Published online 17 May 2011 Nucleic Acids Research, 2011, Vol. 39, No. 16 7161–7178 doi:10.1093/nar/gkr234 G4 Resolvase 1 tightly binds and unwinds unimolecular G4-DNA Banabihari Giri1, Philip J. Smaldino 1 , Ryan G. Thys1, Steven D. Creacy1, Eric D. Routh1, Roy R. Hantgan 2 , Simon Lattmann 3 , Yoshikuni Nagamine 3 , Steven A. Akman1,* and James P. Vaughn1 1Department of Cancer Biology and the Comprehensive Cancer Center of Wake Forest University School of Medicine, Winston-Salem, 2Department of Biochemistry, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA and 3The Friedrich Miescher Institute For Biomedical Research, Novartis Research Foundation, Maulbeerstrasse-66, CH-4058 Basel, Switzerland Received August 16, 2010; Revised December 27, 2010; Accepted April 4, 2011 ABSTRACT It has been previously shown that the DHX36 gene product, G4R1/RHAU, tightly binds tetramolecular G4-DNA with high affinity and resolves these struc- tures into single strands. Here, we test the ability of G4R1/RHAU to bind and unwind unimolecular G4-DNA. Gel mobility shift assays were used to measure the binding affinity of G4R1/RHAU for uni- molecular G4-DNA-formed sequences from the Zic1 gene and the c-Myc promoter. Extremely tight binding produced apparent Kd’s of 6, 3 and 4 pM for two Zic1 G4-DNAs and a c-Myc G4-DNA, re- spectively. The low enzyme concentrations required for measuring these Kd’s limit the precision of their determination to upper boundary estimates. Similar tight binding was not observed in control non-G4 forming DNA sequences or in single-stranded DNA having guanine-rich runs capable of forming tetra- molecular G4-DNA. Using a peptide nucleic acid (PNA) trap assay, we show that G4R1/RHAU cata- lyzes unwinding of unimolecular Zic1 G4-DNA into an unstructured state capable of hybridizing to a complementary PNA. Binding was independent of adenosine triphosphate (ATP), but the PNA trap assay showed that unwinding of G4-DNA was ATP dependent. Competition studies indicated that uni- molecular Zic1 and c-Myc G4-DNA structures inhibit G4R1/RHAU-catalyzed resolution of tetramolecular G4-DNA. This report provides evidence that G4R1/ RHAU tightly binds and unwinds unimolecular G4-DNA structures. INTRODUCTION The nucleic acid base guanine has the capacity to base pair with itself, enabling the formation of alternative nucleic acid structures that differ from the canonical Watson– Crick base paired double helix. G4-DNAs (also termed G quadruplex DNAs) have guanines that form Hoogsteen-bonded quartets that coordinately bind a single monovalent cation at the O6 position of each guanine of the quartet (Figure 1A). Guanine quartets form the fundamental building block of G4-nucleic acids (1). Nucleic acid sequences with four appropriately spaced runs of usually three or more guanine bases can form structures of stacked guanine quartet subunits, although in some cases, e.g. the G15D thrombin aptamer (2), as few as four runs of two guanine bases can form this structure. Nucleic acid structures containing stacked guanine quartets demonstrate unusual thermal stability (3). Although all G4-DNAs have the common stacked guanine quartet motif, they are heterogeneous with regard to the number of individual nucleic acid strands involved and the paired direction of the phosphodiester backbones of the four strands of the structure. Stable G4-nucleic acids may contain intra- and/or interstrand hydrogen bonds along with parallel and/or antiparallel strand orientations. Sequence, concentration of strands, and the ionic environment all inﬂuence the type of G4-nucleic acid structures formed (4–6). Some sequences (especially fully intramolecular G4-DNA) can form poly- morphic structures that are stable under physiological conditions, and, in general, intramolecular G4-DNA must be characterized empirically because ab initio know- ledge has not developed sufﬁciently to predict structures from sequence and solution conditions alone (7). *To whom correspondence should be addressed. Tel: +336 716 0231; Fax: +336 716 0255; Email: sakman@wfubmc.edu Correspondence may also be addressed to James P. Vaughn. Tel: +336 716 7122; Fax: +336 716 0255; Email: jvaughn@wfubmc.edu The authors wish it to be known that, in their opinion, the ﬁrst two authors should be regarded as joint First Authors. The Author(s) 2011. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.