurines within this oligonucleotide is quenched 2-fold upon its hybridization to a complementary strand. Unwinding of this substrate by the T4 dda helicase restores the fluorescence of the 2-aminopurines and is easily followed using stopped-flow or steady-state fluorescence spectroscopy. The fluorescence-based assay provides rate data comparable to that obtained from conventional discontinuous assays using labeled substrates and additionally furnishes a means for following a single turnover. This assay should prove useful for defining the mechanism by which helicases unwind duplex DNA.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 6644-6648 |
| Number of pages | 5 |
| Journal | Proceedings of the National Academy of Sciences of the United States of America |
| Volume | 91 |
| Issue number | 14 |
| DOIs | |
| State | Published - Jul 5 1994 |
| Externally published | Yes |
Keywords
- 2-aminopurine
- dda helicase
- stopped-flow spectroscopy
ASJC Scopus subject areas
- General