G4 Resolvase 1 Binds Both DNA and RNA Tetramolecular Quadruplex with High Affinity and Is the Major Source of Tetramolecular Quadruplex G4-DNA and G4-RNA Resolving Activity in HeLa Cell Lysates

Abstract

Quadruplex structures that result from stacking of guanine quartets in nucleic acids possess such thermodynamic stability that their resolution in vivo is likely to require specific recognition by specialized enzymes. We previously identified the major tetramolecular quadruplex DNA resolving activity in HeLa cell lysates as the gene product of DHX36 (Vaughn, J. P., Creacy, S. D., Routh, E. D., Joyner-Butt, C., Jenkins, G. S., Pauli, S., Nagamine, Y., and Akman, S. A. (2005) J. Biol Chem. 280, 38117-38120), naming the enzyme G4 Resolvase 1 (G4R1). G4R1 is also known as RHAU, an RNA helicase associated with the AU-rich sequence of mRNAs. We now show that G4R1/RHAU binds to and resolves tetramolecular RNA quadruplex as well as tetramolecular DNA quadruplex structures. The apparent K d values of G4R1/RHAU for tetramolecular RNA quadruplex and tetramolecular DNA quadruplex were exceptionally low: 39 ؎ 6 and 77 ؎ 6 pM, respectively, as measured by gel mobility shift assay. In competition studies tetramolecular RNA quadruplex structures inhibited tetramolecular DNA quadruplex structure resolution by G4R1/RHAU more efficiently than tetramolecular DNA quadruplex structures inhibited tetramolecular RNA quadruplex structure resolution. Down-regulation of G4R1/ RHAU in HeLa T-REx cells by doxycycline-inducible short hairpin RNA caused an 8-fold loss of RNA and DNA tetramolecular quadruplex resolution, consistent with G4R1/RHAU representing the major tetramolecular quadruplex helicase activity for both RNA and DNA structures in HeLa cells. This study demonstrates for the first time the RNA quadruplex resolving enzymatic activity associated with G4R1/RHAU and its exceptional binding affinity, suggesting a potential novel role for G4R1/ RHAU in targeting in vivo RNA quadruplex structures.The nucleobase guanine is unique among bases in nucleic acids because of its great tendency toward self-association. Stable cyclic guanine quartets form by self-assembly through Hoogsteen hydrogen bonding between each of the four guanine molecules (N 1 -H and N 2 -H share hydrogen with O 6 and N 7 of the adjacent guanine). In the presence of physiological monovalent cations these quartets can self-associate into vertical stacks that coordinately bond a cation at the center of the G quartets, and the assemblies are further stabilized through stacking interactions (reviewed in Ref. 1). This vertical assembly of G quartets is known as G-quadruplex; this self-associating phenomenon is responsible for the observation that over 30 different derivatives of guanine can form gels in water (2). These are also the forces that stabilize the formation of quadruplex structures in nucleic acids that are also termed G4-DNA and G4-RNA. Since the initial observation by Sen and Gilbert (3) of the formation of tetramolecular G4-DNA, it has been shown that DNA or RNA molecules possessing runs of as few as three guanines in a row can form a variety of extraordinarily stable quadruplex structures (reviewed in Ref. 4). In fact, guanine quadruplex structures create th...

Evidence That G-quadruplex DNA Accumulates in the Cytoplasm and Participates in Stress Granule Assembly in Response to Oxidative Stress

“…2G) were also found in punctate regions of the cytoplasm; these proteins co-localize with DNA after transfection with G4DNA but not after transfection with ssDNA. In addition to DHX36, which is known to bind quadruplexes (27,41), YB-1 was recently reported to bind to G4RNA and G4DNA (34). Two stress granule markers that were not identified by the proteomics screen, due to low mass spectral coverage of the proteins, were G3BP and eIF3.…”

Translational Repression of the Disintegrin and Metalloprotease ADAM10 by a Stable G-quadruplex Secondary Structure in Its 5′-Untranslated Region

“…Apart from FMRP and hnRNP A, there are several proteins described that are able to interact and modify G-quadruplex structures in DNA and RNA substrates. For instance, specific RNA and DNA helicases are able to unwind G-quadruplex structures (83)(84)(85)(86). Deficiencies of certain G-quadruplex resolving helicases result in abnormal mRNA deadenylation and decay and in perturbed telomere maintenance and cellular DNA replication, leading to cancer (87).…”

The 5′ Guanosine Tracts of Human Telomerase RNA Are Recognized by the G-Quadruplex Binding Domain of the RNA Helicase DHX36 and Function To Increase RNA Accumulation

“…The results above suggest that folding favors inclusion of the 5Ј leader 3-G tracts GGG(1-3), GGG (11)(12)(13), and GGG (15)(16)(17). In alternative folds, the fourth strand could be contributed by GG (8,9), GGG(21-23), or GGGG (25)(26)(27)(28).…”

“…Importantly, unlike DNA, RNA uniformly adopts the parallel-strand orientation of quadruplex in vitro and in vivo (4,8,30,41). DHX36 has the ability to bind and resolve a model intermolecular G-quadruplex RNA (11). We therefore tested whether DHX36 binding requires the hTR 5Ј G tracts with quadruplex-forming potential.…”

“…DHX36 (also known as RHAU and G4R1) is a DEXH box RNA helicase independently discovered as a mediator of AUrich element mRNA degradation and as a resolvase for Gquadruplex DNA in vitro (35,37). Subsequent studies expanded the function of DHX36 to include more global roles in regulating mRNA expression and to include resolvase activity on a model RNA as well as a DNA quadruplex of parallelstrand orientation (11,22,25). A previous mass spectrometry analysis of human telomerase holoenzyme affinity purified by tagged TERT (18) unreliably identified DHX36 and several other helicase domain proteins (unpublished results), with the inconclusive identifications based on only a single peptide sequence (in the case of DHX36) or peptide detection in parallel mock purifications from cells lacking tagged TERT (in the case of RuvBL1 and RuvBL2, the Unigene designations for proteins also known as TIP49 or pontin and TIP48 or reptin, respectively).…”