7166 Nucleic Acids Research, 2011, Vol. 39, No. 16 to RES buffer, 10 mM ATP [or adenosine 50-(b, g-imido)- triphosphate (AMP-PNP) in selected experiments], 10 nM complementary PNA and a dilution series of active recom- binant G4R1/RHAU enzyme that included 0, 28.5, 463 fM, 1.85, 115 and 7.40 pM, each in a 30 ml ﬁnal volume. An identical series of reactions were prepared in which recombinant G4R1/RHAU was held at 98 C for 10 min prior to the reaction to destroy the catalytic activity of the enzyme, or wild-type enzyme was replaced with mutant G4R1/RHAU containing an E!A substitution at amino acid position 335 in the Walker B box that abolishes ATPase activity (40), to serve as nega- tive controls. Additionally, a set of 30 ml reactions was made containing 1 nM of 50-[32P]-labeled Poly A Zic1 DNA 47-mer G4-DNA in K-RES buffer, 10 mM ATP, in the absence of complementary PNA and an identical dilution series of active recombinant G4R1/RHAU as described above. All reactions were incubated at 37 C for 30 min, dropped to 4 C and stopped by adding 5 ml of 0.5 M EDTA and mixing. Reactions were then directly loaded (monitored visually by Schlieren lines) alongside of a set of electrophoretic mobility stand- ards (prepared as described above) and analyzed on a 10% non-denaturing PAGE TBE gel with 10% glycerol. Gels were imaged on a Typhoon 9210 Imager (GE Healthcare). G4-DNA inhibition assay Tetramolecular G4-DNA resolving activity was determined as previously described (33) but with K-RES buffer. 2 nM of 50-TAMRA-labeled tetramolecular G4-DNA and 1 U of recombinant G4R1/RHAU were included per 50-ml reaction. Unlabeled inhibitor unimol- ecular G4-DNA oligonucleotides, including c-Myc 51-mer and Poly A Zic1 DNA 47-mer (Table 1), were added to some reactions in a 0- to128-fold molar excess of inhibitor to labeled tetramolecular G4-DNA substrate. Reactions were allowed to proceed at 37 C for 30 min, stopped by addition of 5 ml of 200 mM EDTA, and analyzed by elec- trophoresis through a 10% non-denaturing polyacryl- amide TBE gel with 10% glycerol. Gels were scanned on a Typhoon 9210 Imager (GE-Healthcare) and images were analyzed using Multi Gauge version 3.0 imaging software (Fuji). Statistical analysis was done in MS Excel 2003 and standard deviations were calculated by the STDEV function. RESULTS Circular dichroism spectropolarimetry was used to determine unimolecular G4-DNA formation in test DNA oligonucleotides Our ﬁrst goal in studying the interaction of G4R1/RHAU with unimolecular G4-DNA was to form G4-DNA struc- tures within DNA oligonucleotides of Zic1 and c-Myc and then to conﬁrm and characterize the presence of these G4-DNA structures. CD spectra, combined with melting curve analysis in buffer containing 50 mM K+ or Li+, indicated the formation of G4-DNA. The differential absorption of circularly polarized light measured by CD is attributable to the presence of chiral structure within the oligonucleotides and has been previ- ously used to characterize G4-DNA in oligonucleotides (41,42). Typically, unimolecular parallel G4-DNA struc- tures (Figure 1D) produce a peak of molar ellipticity at 260 nm, while anti-parallel G4-DNA structures (Figure 1C) produce a peak of ellipticity at 290 nm. The Poly A Zic1 DNA 47-mer and the c-Myc DNA 51-mer both contain G-rich sequences that conform to the formulae for formation of unimolecular G4-DNA (14,15). Table 1 shows all sequences used in this study in the order they are reported in the ‘Results’ section. G4-DNA forming regions are bolded. CD spectra indicate that both the Poly A Zic1 DNA 47-mer and the c-Myc 51-mer (Figure 1E and G) show peaks of molar ellipticity at 260 nm that are suggestive of parallel G4-DNA structures in 50 mM KCl at both 25 C (blue trace) and 37 C (red trace). The thermal stability of this CD signal is expected from G4-DNA, which is typic- ally stable well above 37 C in KCl solution. However, at 95 C (green trace in Figure 1E and G) the molar ellip- ticity signiﬁcantly diminishes at 260 nm, as would be expected for the destabilization of a chiral structure un- folding at the higher temperature and thereby losing its chirality. Note that the CD spectrum of the scrambled Zic1 47-mer sequence (Figure 1I), which is an oligo- nucleotide not expected to have G4-DNA, shows a similar peak of ellipticity at 260 nm at 25 C (blue trace). However, this structure was destabilized at both 37 C and at 95 C, displaying a signiﬁcantly diminished ellipticity at 260 nm for both temperatures (Figure 1I, red trace and green trace). The fact that this structure was not stable at 37 C suggests it is unlikely to possess a stable G4-DNA structure. This was further conﬁrmed by melting studies in the presence of K+ and Li+, discussed below. A hallmark of G4-DNA is stabilization by K+ ions and destabilization by Li+ ions (3,4,7,13,43). Atomic modeling has suggested that K+ coordinately bonds between two stacked G-quartets as an ‘optimal ﬁt’. However, the most accepted explanation of the superior stabilization of G4-DNA by K+ is that it is energetically easier to de- hydrate K+ ions (44). Because of enhanced stability, G4 structures are predicted to have higher melting tempera- tures in K+ solutions than in Li+ solutions. We tested the melting behavior of oligonucleotide structures by moni- toring the changes in peak height of molar ellipticity at 263 nm in the presence of K+ and Li+ with increasing tem- perature (Figure 1F, H and J). Poly A Zic1 DNA 47-mer and the c-Myc 51-mer exhibited the characteristic CD spectral changes for unimolecular G4-DNA structures in the presence of both cations, as expected. The Tm’s, deﬁned as the temperatures of 50% depression of the 263 nm ellipticity peak, were well above 60 C for the G4-DNA structures of both oligonucleotides in K+. However, the Tm in KCl was higher than the Tm in LiCl for each G4-DNA structure. The Poly A Zic1 DNA 47-mer exhibited a Tm in 50 mM KCl of 80 C (Figure 1F, blue trace), while its Tm in 50 mM LiCl was 70 C (Figure 1F, red trace). The c-Myc 51-mer DNA similarly