The DEXH protein product of the DHX36 gene is the major source of tetramolecular quadruplex G4-DNA resolving activity in HeLa cell lysates.
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.
Vaughn
James P
JP
Department of Cancer Biology and the Comprehensive Cancer Center of Wake Forest Medical Center, Winston-Salem, North Carolina 27157, USA.
Creacy
Steven D
SD
Routh
Eric D
ED
Joyner-Butt
Christi
C
Jenkins
G Scott
GS
Pauli
Sandra
S
Nagamine
Yoshikuni
Y
Akman
Steven A
SA
D000255:Adenosine Triphosphate / Q000737:chemistry; D000911:Antibodies, Monoclonal / Q000737:chemistry; D015153:Blotting, Western; D002846:Chromatography, Affinity / Q000379:methods; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D053487:DEAD-box RNA Helicases; D004247:DNA / Q000737:chemistry / Q000235:genetics; D004265:DNA Helicases / Q000235:genetics* / Q000502:physiology*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D054856:G-Quadruplexes; D006147:Guanine / Q000737:chemistry; D006367:HeLa Cells; D006639:Histidine / Q000737:chemistry; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D020365:RNA Helicases / Q000235:genetics* / Q000502:physiology*; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D045522:Recombinases / Q000737:chemistry*; D019809:Streptavidin / Q000737:chemistry; D013379:Substrate Specificity
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D012333:RNA, Messenger; D011994:Recombinant Proteins; D045522:Recombinases; D006639:Histidine; D006147:Guanine; D000255:Adenosine Triphosphate; D004247:DNA; D019809:Streptavidin; C466988:DHX36 protein, human; D004265:DNA Helicases; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases