The DEAD-box protein DDX43 (HAGE) is a dual RNA-DNA helicase and has a K-homology domain required for full nucleic acid unwinding activity

Abstract

The K-homology (KH) domain is a nucleic acid-binding domain present in many proteins but has not been reported in helicases. DDX43, also known as HAGE (helicase antigen gene), is a member of the DEAD-box protein family. It contains a helicase core domain in its C terminus and a potential KH domain in its N terminus. DDX43 is highly expressed in many tumors and is, therefore, considered a potential target for immunotherapy. Despite its potential as a therapeutic target, little is known about its activities. Here, we purified recombinant DDX43 protein to near homogeneity and found that it exists as a monomer in solution. Biochemical assays demonstrated that it is an ATP-dependent RNA and DNA helicase. Although DDX43 was active on duplex RNA regardless of the orientation of the single-stranded RNA tail, it preferred a 5' to 3' polarity on RNA and a 3' to 5' direction on DNA. Truncation mutations and site-directed mutagenesis confirmed that the KH domain in DDX43 is responsible for nucleic acid binding. Compared with the activity of the full-length protein, the C-terminal helicase domain had no unwinding activity on RNA substrates and had significantly reduced unwinding activity on DNA. Moreover, the full-length DDX43 protein, with single amino acid change in the KH domain, had reduced unwinding and binding activates on RNA and DNA substrates. Our results demonstrate that DDX43 is a dual helicase and the KH domain is required for its full unwinding activity.

Keywords: DNA; DNA enzyme; DNA helicase; DNA-protein interaction; RNA; RNA helicase; RNA metabolism; RNA-binding protein.

Figure 1.

Purification and identification of DDX43 protein. A, SDS-PAGE analysis of the eluted DDX43 fractions from a Ni-NTA column. M, marker. Fractions 3–9 are shown. B, chromatographic profile of recombinant DDX43 proteins eluting from a Sephacryl S-300 HR column. Two peaks are indicated. mAU, milliabsorbance units. C, SDS-PAGE analysis of the peaks shown in B. D, Western blot analyses of the proteins shown in C with antibody against DDX43 (left) or His (right). E, SEC-MALS analysis of DDX43 protein shows the calculated molecular mass of the peak and the variation in the molecular weight across the peak for DDX43. The molecular mass for each peak was indicated.

Figure 2.

RNA helicase activity of DDX43 protein. A, a representative image of helicase reactions performed by incubating 0.5 nm 5′-tailed 13-bp duplex RNA substrate with increasing protein concentration (0–3 μm) at 37 °C for 15 min. NE, no enzyme. B, a representative image of helicase reactions performed by incubating 0.5 nm 5′-tailed 13-bp duplex RNA substrate and 150 nm DDX43 protein with increasing time (0–30 min) at 37 °C. C and D, representative images of helicase reactions performed by incubating 0.5 nm 3′-tailed 13-bp duplex RNA substrate (C) and 13-bp blunt end duplex RNA substrate (D) with increasing protein concentration (0–3 μm) at 37 °C for 15 min. E, quantitative analyses of RNA unwinding of DDX43 in panels A, C, and D. Data represent the mean of at least three independent experiments with S.D. indicated by error bars. F, a representative image of helicase reactions performed by incubating a 0.5 nm 5′-tailed 16-bp duplex RNA substrate with increasing protein concentrations (0–3 μm) at 37 °C for 15 min. RNA is in black.

Figure 3.

DNA helicase activity of DDX43 protein. A, a representative image of helicase reactions performed by incubating 0.5 nm 19-bp forked duplex DNA substrate with increasing protein concentrations (0–3 μm) at 37 °C for 15 min. NE, no enzyme. B, a representative image of helicase reactions performed by incubating 0.5 nm 19-bp forked duplex DNA substrate and 150 nm DDX43 protein with increasing time (0–30 min) at 37 °C. C–E, representative images of helicase reactions performed by incubating 0.5 nm 3′-tailed 19-bp duplex DNA substrate (C), 5′-tailed 19-bp duplex DNA substrate (D), or 19 bp blunt end duplex DNA substrate (E) with increasing protein concentrations (0–3 μm) at 37 °C for 15 min. F, quantitative analyses of DNA unwinding of DDX43 in panel A and C. Data represent the mean of at least three independent experiments with S.D. indicated by error bars. G–I, representative images of helicase reactions performed by incubating 0.5 nm 30-bp (G), 40-bp (H), or 50-bp (I) forked duplex DNA substrate with increasing protein concentrations (0–3 μm) at 37 °C for 15 min. J, quantitative analyses of DNA unwinding of DDX43 in panels G–I. Data represent the mean of at least three independent experiments with S.D. indicated by error bars. DNA is in red.

Figure 4.

ATP hydrolysis is required for DDX43 unwinding. A and B, representative images of helicase reactions performed by incubating 0.5 nm 5′-tailed 13-bp duplex RNA substrate (A) or forked 19-bp duplex DNA substrate (B) with 150 nm DDX43 protein at 37 °C for 15 min with different nucleoside triphosphates. NE, no enzyme. C and D, representative images of helicase reactions performed by incubating 0.5 nm 5′-tailed 13-bp duplex RNA substrate (C) or forked 19-bp duplex DNA substrate (D) with ATP or ATP analogs with increasing protein concentrations (0–3 μm). E, representative images of helicase reactions performed by incubating 0.5 nm 19 bp forked dsDNA substrate with increasing protein concentrations of DDX43 (0–3 μm) and 2 mm ATP or ADP-BeF_x at 37 °C for 15 min. F, a representative image of ATP hydrolysis detected by TLC with DDX43 protein (2.5 μm) with M13 ssDNA effector (50 μm). WT, wild type; K292A and D396A are two engineered mutants. G, a representative image (top) of DDX43 protein ATP hydrolysis detected by TLC with indicated nucleic acid cofactor dT₃₀ or rU₃₀ (30 μm) and quantitative analyses of ATP hydrolysis (bottom). Data represent the mean of at least three independent experiments with S.D. indicated by error bars.

Figure 5.

Translocase activity and protein displacement activity of DDX43 protein. A and B, translocation reactions (20 μl) were performed by incubating increasing concentrations of DDX43 protein (0–3 μm) with 0.5 nm 5′-tailed plasmid-triplex substrate (A) or 3′-tailed plasmid-triplex substrate (B) at 37 °C for 15 min under standard translocase assay conditions. ChlR1 (2.4 nm) was used as a positive control, and K292A was an engineered DDX43 mutant. Triangle, heat-denatured DNA substrate control. NE, no enzyme. C and D, representative images of protein displacement reactions performed by incubating 0.5 nm DNA substrate (X12-1-28-BioT37; supplemental Table S1) that had streptavidin bound with increasing protein concentrations of FANCJ (0–2.4 nm) and DDX43 (0–3 μm, C) at 37 °C for 15 min or increasing times (0–45 min, D) at 37 °C with a consistent protein concentration of FANCJ (1.2 nm) or DDX43 (1.5 μm).

Figure 6.

Purification and characterization of DDX43 KH domain. A, schematic representation of full-length DDX43 and its KH domain (top) and purified KH domain proteins (wild type and mutant, 1 μg of protein each loaded) shown on Coomassie-stained SDS-PAGE gel (bottom). NE, no enzyme. B, circular dichroism spectrum of KH domain protein. C, secondary structure prediction of KH domain protein. The motif GRGG is highlighted in red square. D–H, representative EMSA images for KH domain proteins binding with 0.5 nm dT₃₀ ssDNA (D), 19-bp forked duplex DNA (E, two images merged), ssRNA rU₃₀ (F), 5′-tailed 13 bp dsRNA (G), and blunt-end dsDNA (H).

Figure 7.

Purification and characterization of DDX43 helicase core domain. A, SDS-PAGE analysis of the DDX43 helicase domain protein eluted from a Sephacryl S-300 HR column. 1 μg of protein was loaded. NE, no enzyme. B–F, representative images of helicase reactions by incubating increasing DDX43^HD protein (0–3 μm) with 0.5 nm 19-bp forked dsDNA (B), 3′-tailed 19-bp dsDNA (C), 5′-tailed 19-bp dsDNA (D), 19-bp blunt-end dsDNA (E), and 13-bp 5′-tailed dsRNA (F).

Figure 8.

KH domain mutation affects unwinding activity of DDX43. A, SDS-PAGE analysis of the DDX43 full-length G84D mutant protein. NE, no enzyme. B and C, representative images (left) and quantitative analyses (right) of helicase reactions by incubating increasing DDX43^FL-G84D protein (0–3 μm) with 0.5 nm 13-bp 5′-tailed dsRNA (B) or 19-bp forked dsDNA (C). D, a representative EMSA image for DDX43 full-length wild type and G84D mutant proteins binding with 0.5 nm 19-bp forked duplex DNA. E, proposed models for the role of KH domain in DDX43 helicase unwinding for dsRNA (upper) and dsDNA (bottom). RecA1 (HD1), RecA2 (HD2), and KH domains are shown in orange, blue, and dark red, respectively, and linkers are in green. The dashed red line in dsDNA representing the 5′ tail may or may not be there. For the visible purpose, the RNA:RNA bubble is larger than the reality.