Abstract
The RecQ family helicases catalyze the DNA unwinding reaction in an ATP hydrolysis-dependent manner. We investigated the mechanism of DNA unwinding by the Escherichia coli RecQ helicase using a new sensitive helicase assay based on fluorescence cross-correlation spectroscopy (FCCS) with two-photon excitation. The FCCS-based assay can be used to measure the unwinding activity under both single and multiple turnover conditions with no limitation related to the size of the DNA strands constituting the DNA substrate. We found that the monomeric helicase was sufficient to perform the unwinding of short DNA substrates. However, a significant increase in the activity was observed using longer DNA substrates, under single turnover conditions, originating from the simultaneous binding of multiple helicase monomers to the same DNA molecule. This functional cooperativity was strongly dependent on several factors, including DNA substrate length, the number and size of single-stranded 3'-tails, and the temperature. Regarding the latter parameter, a strong cooperativity was observed at 37 degrees C, whereas only modest or no cooperativity was observed at 25 degrees C regardless of the nature of the DNA substrate. Consistently, the functional cooperativity was found to be tightly associated with a cooperative DNA binding mode. We also showed that the cooperative binding of helicase to the DNA substrate indirectly accounts for the sigmoidal dependence of unwinding activity on ATP concentration, which also occurs only at 37 degrees C but not at 25 degrees C. Finally, we further examined the influences of spontaneous DNA rehybridization (after helicase translocation) and the single-stranded DNA binding property of helicase on the unwinding activity as detected in the FCCS assay.
Highlights
Helicases are molecular motor enzymes that unwind and translocate nucleic acids in an ATP hydrolysis-dependent manner [1, 2]
We investigated the mechanism of DNA unwinding by the Escherichia coli RecQ helicase using a new sensitive helicase assay based on fluorescence cross-correlation spectroscopy (FCCS) with two-photon excitation
We found that the cooperative dependence of the unwinding activity on ATP concentration originated indirectly from the cooperative DNA binding mode of RecQ helicase, reconciling apparently conflicting published results concerning the possibility of an active monomeric form, whereas the activity displays a strong cooperativity on ATP concentration (Hill coefficient Ϸ 3), which was interpreted previously as compelling evidence for a multimeric active form [27]
Summary
RecQ Helicase Purification, SSB, and Oligonucleotides—The E. coli RecQ helicases, wild-type and mutant proteins K53A and D146A, were purified as described previously [28, 37]. Fluorescence Cross-correlation Spectroscopy—Dual color FCCS measurements were performed by two-photon excitation using a single laser line on a home-built system (described previously in Ref. 38 for the FCS mode) with a 100-fs pulse, 80-MHz mode-. The fluorescence signals from Alexa Fluor 488 (Alexa488 (Al)) and Texas Red (Te) were collected with the same objective and separated from the excitation by a dichroic mirror (Chroma 700DCSPXR). The output signal from the microscope was further filtered with a Chroma E700SP-2p filter to reject the residual excitation light and split by a dichroic mirror (Chroma 580dcxr). Additional filters (Chroma HQ510/50 and HQ630/ 60m-2p for Alexa488 and Texas Red, respectively) were used to minimize cross-talk (Fig. 1B), and the split fluorescence signal was focused on two avalanche photodiodes (PerkinElmer Life Sciences, SPCM-AQR-14 single photon counting module with less than 90 dark counts/s) mounted at right angle. Assuming a three-dimensional Gaussian distribution of excitation intensity, the cross-correlation function for a free Brownian diffusion process is given by Equation 2,
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