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.

FIGURE 1.

G4R1/RHAU resolving activity on tetramolecular quadruplex DNA and tetramolecular quadruplex RNA substrates is similar and independent of the type of 5′ label. Analysis of G4 quadruplex nucleic acid resolution in the presence of G4R1/RHAU by nondenaturing 10% PAGE demonstrated G4R1/RHAU concentration-dependent resolution. Increased resolution is shown by the appearance of increasing amounts of monomers of RNA (rAGA) or DNA (dAGA). A, lanes 1–7, 0.2 pmol of 5′-³²P-labeled tetramolecular quadruplex RNA substrate (rAGA) each incubated at 37 °C in assay buffer for 30 min with 0 units, 0.07 fmol (0.063 units), 0.28 fmol (0.25 units), 1.12 fmol (1 unit), 4.34 fmol (4 units), 17.4 fmol (16 units), and 69.4 fmol (64 units) G4R1/RHAU, respectively. B, lanes 1–6, 0.2 pmol of 5′-labeled TAMRA quadruplex RNA substrate (rAGA) each incubated at 37 °C in assay buffer for 30 min with 0 units, 0.28 fmol (0.25 units), 1.12 fmol (1 unit), 4.34 fmol (4 units), 17.4 fmol (16 units), and 69.4 fmol (64 units), respectively. C, 0.2 pmol of 5′-[³²P]DNA quadruplex substrate (dAGA) incubated for 30 min at 37 °C with 0 units, 0.07 fmol (0.063 units), 0.28 fmol (.25 units), 1.12 fmol (1 unit), 4.34 fmol (4 units), 17.4 fmol (16 units), and 69.4 fmol (64 units) of G4R1/RHAU, respectively. D, lanes 1–6, 0.2 pmol of 5′-labeled TAMRA quadruplex DNA substrate (dAGA) incubated at 37 °C in assay buffer for 30 min with 0 units, 0.28 fmol (0.25 units), 1.12 fmol (1 unit), 4.34 fmol (4 units), 17.4 fmol (16 units), and 69.4 fmol (64 units), respectively. The specific activity of this preparation of recombinant G4R1/RHAU was 7700 units/μg.

FIGURE 2.

Equilibrium binding gel mobility shift assay of purified recombinant G4R1/RHAU incubated with tetramolecular quadruplex DNA and tetramolecular quadruplex RNA substrates yields apparent K_d values of 77 ± 6 and 39 ± 6 pm, respectively. A, lanes 1–7 show a gel mobility shift assay with increasing concentrations of G4R1/RHAU, 0, 30, 50, 75, 100, and 150 pm incubated with 1 pm 5′-³²P-labeled DNA quadruplex. Lanes 3–7 show one major protein-quadruplex DNA species shifted to a higher molecular weight. B, lanes 1–7 show the mobility shift assay for single-stranded DNA of the randomer d(HHN)₁₁ at concentrations of G4R1/RHAU of 0, 100, 150, 300, 500, 1000, and 3000 pm, respectively. C, lanes 1–7 show a gel mobility shift assay of 1 pm 5′-³²P-labeled tetramolecular RNA incubated with increasing concentrations of G4R1/RHAU, 0, 30, 50, 75, 100, and 150 pm. Three different protein-quadruplex RNA species are seen shifted to higher molecular weights. D, lanes 1–7 show the mobility shift assay for single-stranded RNA of the randomer r(HHN)₁₁ at concentrations of G4R1/RHAU of 0, 100, 150, 300, 500, 1000, and 3000 pm, respectively. E, graphic representation of data from four independent gel mobility shift experiments showing an ordinate of percent bound substrate and an abscissa of pm concentration of G4R1/RHAU. Error bars represent S.D.

FIGURE 3.

A G4R1/RHAU resolution assay with 5′-³²P-labeled tetramolecular quadruplex DNA substrate is inhibited most efficiently by unlabeled tetramolecular quadruplex RNA and is not inhibited by unlabeled tRNA. A, representative nondenaturing 10% acrylamide gels of G4R1/RHAU resolution assays of labeled G4-DNA substrate in the presence of 0, 1, 2, 4, 8, 16, 32, and 64 m eq of unlabeled tetramolecular G4-DNA (lanes 1–8), unlabeled tetramolecular G4-RNA (lanes 9–15), and unlabeled tRNA (lanes 16–23). B, graphic representation of four independent experiments showing inhibition by unlabeled tetramolecular G4-DNA, tetramolecular G4-RNA, and tRNA of the resolution of labeled tetramolecular G4-DNA substrate. Error bars represent S.D.

FIGURE 4.

A G4R1/RHAU resolution assay with 5′-³²P-labeled tetramolecular quadruplex RNA substrate resolution is inhibited most efficiently by unlabeled tetramolecular G4-RNA, inhibited less effectively by G4-DNA, and not effectively inhibited by unlabeled tRNA. A, representative nondenaturing 10% acrylamide gels of G4R1/RHAU resolution assays of labeled G4-RNA substrate in the presence of 0, 1, 2, 4, 8, 16, 32, and 64 m eq of unlabeled tetramolecular G4-DNA (lanes 1–8), unlabeled tetramolecular G4-RNA (lanes 9–15), and unlabeled tRNA (lanes 16–23). B, graphic representation of four independent experiments showing inhibition of the resolution of labeled tetramolecular G4-RNA substrate by unlabeled tetramolecular quadruplex DNA or tetramolecular quadruplex RNA and two independent experiments of inhibition by unlabeled tRNA. Error bars represent S.D.

FIGURE 5.

HeLa cells with a doxycycline (dox)-inducible shRNA to G4R1/RHAU show loss of the majority of quadruplex resolving activity for both DNA and RNA tetramolecular quadruplex upon down-regulation of G4R1/RHAU by shRNA. A, lanes 1–3 of a Western blot of identical loadings of 10 μg of lysate from HeLa cells not induced ((–)dox) for shRNA show high levels of G4R1/RHAU when probed with a G4R1/RHAU monoclonal antibody. Lanes 4–6 show identical loadings of 10 μg of protein after 5 days of induction for shRNA ((+)dox); G4R1/RHAU is substantially down-regulated with dox treatment. Actin levels were also detected by monoclonal antibody and were used as a loading control. B, nondenaturing gel electrophoresis of tetramolecular quadruplex DNA incubated with increasing concentrations of HeLa lysates from (–)dox and (+)dox cells for 30 min at 37 °C. Lane 1 shows the effect of no lysate. Lanes 2–5 show results for 1.5, 3.0, 6.0, and 12.0 μg of (–)dox lysate, respectively. Lanes 6–9 show results for (+)dox lysates for 1.5, 3, 6, and 12.0 μg, respectively. C, nondenaturing gel electrophoresis of tetramolecular quadruplex RNA incubated with increasing concentrations of HeLa lysates from (–)dox and (+)dox cells for 30 min at 37 °C. Lane 1 shows the effect of no lysate. Lanes 2–5 show results for 1.5, 3.0, 6.0, and 12.0 μgof (–)dox lysate, respectively. Lanes 6–9 show results for (+)dox of 1.5, 3.0, 6.0, and 12.0 μg of (+)dox lysate, respectively. (+)Dox lysates show diminished resolution activity compared with (–)dox lysates of the identical protein concentration. The reduction in tetramolecular quadruplex resolving activity was 8-fold for both RNA (S.D. = 0.30) and DNA (S.D. = 0.21) for (+)dox lysates measured at 1.5, 3.0, and 6.0 μg of protein.