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

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.