7176 Nucleic Acids Research, 2011, Vol. 39, No. 16 for G4R1/RHAU observed when the oligonucleotide pos- sesses G4-DNA structure. These observations indicate that the presence of G4-DNA structure is required by G4R1/RHAU binding and that the G4R1/RHAU binding site within Poly A Zic1 DNA 47-mer is localized to within its unimolecular G4-DNA forming sequence. G4R1/RHAU speciﬁcally unwinds G4-DNA, allowing a complementary PNA to hybridize within the G4- forming sequence. This ‘PNA trap’ approach has been used previously to study the kinetics of unimolecular G4-DNA opening and folding (46). This study represents the ﬁrst use of a PNA trap to demonstrate enzymatic un- winding of unimolecular G4-DNA. However, at least three other methods have been used to study unimolecular G4-DNA unwinding by proteins; these approaches include CD spectroscopy, ﬂuorescence quenching and surface plasmon resonance (SPR) (49–54). All of these approaches are relatively insensitive in comparison to the PNA trap method that can demonstrate resolution of 1 pmol of unimolecular G4-DNA by 0.5 pmol of enzyme in a 30 ml reaction volume. In comparison to these previously reported techniques for assessing reso- lution of unimolecular G4-DNA, the PNA trap assay retains the advantages of: (i) direct observation of the resolved DNA; (ii) enhanced sensitivity of detection of enzyme-catalyzed G4-DNA resolution; and (iii) the ability to demonstrate complete unwinding events. One disadvantage that we have observed with the PNA trap is that, in the presence of Mg2+, PNA can impede binding of G4-DNA to G4R1/RHAU (data not shown), possibly slowing the kinetics of the unwinding reaction. Blocking of the G4-DNA:G4R1/RHAU binding interaction does not occur in the absence of free Mg2+ ions (Supplementary Figure S6, lanes 14 and 15). Comparison with other proteins such as RPA, BLM and T-Ag (52–54) that have been characterized for binding unimolecular G4-DNA highlights the unique G4-DNA binding properties of G4R1/RHAU. First, G4R1/RHAU shows signiﬁcantly more speciﬁcity in binding G4-DNA than unstructured single-stranded DNA (Figure 3). In contrast, RPA (52) and T-Ag (54) do not preferentially bind G4 versus unstructured single-stranded DNA. With regard to BLM (52), there is current debate whether this enzyme has a greater speciﬁ- city for G4-DNA structures versus Watson–Crick duplex DNA. G4R1/RHAU also displays a much tighter binding afﬁnity for unimolecular G4-DNA (Kd in the low pM range) than does RPA (Kd in the low mM range) (52), or BLM (Kd of 4 nM for binding of tetramolecular G4 DNA (55). The SPR technique could not accurately report a Kd for G4-DNA binding by T antigen; however, the working concentration range for T antigen interaction was in the low nanomolar range (54). It is also of interest that G4R1/RHAU tightly binds unimolecular G4-DNA structures without a requirement for divalent cations or ATP. In contrast, RPA, BLM and T-Ag require divalent cations for G4-DNA binding (52–54); T-Ag requires both Mg2+ and ATP (54). Although ATP is not required for G4 DNA binding by G4R1/RHAU, the PNA trap demon- strates that ATP is required for G4R1/RHAU-catalyzed G4-DNA resolution. We tested two unimolecular-G4-DNA-containing oligo- nucleotides to determine how well each oligonucleotide could inhibit the ability of G4R1/RHAU to unwind a tetramolecular G4-DNA substrate. The stoichiometric ratio of c-Myc oligonucleotidecompetitor:G4R1/RHAU concentration (50% inhibition at an inhibitor:substrate molar ratio of 0.06:1) is consistent with a simple competi- tive binding model of inhibition. However, the poor inhib- ition of resolution of tetramolecular G4-DNA substrate by Poly A Zic1 G4-DNA, relative to the 10-fold lower Kd of G4R1/RHAU for unimolecular versus tetra- molecular G4-DNA is not consistent with this model. The explanation for why inhibition of G4R1/RHAU catalyzed unwinding of tetramolecular G4-DNA by Poly A Zic1 G4-DNA is weak despite its tighter binding to G4R1/RHAU is currently unknown; however, a possibil- ity that may be considered (among many) is that the efﬁ- ciency of inhibition is inﬂuenced by the catalytic unwinding rate of the G4-DNA. The unlabeled competing unimolecular G4-DNAs are substrates competing for the enzyme active sites with labeled tetramolecular G4-DNA substrate. It is possible that Poly A Zic1 G4-DNA is being catalytically unwound by and released as single-stranded DNA from G4R1/RHAU at a faster rate than is the tetramolecular G4-DNA, thereby making Poly A Zic1 DNA a less efﬁcient inhibitor. In contrast, G4R1/ RHAU may unwind c-Myc more slowly, in which case the enzyme spends a greater residence time engaged cata- lytically to the c-Myc 51-mer, making it a better competitor. G4R1/RHAU is the protein product of the DHX36 gene, a member of the DEAH-box family of helicases (36). Some members of the DEAH-box family of helicases are known to have helicase activity on duplex RNA or DNA substrates and in some cases both RNA and DNA. The closest characterized enzyme homolog to G4R1/RHAU is the nuclear DNA helicase II (NDH II), which is also known as DHX9 or RNA helicase A. NDH II appears to have roles in both DNA and RNA metab- olism (56). A number of lines of evidence have already suggested that G4R1/RHAU is involved with RNA pro- cessing. G4R1/RHAU was originally identiﬁed by afﬁnity chromatography associated with mRNA of the AU-rich element derived from the urokinase plasminogen activator gene (40). G4R1/RHAU was shown to interact with PARN, the exosome, and target RNAs to enhance decay of ARE(uPA)-mRNAs (40). More recently is has been shown that G4R1/RHAU also accumulates in stress granules after arsenite stress, suggesting it is involved in modulating changes in RNA metabolism in response to stress (57). However, the majority of G4R1/RHAU-GFP fusion protein is observed in the nucleus (58) suggesting the potential of DNA interactions and the potential of early transcript RNA processing interactions as roles for the enzyme. It should be noted that RNA readily forms unimolecular G4 structures; G4R1/RHAU may also have a role in processing unimolecular G4-RNA in cells as well, and we have previously shown that it can recognize and unwind tetramolecular G4-RNA (37). However, prelimin- ary experiments with RNA in our laboratory have sug- gested that, although G4R1/RHAU does not tightly bind