Ensemble and single-molecule fluorescence-based assays to monitor DNA binding, translocation, and unwinding by iron–sulfur cluster containing helicases

Abstract

Many quantitative approaches for analysis of helicase-nucleic acid interactions require a robust and specific signal, which reports on the presence of the helicase and its position on a nucleic acid lattice. Since 2006, iron-sulfur (FeS) clusters have been found in a number of helicases. They serve as endogenous quenchers of Cy3 and Cy5 fluorescence which can be exploited to characterize FeS containing helicases both in ensemble-based assays and at the single-molecule level. Synthetic oligonucleotides site-specifically labeled with either Cy3 or Cy5 can be used to create a variety of DNA substrates that can be used to characterized DNA binding, as well as helicase translocation and unwinding. Equilibrium binding affinities for ssDNA, duplex and branched DNA substrates can be determined using bulk assays. Identification of preferred cognate substrates, and the orientation and position of the helicase when bound to DNA can also be determined by taking advantage of the intrinsic quencher in the helicase. At the single-molecule level, real-time observation of the helicase translocating along DNA either towards the dye or away from the dye can be used to determine the rate of translocation by the helicase on ssDNA and its orientation when bound to DNA. The use of duplex substrates can reveal the rate of unwinding and processivity of the helicase. Finally, the FeS cluster can be used to visualize protein-protein interactions, and to examine the interplay between helicases and other DNA binding proteins on the same DNA substrate.

Two steps forward, one step back: Determining XPD helicase mechanism by single-molecule fluorescence and high-resolution optical tweezers

“…By altering the distance between bound helicase and DNA-tethered fluorescent dye (for example Cy3 or Cy5), the quenching signal can be calibrated: in the case of XPD, a change in the helicase position by 1 nucleotide (or 1-bp) corresponds to a 3% change in the Cy3 fluorescence intensity ([54] and our unpublished data) (Figure 2C). Cy3 quenching, therefore, can be used as a proximity indicator in analysis of XPD binding to and translocation along ssDNA, as well as in monitoring duplex separation or nucleoprotein remodeling by XPD helicase and related enzymes [62]. Furthermore, FeS cluster-mediated fluorescence quenching can be combined with Förster resonance energy transfer (FRET) [63, 64] between a pair of fluorophores thereby expanding the translocation and unwinding measurements to pseudo three color analyses of complex nucleoprotein interactions [54, 62].…”

“…Cy3 quenching, therefore, can be used as a proximity indicator in analysis of XPD binding to and translocation along ssDNA, as well as in monitoring duplex separation or nucleoprotein remodeling by XPD helicase and related enzymes [62]. Furthermore, FeS cluster-mediated fluorescence quenching can be combined with Förster resonance energy transfer (FRET) [63, 64] between a pair of fluorophores thereby expanding the translocation and unwinding measurements to pseudo three color analyses of complex nucleoprotein interactions [54, 62]. …”

“…Single-molecule fluorescence imaging by TIRFM is ideally suited for visualizing movement of slow and marginally processive XPD helicase [54, 62]. The minimal single-color TIRFM experiment is sufficient to follow, via the FeS-mediated fluorescence quenching, XPD binding to and movement on the surface-tethered fluorescently-labeled DNA molecules [54, 62] (Figure 2D).…”

The N-terminal Domain of the Drosophila Mitochondrial Replicative DNA Helicase Contains an Iron-Sulfur Cluster and Binds DNA

“…However, the absence of the iron-sulfur cluster in the double alanine variants in the ZBD does not permit a direct comparison of their DNA-binding affinities to the Fe-Scontaining forms, because they must be exhibiting fluorescence quenching by a mechanism other than the Förster resonance transfer mechanism, which predominates in the latter (31). Nonetheless, the C68A/C71A and C102A/C105A variants do exhibit quenching (Fig.…”

Close encounters for the first time: Helicase interactions with DNA damage

“…Under single-turnover conditions, we determined that RECQ1 unwinds the DNA substrate with the isopropyl PTE lesion in the translocating strand at a significantly lower rate compared to the control (undamaged) substrate or the substrate harboring the PTE adduct in the non-translocating strand [23]. In the future, single-molecule fluorescence-based assays, discussed in a Methods paper from the Spies lab [24], will be a useful approach to study mechanistic aspects of helicase interaction with damaged DNA substrates.…”