G4-DNA species. Unimolecular G4-DNA formation was ﬁrst observed by Williamson et al. (10) in oligodeoxyri- bonucleotide sequences of the Oxytrichia 30 telomeric single-stranded overhang. Unimolecular G4-DNA struc- tures have both thermal stability and low ﬁrst-order kinetic barriers of formation (11). Furthermore, unimol- ecular G4-DNA is commonly stabilized best by a K+ containing ionic environment (3,12,13), as found in mam- malian nuclei. Many of these structures can also be stabilized in an equimolar Na+ environment, albeit to a lesser extent as evidenced by a lowered Tm. Two bio- informatics studies have suggested a canonical sequence predicting unimolecular G4-DNA formation that requires four runs of at least three tandem Gs with three loop regions (Ns) in between the G runs; the canonical putative quadruplex sequence (PQS) is summarized in the formula: d(G3–5N1–6G3–5N1–6G3–5N1–6G3–5) (14,15). These two studies found approximately 375 000 PQS in the human genome using distinctly different sequence search approaches (14,15). These determinations are probably underestimates of the true number of PQS in the human genome, since stable DNA aptamers, e.g. the thrombin aptamer (2), have been found with four guanine runs of as few as two tandem guanines. The most direct evidence of the presence of G4-nucleic acid structures in vivo comes from studies of unimolecular and bimolecular G4-DNA structures (16,17). Commonly, telomere sequences have a 30 overhang that remains single stranded, and most telomere sequences meet the canonical sequence requirements to form intramolecular G4-DNA structures. Speciﬁc antibodies to G4-DNA were observed to bind to telomeres of the macronuclear chromosomes of the ciliate Stylonychia lemnae. This work included import- ant controls to address the issue of whether the antibody was inducing G4-DNA structures (18,19). A carbazole derivative dye exhibited a G4-DNA binding-dependent ﬂuorescence emission wavelength shift when staining the telomeres of human mitotic chromosomes, suggesting the presence of antiparallel unimolecular G4-DNA (20). An inducible gene containing the immunoglobin Sm and Sg3 switch regions cloned into an Escherichia coli plasmid produced G4-DNA structures within the non-template strand of the D-loop created upon transcription induction (21). Furthermore, a number of DNA sequences have been shown in vitro to readily form unimolecular G4-DNA structures under physiological salt conditions. These PQS are located within key control regions of im- portant genes, including the aforementioned immuno- globin switch region (8), the d(pCGG) repeats of the fragile X mental retardation gene (22) and the promoter regions of a number of proliferation-associated genes such as c-Myc (23,24), PDGF-A (25), RET (26), the diabetes susceptibility locus (27) and the human insulin gene (28). Also, within transcribed regions of genes, unimolecular PQS have been found in 50-UTRs of genes such as Zic1 and NRAS (29,30), and also near intron splicing and poly (A) addition sites of genes (31,32). Although there is interest in the consequences of G4-RNA formation within these gene transcripts (29,30), it is also important to note that the non-template strand of these transcribed genes is capable of forming G4-DNA, which becomes more Nucleic Acids Research, 2011, Vol. 39, No. 16 7163 probable as transcription increases. Such transcription- associated G4-DNA formation has been observed in the non-template strand of transcribed sequences of immunoglobin Sm and Sg3 switch regions in a constructed plasmid (21). Considering that the melting temperature of most G4-DNAs that conform to the PQS sequence are pre- dicted to be above 41 C under physiological salt condi- tions, it has been hypothesized by a number of researchers that, once G4-DNAs form in vivo, they must be resolved by enzymes that speciﬁcally recognize and unwind such structures. In this regard, we identiﬁed the ﬁrst human tetramolecular G4-DNA resolving activity from cell lysates and began to characterize the nucleoside triphosphate (NTP) utilization requirement of this activity (33). Subsequently, a number of genes related to genomic instability syndromes have been demonstrated to code for enzymes possessing G4-DNA unwinding activity in vitro, including the RecQ homologs BLM (34) and WRN (22) and the FANCJ protein (35). However, further puriﬁcation, mass spectrometric identiﬁcation and recombinant expression studies revealed that the original G4-DNA resolving activity that we observed in human cell lysates did not reside in one of the above men- tioned proteins; rather, it resided in the DHX36 gene product G4R1/RHAU (36). Furthermore, G4R1/RHAU was observed to be responsible for the major tetramolecular G4-DNA resolving activity in HeLa cells (36,37), which conformed to the expectations of isolating a major activity through a biochemical approach rather than a genetic one. G4R1/RHAU is the protein product of the DHX36 gene, a member of the DEAH box family (36). It is a 115 kDa protein containing a highly conserved helicase core that contains sequence motifs, e.g. the Walker A and Walker B box motifs, in common with RecQ heli- cases. Full-length recombinant G4R1/RHAU has been shown to bind tightly to tetramolecular G4-DNA and G4-RNA with Kd’s of 77 ± 6 pM and 39 ± 6 pM, respect- ively, and to efﬁciently unwind these tetramolecular struc- tures into monomers in the presence of adenosine triphosphate (ATP) (37). G4R1/RHAU requires an un- structured loading region of at least seven bases 30 of the G4-DNA structure for efﬁcient resolution of tetramolecular molecules; no such extension of nucleotides were required at the 50-end of the G4-DNA structure for unwinding (S. Creacy, S. Akman and J. Vaughn, unpub- lished results). These results suggest that the enzyme loads on the 30-end of tetramolecular G4-DNA structures and translocates in the 30 to 50 direction. Recent deletion studies have shown that about a 100 amino acid N-terminal region of the protein is responsible for most of its binding afﬁnity to tetramolecular G4 structures (38). However, our previous work did not address the question of whether G4R1/RHAU can bind and unwind the single- stranded molecules of unimolecular G4-DNA, which are the G4-DNA structures most expected to commonly form in cells. In this study, we assessed the binding and reso- lution activity of full-length recombinant human G4R1/ RHAU upon unimolecular G4-DNA structure, and found that G4R1/RHAU binds tightly to this structure.