RNA Helicase

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Recombinant DHX33 protein possesses dual DNA-RNA helicase activity Xingshun Wang, Wei Ge, and Yandong Zhang Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00166 • Publication Date (Web): 05 Jun 2018 Downloaded from http://pubs.acs.org on June 6, 2018

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Biochemistry

Recombinant DHX33 protein possesses dual DNA-RNA helicase activity Running title: DHX33 unwinds both DNA and RNA duplexes Xingshun Wang 1，2, Wei Ge2 and Yandong Zhang1* 1. Department of Biology, Southern University of Science and Technology, Shenzhen, Guangdong, P.R.China 2. Faculty of Health Sciences, University of Macau, Macau, China

Key words: DHX33, RNA helicase, helicase activity, ATPase

*To whom correspondence should be addressed: Dr. Yandong Zhang Department of Biology Southern University of Science and Technology 1088 Xueyuan Blvd, Xili Nanshan District, Shenzhen Guangdong, P.R.China E-mail: [email protected] Telephone: 86-755-88018422

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Abstract RNA helicase DHX33 has been shown to participate in a variety of cellular activities including ribosome biogenesis, protein translation as well as gene transcription. We and others further discovered that DHX33 is highly expressed in several types of human cancers and plays important roles in promoting cancer cell proliferation. To better understand the molecular mechanism for DHX33 in exerting its biological functions, we purified recombinant DHX33 and performed biochemical studies in vitro. DHX33 protein was found to have ATPase activity which is dependent on DNA or RNA duplexes. The ATPase activity of DHX33 is coupled with its RNA/DNA unwinding activity. If a key residue in ATP binding site was mutated, the mutant DHX33 could not unwind DNA/RNA duplexes. Furthermore, deletion mutant of a RKK motif previously identified to be involved in ribosome DNA binding could still unwind DNA duplexes, albeit in reduced efficiency. In summary, our study reveals that purified DHX33 protein possesses unwinding activity toward DNA and RNA duplexes.

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Biochemistry

Introduction DEAD/H-box helicase proteins belong to the large Superfamily 2 (SF2) of helicases which primarily remodel RNA structures 1. Although these proteins are called RNA helicases, a few DEAD/H box proteins have been found to unwind DNA duplexes and influence DNA activity, such as gene transcription 2. All DEAD/H-box proteins contain eight conserved motifs distributed on two RecA-like domains 3. DEAD box proteins have an extra Q motif in the N-terminal region while DEAH box proteins have not 4. These motifs are important in binding to NTP and RNA substrates to cause strand unwinding, annealing, clamping or protein displacement 5. DEAD box proteins can catalyze local RNA duplex unwinding in a non-processive manner, while most DEAH box proteins preferentially load onto single strand RNA to unwind duplexes from the 3’-tail in a processive manner

6

. Through changing the

conformation of RNA, DEAD/H box proteins modify the interaction of these RNAs with proteins 7. They participate in essentially all aspects of RNA metabolism, including gene transcription, pre-mRNA splicing, ribosome biogenesis, nuclear export, RNA decay and mRNA translation 8. DHX33 protein is one member of this large protein family, and it was identified by our research group and others to be an important player in cell proliferation and innate immunity translation

12, 13

9-12

. DHX33 functions in ribosome biogenesis

11, 13

, protein

and gene transcription 10. Several lines of evidence also indicate that

DHX33 is involved in cancer development

10, 14-16

. It can be regulated by many

oncogenes and tumor suppressors such as Ras, PI3K, ARF, NF-1, Myc

10, 16, 17

.

Inhibition of DHX33 gene results in cancer cell death and marked reduced cell 3 / 26


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proliferation10, 13, 16, 17. Despite these findings with regard to its biological function and its role in human cancers, little is known about its biochemical properties and mechanisms at the molecular level. Several members of the RNA helicases have been purified and found to be able to unwind RNA or DNA duplexes in vitro; however, no one has characterized the enzymatic activity of DHX33. Additionally, given its important role in promoting cancer development and the fact that DHX33 helicase activity is required for its functions in cells, to find out the inhibitors of DHX33 might provide one potential therapy for treating cancer patients. In the present study, we purified DHX33 protein into homogeneity and found that DHX33 protein could unwind both RNA and DNA duplexes in an ATP-dependent manner in vitro. The ATPase activity of DHX33 is coupled to its helicase activity of DHX33.

Materials and Methods Plasmids The pET32M-3C vector was mutagenized to delete the internal 6X His tag by site-directed mutagenesis (Agilent Technologies). DNA sequencing was performed to make sure it is correct. The open reading frame of mouse DHX33 was then subcloned into the BamHI/HindIII site in this modified pET32M-3C vector. The plasmid should encode a DHX33 fusion protein with a thioredoxin tag at the N-terminus and a 6X His tag at the C-terminus. To generate K94N mutant DHX33 protein, a PCR-based site-directed mutagenesis was carried out with the pET32M-3C-DHX33 plasmid as a 4 / 26


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Biochemistry

template. To generate DNA binding domain mutant DHX33 (Δ540-542RKK), similar PCR-based site-directed mutagenesis was performed. Sequencing was performed to confirm the correct sequences. Recombinant protein purification Plasmid pET32M-3C-DHX33 (WT or mutants) was transformed into E. coli strain BL-21pLysS (DE3), and 0.5 mmol/L isopropyl-L-thio-B-D-galactopyranoside was used to induce the recombinant protein expression at 16oC for 16 hours. Cells were pelleted, resuspended in cell lysis buffer (50mM Tris-HCl (pH7.2), 150mM NaCl, 1% Triton X-100, and 50mM imidazole supplemented with protease inhibitors). Cells were then sonicated and centrifuged at 13,000rpm for 25 min. Supernatants were incubated with Tris buffer-equilibrated Nickel- nitrilotriacetic acid beads followed by extensive washing. Protein was then eluted with 300mmol/L imidazole in Tris buffer followed by dialysis against Tris buffer without imidazole at 4°C overnight. Analysis of ATPase activity in vitro Recombinant DHX33 protein (0.25 µg) was incubated in binding buffer [25 mM 4-MOPS (pH7.0), 5mM ATP, 2mM DTT, 3mM MnCl2, and 100 µg/ml of BSA] alone or with 2.5 µg annealed DNA/RNA duplexes for 60 minutes at 37°C. For negative controls, the above buffer without ATP should be used in the enzymatic reactions. ATP levels were counted on a plate reader (Enspire, Perkin-Elmer). Kinase-Glo reagent (Promega, Madison, WI) was then incubated with the reaction mixture at 1:1 ratio for 10 minutes. Kinase-Glo reagent binds to the remaining ATP, so reduction in counts indicates ATPase activity. 5 / 26


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DNA/RNA duplex annealing The sequences of the DNA/RNA oligo were derived from a previous protocol 18. To generate DNA or RNA duplex with 3'-end tail, the following DNA oligoes were used: A biotin-labeled DNA/RNA oligo at the 5'-end has the following sequence: 5'-GCTGACCCTGCTCCCAATCGTAATCTATAG-3'; DNA/RNA

oligo

at

the

5'-end

has

while the

a

DIG-labeled

following

sequence:

5'-CGATTGGGAGCAGGGTCAGC-3'. These two strands were heated to 95oC, and then slowly cooled down to room temperature. The annealed DNA/RNA duplex is a partially annealing duplex with a 3'-tail. To generate DNA or RNA duplex with 5'-end tail, a DIG-labeled DNA or RNA oligo at the 5'-end has the following sequence: 5'TAGGTGACACTATAGATTAC-3'. While the biotin-labeled DNA or RNA oligo is the same as above. Similarly, annealing was performed for this DIG-labeled DNA or RNA oligo with Biotin labeled DNA or RNA oligo as described above. Bioluminescence analysis for RNA/DNA unwinding activity The protocol was modified based on a previously published method

18

. Neutravidin

was coated on 96 well plates at a final concentration of 10µg/ml (100µl/well) which is dissolved in 0.5M sodium carbonate buffer (pH9.3) overnight at 4oC. After washing with PBS for three times, the plates were slammed onto paper towels to drain out all residual buffer. The plates were then air-dried at room temperature. The neutravidin coated plates were subsequently blocked with 100µl of 0.1% (w/v) BSA (dissolved into regular PBS) at 22oC for 2 hours. Plates were then washed three times with PBS (200 µl/well), air-dried at room temperature. Annealed DNA duplex (2.5ng) in 1M 6 / 26


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Biochemistry

PBS (pH7.0) containing 1 M NaCl was then added and incubated at 22oC for 4 hours. This is followed by washing with 200 µl regular PBS (pre-warmed to 37oC) and with 200 µl of 50mM Tris-HCl (pH7.5) containing 50mM NaCl (pre-warmed to 37oC). Helicase reactions are initiated upon addition of 90µl of the reaction mix (0.25µg of purified full-length DHX33 protein, 25 mM 4-MOPS (pH7.0), 5mM ATP, 2mM DTT, 3mM MnCl2, and 100 µg/ml of BSA). Reactions should be carried out for 60 minutes at 37oC.

The plates were then washed twice with 150 mM NaCl and dried at room

temperature for 15 minutes. The plates were further washed with buffer (0.1M maleic acid, 0.15 M NaCl, 0.3% Tween-20, pH7.5). Then each well was filled up with blocking solution (10% BSA (w/v) in 0.1M maleic acid, 0.15M NaCl (pH7.5)) for 30 minutes followed by incubation with 20 µl antibody solution (anti-DIG-AP, Roche, in blocking buffer) for 30 minutes. After washes with 100 µl of detection buffer (0.1 M Tris-HCl, 0.1M NaCl, pH9.5), 1 µl of chemiluminescence substrate (CSPD-0.25mM) was then applied into each well and the plates were incubated for 5 minutes at 17oC. The plates were then drained and incubated at 37oC for 30 minutes. The remaining DIG-AP-label in each well was counted for 10 minutes against controls by a luminescence multi-well plate reader (Enspire, Perkin-Elmer). Western blot Approximately 25µg of proteins were denatured in SDS sample buffer at 95oC for 6 minutes, then resolved on 10% SDS-PAGE and transferred to PVDF membrane. After blocking with TBST containing 5% non-fat milk powder at room temperature for 30 minutes, the membrane was incubated with primary antibody (anti-DHX33, mouse 7 / 26


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monoclonal, Santa Cruz, B-4). After washing with PBS for three times, membrane was further incubated with secondary antibody (goat anti-mouse-HRP) at room temperature for 40 minutes. The membrane was then detected by ECL (Promega). Circular Dichroism The CD spectrum was recorded by Chirascan spectrometer (Applied Photophysics， U.K.). The absorbance wavelength ranges from 195nm to 260nm. DHX33 protein (~0.2mg/ml in 50mM Tris-HCl, pH7.5, 100mM NaCl) was loaded into a 1 mm quartz cell. The data were analyzed using the program Origin7.0. Statistical analysis Data is presented as the mean ± SD. Statistical significance was determined using the Student’s t test, with a p value < 0.05 considered significant.

Results Purification of recombinant DHX33 protein Previously, we tried to overexpress DHX33 in E. coli, we found DHX33 hardly soluble for purification. Most of the induced DHX33 existed in the inclusion bodies due to misfolding. To enhance the efficiency of correct folding, we engineered a thioredoxin tag at the N-terminus of DHX33 (Figure 1A).

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Biochemistry

Figure 1. Purification of DHX33 protein. (A). Construction of a thioredoxin (Trx) tagged DHX33. Thioredoxin is at the N-terminus of DHX33 while 6X His tag is at the C-terminus of DHX33. (B). The recombinant protein was induced by IPTG (at 1mM final concentration) in E.coli. Right lane- protein marker; Middle lane-whole cell lysate from E coli without IPTG induction; Left lane- whole cell lysate from E coli with IPTG induction. (C). The DHX33 recombinant protein was purified through Ni-NTA column into homogeneity. Five eluents from the purification demonstrate single band in SDS-PAGE gel analysis (by Commassie blue staining). (D). The purified fractions were further analyzed by western blot with anti-DHX33 antibody. Both samples contain DHX33 proteins.

In order to study whether DHX33 can function as a helicase to unwind RNA/DNA in vitro, we induced the expression of DHX33 and then purified the protein into 9 / 26


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homogeneity, albeit with very low efficiency (Figure 1B-C). To make sure this purified protein folded properly, we ran circular dichroism spectra as shown in Figure S1 (supplemental figure). We found that the DHX33 protein folded well with ~70.1% α-helices, ~11.7% β-sheets and ~14.2% random coil. We further confirmed the identity of the purified DHX33 protein by western blot (Figure 1D). DHX33 hydrolyzes ATP in the presence of DNA/RNA duplexes

According to

previous reports, DEAD/DEAH box proteins have conserved domains that could bind and hydrolyze ATP in order to modify RNA structure 8. To analyze whether the purified protein hydrolyzes ATP intrinsically, we performed ATP consumption analysis. As shown in Figure 2A,

Figure 2. DHX33 protein is not an intrinsic ATPase . (A) A standard curve was generated to correlate the amount of ATP with chemiluminescence signal from Kinase-Glo reagent. The linear range is from 0.1M to 1.5 M. (B)

DHX33 could not hydrolyze ATP without substrates. ATP

was used at a final concentration of 1M in the reaction mixture. DHX33 protein was used at an amount from 0 to 560ng. No change in ATP levels was observed as shown by the chemiluminescence signals.

we set up a standard curve to correlate the ATP concentration with luminescence 10 / 26


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Biochemistry

signal. We incubated different amounts of purified DHX33 recombinant protein in the presence of the reagent, and then detected the change of luminescence signals. As shown in Figure 2B, when we increased the DHX33 protein levels, the reduction of ATP signals could not be detected. This indicates that DHX33 does not have any ATPase activity intrinsically. We then analyze the ATPase activity of DHX33 in the presence of DNA/RNAs. Firstly, we anneal two totally complementary oligo DNA molecules; the annealed DNA duplex is blunt ended. The annealing efficiency was detected by gel electrophoresis as shown in Figure 3A.

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Figure 3. Duplexes of DNA/RNA or heteroduplexes of RNA/DNA stimulate the ATPase activity of DHX33. (A). Gel electrophoresis was performed to analyze the annealing efficiency for two single strand DNA oligoes. Ds - double strand; ss - single strand. The sequences for the two oligoes are shown below. (B). Changes of the chemiluminescence signals from the Kinase-Glo reagent after the same amount of DHX33 was co-incubated with different DNAs. ATP was used at a final concentration of 1 M in the reaction. Only double-stranded DNA can stimulate DHX33 ATPase activity, while single-stranded DNA could not. **, n=3, P3' direction. Finally, we performed experiments with shorter and longer duplexes to analyze the processivity of DHX33, as shown in the supplemental Figure S3, we found the longer duplex could not be efficiently separated, while the shorter duplex could. ATP hydrolysis is coupled to DHX33 unwinding

To investigate whether the

ATPase activity and helicase activity are coupled together, we purified mutant DHX33 recombinant protein which has its K94 mutated into N. Previously, it was reported that the “K” in the “GSGKT/S” motif is critical in ATP binding and hydrolysis. As shown in Figure 5A,

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Figure 5. ATP hydrolysis is coupled to DHX33 unwinding . (A)

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The K94N mutant DHX33

recombinant protein can be induced by IPTG (1mM for its final concentration) in E.coli. Lane 1protein marker; Lane 2-whole cell lysate from E coli without IPTG induction; Lane 3 to lane 6 represent the whole cell lysates from 4 different clones of E coli after IPTG induction. (B) The K94N mutant of DHX33 recombinant protein was purified through Ni-NTA column. Two eluents from the purification demonstrate triple bands in SDS-PAGE gel analysis (by Commassie blue staining). The top band belongs to DHX33 protein, while the middle band and the bottom band belong to molecular chaperons through mass spectrometry analysis. (C) The purified fractions were further analyzed by western blot with anti-DHX33 antibody. Both samples 18 / 26


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Biochemistry

contain DHX33 proteins. (D) Incubation of K94N mutant DHX33 with either double-stranded DNA or single-stranded DNA did not cause the consumption of ATP. Experiments were repeated for three times, no statistical significance was found. (E)

Results of helicase reaction in the

presence of DNA duplex after addition of the K94N mutant DHX33 recombinant protein. All reaction mixtures contain the same amount of DNA substrates. Experiments were repeated for three times, no statistical significance was found.

we successfully induced the mutant DHX33 protein expression. We further purified the recombinant protein (Figure 5B). For this purified protein, we found it interacted with two molecular chaperons (as confirmed by mass spectrometry, data are shown in the supplemental Table S1). We confirmed the purified protein to be DHX33 by western blot analysis (Figure 5C). We then performed ATPase and helicase analysis. As shown in Figure 5D, we found that the mutant DHX33 could not hydrolyze ATP as compared to the wild type DHX33 control. We further analyzed the helicase activity for this mutant, and found that it could not unwind DNA/ RNA duplexes (Figure 5E, supplemental Figure S4A), either. Our results show that ATP binding and hydrolysis are critical in the unwinding activity of DHX33, and these two activities are coupled together. Motif 540-542 is contributing to the helicase activity of DHX33

We have

previously found that DHX33 is able to bind to ribosome DNA via a critical motif in the C-terminus11. To find out whether this motif is important in the helicase activity of DHX33 via DNA binding, we performed site-directed mutagenesis to delete three critical residues 540-542, RKK. As shown in Figure 6A-C, 19 / 26


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Figure 6. A RKK motif involved in DNA binding is contributing to DHX33 helicase activity. (A). The ∆540-542RKK mutant DHX33 recombinant protein can be induced by IPTG (0.5 mM) in E.coli. Lane 1- protein marker; Lane 2-whole cell lysate from E coli without IPTG induction; Lane3 represents whole cell lysate from E. coli after IPTG induction. (B). The ∆540-542RKK mutant DHX33 recombinant protein was purified through Ni-NTA column. Eluent from the purification demonstrates triple bands in SDS-PAGE gel analysis (by Commassie blue staining). The top band belongs to DHX33 protein; similarly, as identified by mass spectrometry, the middle band and the bottom band belong to molecular chaperons. As a comparison, wild type DHX33 was purified in a similar way. Both proteins show similar purity by Commassie Blue Staining on 20 / 26


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Biochemistry

the SDS-PAGE gels. (C). The purified fractions for the∆540-542RKK mutant protein and wild type DHX33 protein were further analyzed by western blot with anti-DHX33 antibody. Both samples contain DHX33 protein. (D). Incubation of ∆540-542RKK mutant DHX33 with double-stranded DNA but not single-stranded DNA caused ATP consumption. *, n=3, P

RNA Helicase

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