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 language | English (US) |
|---|---|
| Pages (from-to) | 7161-7178 |
| Number of pages | 18 |
| Journal | Nucleic Acids Research |
| Volume | 39 |
| Issue number | 16 |
| DOIs | |
| State | Published - Sep 1 2011 |
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G4 Resolvase 1 tightly binds and unwinds unimolecular G4-DNA. / Giri, Banabihari; Smaldino, Philip J.; Thys, Ryan G.; Creacy, Steven D.; Routh, Eric D.; Hantgan, Roy R.; Lattmann, Simon; Nagamine, Yoshikuni; Akman, Steven A.; Vaughn, James P.
In: Nucleic Acids Research, Vol. 39, No. 16, 01.09.2011, p. 7161-7178.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - G4 Resolvase 1 tightly binds and unwinds unimolecular G4-DNA
AU - Giri, Banabihari
AU - Smaldino, Philip J.
AU - Thys, Ryan G.
AU - Creacy, Steven D.
AU - Routh, Eric D.
AU - Hantgan, Roy R.
AU - Lattmann, Simon
AU - Nagamine, Yoshikuni
AU - Akman, Steven A.
AU - Vaughn, James P.
PY - 2011/9/1
Y1 - 2011/9/1
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=80052445273&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=80052445273&partnerID=8YFLogxK
U2 - 10.1093/nar/gkr234
DO - 10.1093/nar/gkr234
M3 - Article
C2 - 21586581
AN - SCOPUS:80052445273
VL - 39
SP - 7161
EP - 7178
JO - Nucleic Acids Research
JF - Nucleic Acids Research
SN - 0305-1048
IS - 16
ER -