The DEXH Protein Product of the DHX36 Gene Is the Major Source of Tetramolecular Quadruplex G4-DNA Resolving Activity in HeLa Cell Lysates

Abstract

G4-DNA is a highly stable alternative DNA structure that can form spontaneously in guanine-rich regions of single-stranded DNA under physiological conditions. Since a number of biological processes create such single-stranded regions, G4-DNA occurrence must be regulated. To date, resolution of tetramolecular G4-DNA into single strands (G4-resolvase activity) has been observed only in recombinant RecQ DNA helicases. We previously reported that human cell lysates possess tetramolecular G4-DNA resolving activity (Harrington, C., Lan, Y., and Akman, S. (1997) J. Biol Chem. 272, 24631-24636). Here we report the first complete purification of a major non-RecQ, NTP-dependent G4-DNA resolving enzyme from human cell lysates. This enzyme is identified as the DEXH helicase product of gene DHX36 (also known as RHAU). G4-DNA resolving activity was captured from HeLa cell lysates on G4-DNA affinity beads and further purified by gel filtration chromatography. The DHX36 gene product was identified by mass spectrometric sequencing of a tryptic digest from the protein band on SDS-PAGE associated with activity. DHX36 was cloned within a His 6 -tagging vector, expressed, and purified from Escherichia coli. Inhibition and substrate resolution assays showed that recombinant DHX36 protein displayed robust, highly specific G4-DNA resolving activity. Immunodepletion of HeLa lysates by a monoclonal antibody to the DHX36 product removed ca. 77% of the enzyme from lysates and reduced G4-DNA resolving activity to 46.0 ؎ 0.4% of control, demonstrating that DHX36 protein is responsible for the majority of tetramolecular G4-DNA resolvase activity.G4-DNA is an alternative highly stable DNA structure forming within runs of guanine bases. It has been amply described previously (1). G4-DNA structures have the potential to disrupt normal duplex DNA; therefore, it might be expected that the genome would have minimized the usage of runs of deoxyguanosine. On the contrary, a growing body of data support the hypothesis that formation of G4-DNA in vivo is a recognized structural motif of specialized utility for key biological processes. Recent studies with a fluorescent G4-DNAbinding ligand, as well as a specific G4-DNA-binding protein, support the presence of G4-DNA structures located at human telomeres in vivo (2, 3). In addition to the aforementioned telomeres, other guanine-rich regions in human DNA readily form G4-DNA structures in vitro and make up specific genetic control elements, including the immunoglobin heavy chain switch region (4), guanine-rich regions of ribosomal DNA (5), the d(pCGG) repeats of the fragile X mental retardation gene (6), promoters of proliferation-associated genes, such as the c-MYC (7, 8), PDGF-A (9), RET (10), and the diabetes susceptibility locus IDDM2 promoter (11). It has been shown that a unimolecular G4-DNA structure has a repressor function in the c-MYC promoter (8). Compounds that stabilize G4-DNA in vivo have generated much interest because of their antitumor activity, suggesting that G4-DNA structures might be ...

Aspartate-glutamate-alanine-histidine box motif (DEAH)/RNA helicase A helicases sense microbial DNA in human plasmacytoid dendritic cells

“…These proteins are highly conserved from viruses and bacteria to humans (26). Although initially classified as RNA helicases, many members of the DExD/H helicase family, including DHX36 and DHX9, were found to display DNA helicase activity (27,28). Sequence analyses revealed that DHX36 and DHX9 are in the same DEAH/RHA helicase subfamily, which is close to but is clearly separated from the RIG-I-like helicase subfamily (Fig.…”

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.…”

“…The most highly enriched protein in the G4DNA sample was the DHX36/RHAU/G4R1 RNA helicase, previously identified as the major source of G4DNA binding and unwinding activity in the cell (27)(28)(29). DHX36 is a multifunctional enzyme involved in transcription (30) and translation (31) and has been suggested to serve as a sensor for DNA in the cytoplasm (32).…”

“…DHX36, a member of this family, is capable of binding and unwinding RNA, which combined with its ability to be recruited to stress granules (33) likely plays a role in regulating translation. It is an essential gene (94) and is the primary enzyme responsible for unwinding G4DNA in cells (27). Another cytosolic RNA helicase, DDX41 (95), binds to DNA and activates STING, which ultimately leads to the activation of NF-kB and interferon regulatory factor 3 (IRF3).…”

Binding of G-quadruplexes to the N-terminal Recognition Domain of the RNA Helicase Associated with AU-rich Element (RHAU)

“…RNA helicase associated with AU-rich element (RHAU 9 ; also known as DHX36 and G4R1) has been recognized as a major source of quadruplex-resolving activity in cell lysates (17,18). RHAU belongs to the DEX(H/D) family of RNA helicases, a large group of enzymes that remodel nucleic acid structures in an ATP-dependent manner (19).…”