| Autor: |
Raney, K D, Sowers, L C, Millar, D P, Benkovic, S J |
| Zdroj: |
Proceedings of the National Academy of Sciences of the United States of America; July 1994, Vol. 91 Issue: 14 p6644-6648, 5p |
| Abstrakt: |
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 flourescence-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. |
| Databáze: |
Supplemental Index |
| Externí odkaz: |
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