Figure 4. Equilibrium binding GMSA of puriﬁed recombinant G4R1/ RHAU incubated with Poly A Zic1 DNA 47-mer hybridized to a Watson–Crick complementary PNA shows little binding by the enzyme when G4-DNA structure is inhibited from forming. (A) Schematic drawing of G4R1/RHAU binding an oligonucleotide con- taining a G4-DNA structure. (B) Schematic drawing showing that hy- bridization of a complementary PNA to the G4-DNA-forming sequence inhibits high afﬁnity binding by G4R1/RHAU. The GMSA shown in (C) suggests that schematics (A) and (B) are correct. (C) Phosphorimage of a representative (of three repetitions) nondenaturing gel electropherogram of a GMSA; the top triangle indicates the direc- tion of increased G4R1/RHAU concentration. Electrophoretic mobility standards for the two Poly A Zic1 47-mer G4-DNA isomers and the DNA:PNA hybrid were created by incubating 1 nM 50-[32P]-labeled Poly A Zic1 DNA 47-mer DNA in K-RES buffer (with MgCl2 and ATP) with 0 nM PNA (lane 1, showing fastest mobility G4-DNA isomer), 10 nM PNA (lane 2, showing the slower mobility G4-DNA isomer) and the PNA:DNA duplex, or 500 nM PNA (lane 3, showing PNA:DNA duplex); lanes 4–12, 1 pM 50-[32P]-labeled PNA:Poly A Zic1 DNA duplex was incubated with increasing concentrations of G4R1/ RHAU (lane 4, 0 pM; lane 5, 5 pM; lane 6, 10 pM; lane 7, 15 pM; lane 8, 30 pM; lane 9, 50 pM; lane 10, 100 pM; lane 11, 150 pM; lane 12, 300 pM); lane 13, 1 pM 50-[32P]-labeled Poly A Zic1 47-mer G4-DNA in the absence of either complementary PNA or G4R1/RHAU; lane 14, 1 pM 50-[32P]-labeled Poly Zic1 DNA 47-mer G4-DNA incubated with 30 pM G4R1/RHAU in the absence of complementary PNA. upper right spectra). This behavior is expected from a hybrid containing PNA, but not from a G4-DNA struc- ture. The 263 nm peak of molar ellipticity of the upper band did exhibit temperature dependence in the presence of either LiCl or KCl; this temperature dependence may be due to a destabilization caused by salt shielding of a charged lysine at the carboxyl terminal of the PNA. Only the upper, non-G4 PNA:DNA hybrid band was observed after a 500:1 PNA:DNA molar ratio was taken through a boiling and cooling cycle (Figure 4C, lane 3). A Nucleic Acids Research, 2011, Vol. 39, No. 16 7171 slower mobility smear was also observed, which we specu- late may represent a second, less stable PNA:DNA hybrid in which the PNA is Watson–Crick paired with the second longest d(pG) run in the Poly A Zic1 DNA 47-mer. A 10:1 PNA:DNA molar ratio incubated at 55 C for 6 h produced only electrophoretic Band 3 (Figure 4C, lane 4) that was conﬁrmed by CD to represent a PNA:DNA hybrid due to CD signal behavior in the no salt conditions (Supplementary Figure S5). This non-G4 PNA: DNA hybrid was assessed for G4R1/RHAU binding. Figure 4A shows an idealized schematic of G4R1/ RHAU recognizing a G4-DNA site and showing the protein binding to that site, thereby forming a protein– nucleic acid complex that retards the electrophoretic mobility of the nucleic acid. We believe this mechanism explains the observed binding with G4R1/RHAU:G4- DNA-containing oligonucleotide complexes (Figure 2). Figure 4B illustrates the expected outcome regarding the interaction of G4R1/RHAU with a PNA:Poly A Zic1 47-mer DNA hybrid. PNA hybridization inhibits G4- DNA formation, which in turn, prevents G4R1/RHAU binding if the enzyme indeed recognizes G4-DNA struc- ture. Consistent with this predicted outcome, titration of G4R1/RHAU concentrations of up to 300 pM with 1 pM PNA:DNA hybrid did not demonstrate any detectable enzyme–hybrid complex formation, as evidenced by GMSA (Figure 4C, lanes 5–12). In contrast, a comparison of G4 structure-containing Poly A Zic1 DNA 47-mer monomer electrophoresed in the absence (Figure 4C, lane 13) or presence (Figure 4C, lane 14) of 30 pM of G4R1/RHAU clearly demonstrates a slow mobility band in the presence of G4R1/RHAU indicative of a bound enzyme:G4-containing monomer complex. These results demonstrate that the Poly A Zic1 DNA 47-mer has a sub- stantially poorer afﬁnity for G4R1/RHAU when hybrid- ized with PNA. The result is consistent with G4R1/ RHAU recognizing the G4-DNA structure in the Poly A Zic1 DNA 47-mer. It should be noted that the phosphor- image contrast in Figure 4C, lanes 4–14 was enhanced to better detect additional bands due to gel mobility shifts. Additional controls (Supplementary Figure S6) demon- strated that excess unlabeled PNA does not block G4R1/RHAU binding to G4-DNA and that PNA:DNA hybrids do not bind G4R1/RHAU when produced without excess unlabeled PNA. Therefore, when G4-DNA struc- ture is lost the enzyme does not bind. A PNA trap assay shows that G4R1/RHAU unwinds G4- DNA structure, enabling it to hybridize to a complementary PNA In the experiments presented above, a complementary PNA was shown to hybridize to the G4-DNA-forming sequence of the Poly A Zic1 DNA 47-mer upon heat de- stabilization of the G4-DNA structure. Hybridization occurred within the G4-DNA forming region of the oligo- nucleotide, producing a PNA:DNA hybrid devoid of G4- DNA structure as determined by CD (Supplementary Figure S5). This ﬁnding led us to consider whether active G4R1/RHAU in the presence of ATP could desta- bilize Poly A Zic1 G4-DNA (i.e. open by unwinding the