We describe a fluorescence assay that can be used to monitor helicase-catalyzed unwinding of duplex DNA continuously in real time. The assay is based on the observation that fluorescence resonance energy transfer (FRET) occurs between donor (fluorescein) and acceptor (hexachlorofluorescein) fluorophores that are in close proximity due to their covalent attachment to the 3' and 5' ends of the complementary strands of a duplex oligodeoxynucleotide. FRET results in a reduction in the fluorescence emission intensity of fluorescein in the duplex DNA substrate relative to that observed for fluorescein-labeled single stranded DNA. Therefore, an enhancement of fluorescein fluorescence (lambda ex = 492 nm; lambda em = 520 nm) occurs upon helicase-catalyzed unwinding of the duplex DNA and separation of the complementary strands. The fluorescence assay is extremely sensitive, allowing DNA unwinding reactions to be monitored continuously at DNA concentrations as low as 1 nM in a fluorescence stopped-flow experiment. We demonstrate the use of this DNA substrate in pre-steady state, single turnover studies of duplex DNA unwinding catalyzed by the Escherichia coli Rep helicase, monitored by fluorescence stopped flow. We show that the fluorescence enhancement monitors Rep-catalyzed DNA unwinding by comparisons with identical kinetic studies carried out using rapid chemical quench-flow techniques. Single turnover kinetic studies performed at 1 nM DNA as a function of excess Rep concentration show that Rep-catalyzed unwinding of an 18 base pair duplex containing a 3'-ss-(dT)20 tail is biphasic and can be described by the sum of two exponential terms. The observed rate constant of the first phase is independent of [Rep] (20-300 nM) and measures the rapid single turnover, unwinding of the duplex DNA by Rep dimers bound in productive complexes (1.3 +/- 0.2 s-1; 23 +/- 3 base pairs s-1 at 25.0 degrees C). The observed rate constant for the second phase increases linearly with [Rep], reflecting DNA unwinding that is limited by a Rep binding event occurring with a bimolecular rate constant of (1.8 +/- 0.1) x 10(5) M-1 s-1, which may reflect the rate constant for Rep dimerization on DNA. Kinetic competition studies indicate that both Rep subunits are bound stably to the DNA substrate in the productive complex that is unwound in the fast phase. The results of these kinetic studies are consistent with an active, rolling mechanism for Rep-catalyzed unwinding of DNA [Wong, I., & Lohman, T. M., (1992) Science 256, 350].(ABSTRACT TRUNCATED AT 400 WORDS)