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

Abstract

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 Kd. 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 Kd = 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 Kd = 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.

The G-quadruplex (G4) resolvase DHX36 efficiently and specifically disrupts DNA G4s via a translocation-based helicase mechanism

“…DHX36 can efficiently disrupt both intramolecular and tetramolecular G4s (17,25,26). Disruption of a tetramolecular G4 DNA was shown to require ATP and a 3′ extension (25,27). The disruption rate was shown to be greater for a G4 with fewer G-quartets, suggesting that less stable G4s are disrupted more efficiently by DHX36 (26).…”

“…A distinguishing feature of DEAH family helicases is that they typically require such an extension because the protein loads onto the single-stranded segment and then translocates 3′ to 5′ for helix unwinding. It was shown recently that DHX36 indeed requires a 3′ extension for efficient G4 disruption (27,42). However, little is known about the dependence of G4 disruption efficiency on the length of this 3′ extension, and the mechanistic origin of this requirement has not been systematically explored.…”

“…Extension sequences have been shown to influence equilibrium binding to G4 DNA structures (25,27), but potential effects of these sequences on G4 disruption kinetics have not been explored.…”

Distinct RNA-unwinding mechanisms of DEAD-box and DEAH-box RNA helicase proteins in remodeling structured RNAs and RNPs

“…Instead of binding directly to structured RNA elements, DEAH-box helicases require 3′ single-stranded regions for unwinding activity (50, 52, 53). Unlike DEAD-box proteins, which are highly specific for dsRNA, some members of the DEAH-box family can act on both DNA and RNA, leading to unwinding of helices and, for some DEAH-box proteins, four-stranded G-quadruplex structures (54, 55).…”

G-quadruplexes unfolding by RHAU helicase

“…In addition, to the best of our knowledge and with the exception of a study carried out using single molecule FRET, only gel experiments have been used to characterize the G4/full-length RHAU interactions. [17,25,28,29,24,30] In the present work, we use a high-throughput real-time fluorescent G4-based helicase assay, initially developed for the yeast Saccharomyces cerevisiae Pif1 helicase, to characterize G4/RHAU interaction. [31] We demonstrate that RHAU is active in comparable conditions as Pif1 but unlike it, RHAU activity towards a G4 substrate such as the c-myc oncogene promoter sequence is not sensitive to changes in potassium concentration.…”