G4 Resolvase 1 tightly binds and unwinds unimolecular G4-DNA

Banabihari Giri, Philip J. Smaldino, Ryan G. Thys, Steven D. Creacy, Eric D. Routh, Roy R. Hantgan, Simon Lattmann, Yoshikuni Nagamine, Steven A. Akman, James P. Vaughn

Research output: Contribution to journalArticle

44 Scopus citations

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 Kd's of 6, 3 and 4pM for two Zic1 G4-DNAs and a c-Myc G4-DNA, respectively. The low enzyme concentrations required for measuring these Kd'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.

Original languageEnglish (US)
Pages (from-to)7161-7178
Number of pages18
JournalNucleic Acids Research
Volume39
Issue number16
DOIs
StatePublished - Sep 1 2011

ASJC Scopus subject areas

  • Genetics

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    Giri, B., Smaldino, P. J., Thys, R. G., Creacy, S. D., Routh, E. D., Hantgan, R. R., Lattmann, S., Nagamine, Y., Akman, S. A., & Vaughn, J. P. (2011). G4 Resolvase 1 tightly binds and unwinds unimolecular G4-DNA. Nucleic Acids Research, 39(16), 7161-7178. https://doi.org/10.1093/nar/gkr234