7174 Nucleic Acids Research, 2011, Vol. 39, No. 16 Figure 6. G4R1/RHAU-catalyzed resolution of 50-TAMRA-labeled tetramolecular G4-DNA substrate is inhibited most efﬁciently by unlabeled unimolecular c-Myc 51-mer G4-DNA. (A, C and E) Phosphorimage of representative (of three repetitions) nondenaturing gel electropherograms; the top triangles indicate increasing concentrations of unlabeled (A) Poly A Zic1 47-mer G4-DNA, (C) c-Myc 51-mer G4-DNA, or (E) tetramolecular Z33 G4-DNA added to G4R1/RHAU-catalyzed resolving reactions of 50-TAMRA-labeled tetramolecular Z33 G4-DNA substrate. Lane 1 of panels A, C and E: 2 nM 50-TAMRA-labeled tetramolecular Z33 G4-DNA substrate in the absence of enzyme or unlabeled unimolecular G4-DNA. Lanes 2–12 of panels A, C and E: 2 nM 50-TAMRA-labeled tetramolecular Z33 G4-DNA substrate after incubation with 1 unit of G4R1/ RHAU plus increasing concentrations of unlabeled inhibitor G4-DNA. (B, D and F) Graphic representation of concentration-dependent inhibition of G4R1/RHAU-catalyzed resolution of 50-TAMRA-labeled tetramolecular Z33 G4-DNA substrate by unlabeled (B) Poly A Zic1 47-mer G4-DNA, (D) c-Myc 51-mer G4-DNA, or (F) tetramolecular Z33 G4-DNA. Inhibition is represented by the percentage of maximal resolution (error bars are Mean ± SD, n = 3 independent experiments). Inhibition of 50% is observed at a 6.25:1 unimolecular Poly A Zic1 DNA 47-mer G4-DNA:Z33 tetramer G4-DNA molar ratio, at a 0.06:1 unimolecular c-Myc DNA 51-mer:Z33 tetramer G4-DNA molar ratio, and at a 1:1 unlabeled Z33 tetramer G4-DNA:labeled Z33 tetramer G4-DNA molar ratio. of unwinding of the tetramolecular structure was analyzed by non-denaturing gel electrophoresis (Figure 6A, C and E). The substrate target was preformed tetramolecular G4-DNA of >95% purity (Figure 6A, lane 1; Figure 6C, lane 1; Figure 6E, lane 1). Sufﬁcient G4R1/RHAU was present in each reaction to produce 50% resolution of tetramer into monomer in the absence of competitor (Figure 6A, lane 2; Figure 6C, lane 2; Figure 6E, lane 2), corresponding to a G4R1/RHAU concentration of 7.5 pM. We have previously deﬁned 1 U of enzyme to be