title: G4 resolvase 1 tightly binds and unwinds unimolecular G4-DNA.
abstract: It has been previously shown that the DHX36 gene product, G4R1/RHAU, tightly binds tetramolecular G4-DNA with high affinity and resolves these structures into single strands. Here, we test the ability of G4R1/RHAU to bind and unwind unimolecular G4-DNA. Gel mobility shift assays were used to measure the binding affinity of G4R1/RHAU for unimolecular G4-DNA-formed sequences from the Zic1 gene and the c-Myc promoter. Extremely tight binding produced apparent K(d)'s of 6, 3 and 4 pM for two Zic1 G4-DNAs and a c-Myc G4-DNA, respectively. The low enzyme concentrations required for measuring these K(d)'s limit the precision of their determination to upper boundary estimates. Similar tight binding was not observed in control non-G4 forming DNA sequences or in single-stranded DNA having guanine-rich runs capable of forming tetramolecular G4-DNA. Using a peptide nucleic acid (PNA) trap assay, we show that G4R1/RHAU catalyzes unwinding of unimolecular Zic1 G4-DNA into an unstructured state capable of hybridizing to a complementary PNA. Binding was independent of adenosine triphosphate (ATP), but the PNA trap assay showed that unwinding of G4-DNA was ATP dependent. Competition studies indicated that unimolecular Zic1 and c-Myc G4-DNA structures inhibit G4R1/RHAU-catalyzed resolution of tetramolecular G4-DNA. This report provides evidence that G4R1/RHAU tightly binds and unwinds unimolecular G4-DNA structures.
authors.affiliation: Department of Cancer Biology and the Comprehensive Cancer Center of Wake Forest University School of Medicine, Winston-Salem, USA.
authors.forename: Banabihari
authors.initials: B
authors.lastname: Giri
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Smaldino
authors.forename: Ryan G
authors.initials: RG
authors.lastname: Thys
authors.forename: Steven D
authors.initials: SD
authors.lastname: Creacy
authors.forename: Eric D
authors.initials: ED
authors.lastname: Routh
authors.forename: Roy R
authors.initials: RR
authors.lastname: Hantgan
authors.forename: Simon
authors.initials: S
authors.lastname: Lattmann
authors.forename: Yoshikuni
authors.initials: Y
authors.lastname: Nagamine
authors.forename: Steven A
authors.initials: SA
authors.lastname: Akman
authors.forename: James P
authors.initials: JP
authors.lastname: Vaughn
mesh_terms: D002942:Circular Dichroism; D053487:DEAD-box RNA Helicases / Q000378:metabolism*; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D054856:G-Quadruplexes; D016259:Genes, myc; D006801:Humans; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D020135:Peptide Nucleic Acids / Q000737:chemistry; D045522:Recombinases / Q000378:metabolism
chemical_list: D009838:Oligodeoxyribonucleotides; D020135:Peptide Nucleic Acids; D045522:Recombinases; D004247:DNA; C466988:DHX36 protein, human; D053487:DEAD-box RNA Helicases