G4 Resolvase 1 tightly binds and unwinds unimolecular G4-DNA

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 4 pM 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 structure

The DEAH-box RNA helicase RHAU binds an intramolecular RNA G-quadruplex in TERC and associates with telomerase holoenzyme

Guanine-quadruplexes (G4) consist of non-canonical four-stranded helical arrangements of guanine-rich nucleic acid sequences. The bulky and thermodynamically stable features of G4 structures have been shown in many respects to affect normal nucleic acid metabolism. In vivo conversion of G4 structures to single-stranded nucleic acid requires specialized proteins with G4 destabilizing/unwinding activity. RHAU is a human DEAH-box RNA helicase that exhibits G4-RNA binding and resolving activity. In this study, we employed RIP-chip analysis to identify en masse RNAs associated with RHAU in vivo. Approximately 100 RNAs were found to be associated with RHAU and bioinformatics analysis revealed that the majority contained potential G4-forming sequences. Among the most abundant RNAs selectively enriched with RHAU, we identified the human telomerase RNA template TERC as a true target of RHAU. Remarkably, binding of RHAU to TERC depended on the presence of a stable G4 structure in the 5′-region of TERC, both in vivo and in vitro. RHAU was further found to associate with the telomerase holoenzyme via the 5′-region of TERC. Collectively, these results provide the first evidence that intramolecular G4-RNAs serve as physiologically relevant targets for RHAU. Furthermore, our results suggest the existence of alternatively folded forms of TERC in the fully assembled telomerase holoenyzme

Role of the amino terminal RHAU-specific motif in the recognition and resolution of guanine quadruplex-RNA by the DEAH-box RNA helicase RHAU

Under physiological conditions, guanine-rich sequences of DNA and RNA can adopt stable and atypical four-stranded helical structures called G-quadruplexes (G4). Such G4 structures have been shown to occur in vivo and to play a role in various processes such as transcription, translation and telomere maintenance. Owing to their high-thermodynamic stability, resolution of G4 structures in vivo requires specialized enzymes. RHAU is a human RNA helicase of the DEAH-box family that exhibits a unique ATP-dependent G4-resolvase activity with a high affinity and specificity for its substrate in vitro. How RHAU recognizes G4-RNAs has not yet been established. Here, we show that the amino-terminal region of RHAU is essential for RHAU to bind G4 structures and further identify within this region the evolutionary conserved RSM (RHAU-specific motif) domain as a major affinity and specificity determinant. G4-resolvase activity and strict RSM dependency are also observed with CG9323, the Drosophila orthologue of RHAU, in the amino terminal region of which the RSM is the only conserved motif. Thus, these results reveal a novel motif in RHAU protein that plays an important role in recognizing and resolving G4-RNA structures, properties unique to RHAU among many known RNA helicases

Ribonuclease 7 polymorphism rs1263872 reduces antimicrobial activity and associates with pediatric urinary tract infections

Ribonuclease 7 (RNase 7) is an antimicrobial peptide that prevents urinary tract infections (UTI); however, it is yet unknown how RNASE7 genetic variations affect its antimicrobial activity and its mitigation of UTI risk. This study determined whether the RNASE7 SNP rs1263872 is more prevalent in children with UTI and defined how rs1263872 affects RNase 7’s antimicrobial activity against uropathogenic E. coli (UPEC). We performed genotyping for rs1263872 in 2 national UTI cohorts, including children enrolled in the Randomized Intervention for Children with Vesicoureteral Reflux trial or the Careful Urinary Tract Infection Evaluation study. Genotypes from these cohorts were compared with those of female controls with no UTI. To assess whether rs1263872 affects RNase 7’s antimicrobial activity, we generated RNase 7 peptides and genetically modified urothelial cultures encoding wild-type RNase 7 and its variant. Compared with controls, girls in both UTI cohorts had an increased prevalence of the RNASE7 variant. Compared with the missense variant, wild-type RNase 7 peptide showed greater bactericidal activity against UPEC. Wild-type RNase 7 overexpression in human urothelial cultures reduced UPEC invasive infection compared with mutant overexpression. These results show that children with UTI have an increased prevalence of RNASE7 rs1263872, which may increase UTI susceptibility by suppressing RNase 7’s antibacterial activity

Mutational Dissection of Telomeric DNA Binding Requirements of G4 Resolvase 1 Shows that G4-Structure and Certain 3’-Tail Sequences Are Sufficient for Tight and Complete Binding

<div><p>Ends of human chromosomes consist of the six nucleotide repeat d[pTTAGGG]_n known as telomeric DNA, which protects chromosomes. We have previously shown that the DHX36 gene product, G4 Resolvase 1 (G4R1), binds parallel G-quadruplex (G4) DNA with an unusually tight apparent K_d. Recent work associates G4R1 with the telomerase holoenzyme, which may allow it to access telomeric G4-DNA. Here we show that G4R1 can tightly bind telomeric G4-DNA, and in the context of the telomeric sequence, we determine length, sequence, and structural requirements sufficient for tight G4R1 telomeric binding. Specifically, G4R1 binds telomeric DNA in the K⁺-induced “3+1” G4-topology with an apparent K_d = 10 ±1.9 pM, a value similar as previously found for binding to unimolecular parallel G4-DNA. G4R1 binds to the Na⁺-induced “2+2” basket G4-structure formed by the same DNA sequence with an apparent K_d = 71 ± 2.2 pM. While the minimal G4-structure is not sufficient for G4R1 binding, a 5’ G4-structure with a 3’ unstructured tail containing a guanine flanked by adenine(s) is sufficient for maximal binding. Mutations directed to disrupt G4-structure similarly disrupt G4R1 binding; secondary mutations that restore G4-structure also restore G4R1 binding. We present a model showing that a replication fork disrupting a T-loop could create a 5’ quadruplex with an opened 3’tail structure that is recognized by G4R1.</p></div

Schematic depiction of: (A) telomere in a T-loop; (B) T-loop with an incoming replication fork allowing G4-DNA to form at the 5’ side of the 3’ overhang; (C) displaced unstructured 3’ end plus quadruplex region that would allow tight binding by G4R1.

<p>G4R1 is depicted associated with the telomerase holoenzyme which also includes: hTR (TERC), hTERT, and Dyskerin.</p

Tight G4R1 binding to the telomeric repeat sequence requires more than the minimal G4-structure found in Tel22.

<p>(<b>A</b>) Gel mobility shift assay (GMSA) with purified recombinant G4R1 and ³²P-end-labeled telomeric G4-DNA sequences (left to right) Tel22, Tel26, Tel30, and Tel33 with indicated G4R1 concentrations. (<b>B</b>) CD wavelength spectra in 50 mM KCl of telomeric G4-DNA sequences (left to right) Tel22, Tel26, Tel30, and Tel33. (<b>C</b>) DNA oligonucleotide sequences used in Fig 2A and 2B.</p

A single guanine mutation disrupts the 5’G4-DNA structure of Tel33mut5 and concomitantly disrupts G4R1 binding, but two secondary mutations that partially restore G4- DNA structure also restore binding.

<p>(<b>A</b>) Schematic depiction of a stable unimolecular G4-DNA structure with three guanine tetrads and two coordinate-bonded K⁺ ions (in purple) (i); G4-DNA structure is completely destabilized by the effects of a single G to A mutation at the central guanine of the 5’ most guanine run. The central guanine tetrad breaks apart and two coordinate bonded K⁺ ions (in purple) are lost (ii); Situations that restore two 5’ guanines in a row or mutations that keep two 5’guanines in a row intact can produce G4-DNA stabilized by two G- tetrads and a single-coordinate K+ ion (in purple). (<b>B</b>) GMSA with indicated concentrations of purified recombinant G4R1 and ³²P-end-labeled mutant telomeric G4-DNA sequences (left to right) Tel33mut5-mutA, Tel33mut5-mutA-pos3G, and Tel33mut5-mutA-pos7G. (<b>C</b>) CD wavelength spectra in 50 mM KCl of mutant telomeric G4-DNA sequences (left to right) Tel33mut5-mutA, Tel33mut5-mutA-pos3G, and Tel33mut5-mutA-pos7G. (<b>D</b>) DNA oligonucleotide sequences used in Fig 7B and 7C.</p