(150, 100 and 75 kDa) were then made using Fuji Multiguage software. Linear regression analysis of the three standard proteins’ titration series gave similar curves. The concentration of rG4R1/RHAU was then extrapolated from the titration series, yielding a ﬁnal con- centration of 223 ± 18 nM (n = 9). To determine the accuracy of the Promega Broad Range Molecular Weight Markers (V849A) that were used to quantify rG4R1 via Coomassie staining, bovine serum albumin (BSA) (Pierce, # 23209) and b-galactosidase (Sigma, # 5635- 1KU) were precisely quantiﬁed via spectrophotometry at 280 nm using extinction coefﬁcients. BSA and b-galactosi- dase were chosen because their histidine, arginine and aromatic amino acid contents are similar to rG4R1. 150 and 300 ng aliquots of each protein (as determined spectro- photometrically) were loaded and run on a typical 5% stacking/12% resolving polyacrylamide gel next to a titra- tion of the Promega Broad Range Molecular Weight markers (31.3, 62.5, 125, 250, 500 ng); these titrations were done in triplicate. Gels were then stained with Coomassie R-250 (50 mg/100 ml of 50:10:40 v/v metha- nol:acetic acid:H2O) and then destained with 30:10:60 v/v methanol:acetic acid:H2O. The three proteins that were originally used to determine rG4R1 concentration via Coomassie staining (75, 100, 150 kDa bands of Promega # V849A) were used to generate three standard curves (n = 9). Amounts of BSA and b-galactosidase were extrapolated and compared to the amounts that were loaded based upon the spectrophotometrically determined values, respectively. We observed that the two methods (spectrophotometric versus in-gel Coomassie determin- ation of protein concentration) differ by 20–49% (<2-fold, data not shown). The apparent Kd of G4R1/ RHAU bound to unimolecular G4-DNA was estimated by GMSA. Recombinant G4R1/RHAU at concentrations of 2–300 pM was incubated with 1 pM 50-[32P]end-labeled G4-DNA in K-Res (100 mM KCl, 10 mM NaCl, 3 mM MgCl2, 50 mM Tris acetate, pH 7.8, 70 mM glycine, 0.012% bovine a-lactalbumin, 10% glycerol) buffer with 10 mM EDTA at 37 C for 30 min. Binding mixtures were then loaded and analyzed by 10% non-denaturing PAGE. Electrophoresis was performed at 70 V for 15 h in a cold room (7 C). Gels were imaged on a Typhoon 9210 Imager (GE Healthcare). Band densities were analyzed using Multi Gauge (Fuji) software and statistical analysis was performed with MS Excel. The apparent Kd value was estimated from each model as the concentration of enzyme at which 50% of target DNA substrate was bound for three separate experiments for each DNA. In selected experiments in which data were obtained at closely spaced 0.5–1 pM intervals over a broad range of DNA substrate concentrations, the data were ﬁtted to a saturable single site binding model: Bmax½Substrate B ¼ Kd+½Substrate 1 ð Þ Here Bmax is the fractional degree of saturation of receptor with ligand at large excess, and Kd is the equilibrium afﬁnity constant expressed in molarity. Nonlinear Nucleic Acids Research, 2011, Vol. 39, No. 16 7165 regression analysis (SigmaPlot 11.0, Systat, San Jose, CA, USA) yielded Bmax, Kd and the standard deviation of these ﬁtted parameters. A linear regression analytic ﬁt of 1/[Bound Substrate] as a function of 1/[G4R1/RHAU] was also performed. koff determination A 1500 ml solution of 1 pM 50-[32P]-Poly A Zic1 47-mer G4-DNA and 200 pM G4R1/RHAU was incubated at 37 C for 30 min under standard GMSA conditions (described above). Following incubation, a 200 ml sample of the binding mixture was removed and placed at 80 C to be used as a positive binding control. At this point, 20 mM unlabeled Poly A Zic1 47-mer G4-DNA was added to the binding mixture and maintained at 37 C. At 0, 1, 2, 24, 48 and 72 h following the addition of un- labeled competitor, 200 ml samples of the binding mix were removed and placed at 80 C. As a negative control a reaction in which 20 nM of unlabeled Poly A Zic1 47-mer G4-DNA was added to 1 pM 50-[32P] end- labeled-Poly A Zic1 47mer G4-DNA prior to incubation with G4R1/RHAU was included to demonstrate that 20 nM of unlabeled Poly A Zic1 47-mer G4-DNA is suf- ﬁcient to block detection of binding of 50-[32P] end- labeled-Poly A Zic1 47mer G4-DNA to G4R1/RHAU. Following the completion of the 72 h time point, binding mixtures were simultaneously thawed and analyzed by 10% non-denaturing PAGE. Electrophoresis was per- formed at 70 V for 15 h in a cold room (7 C). Gels were imaged on a Typhoon 9210 Imager (GE Healthcare). Band densities were analyzed using Multi Gauge (Fuji) software. Dimethyl sulfate treatment of poly A Zic1 DNA Poly A Zic1 DNA 47-mer oligonucleotide was treated with dimethyl sulfate using a slight modiﬁcation of the method of Maxam and Gilbert (39). A Total of 250 pM poly A Zic1 DNA 47-mer was added to a 50 ml reaction volume containing 50 mM sodium cacodylate, pH 8.0, 1 mM EDTA. Samples were chilled on ice for 5 min followed by heating at 95 C for 5 min, after which 2 ml (1:4 ethanol v/v) dimethyl sulfate was added and incubated at 37 C for 30 min. PNA hybridization and PNA trap assay Electrophoretic mobility standards for the two isomers of Poly A Zic1 DNA 47-mer G4-DNA (Figure 5 and Supplementary Figure S4, Bands 1 and 2) and the Poly A Zic1 DNA 47-mer:PNA hybrid (Figure 5 and Supplementary Figure S4, Band 3) were prepared by adding 100 pM or 1 nM 50-[32P]-labeled Poly A Zic1 DNA 47-mer G4-DNA to K-RES buffer, 10 mM ATP and 0, 10, 100,500 nM, or 10 mM PNA (with Watson– Crick complementarity to the internal Poly A Zic1 DNA 47-mer PQS) in a 30 ml ﬁnal volume. Solutions were held at 98 C for 10 min and then cooled to room temperature. In order to assess G4R1/RHAU-catalyzed unwinding of unimolecular G4-DNA by trapping of the open product with a complementary PNA, 100 pM or 1 nM 50-[32P]- labeled Poly A Zic1 DNA 47-mer G4-DNA was added