| 20167274 |
Pugh RA, Honda M, Spies M: Ensemble and single-molecule fluorescence-based assays to monitor DNA binding, translocation, and unwinding by iron-sulfur cluster containing helicases. Methods. 2010 Feb 16.
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 cluster 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. |
89(1,1,1,9) |