title: 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.
authors.affiliation: Department of Cancer Biology, Wake Forest School of Medicine, Winston-Salem, North Carolina, 27157, United States of America.
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Smaldino
authors.affiliation: Department of Cancer Biology, Wake Forest School of Medicine, Winston-Salem, North Carolina, 27157, United States of America.
authors.forename: Eric D
authors.initials: ED
authors.lastname: Routh
authors.affiliation: Department of Cancer Biology, Wake Forest School of Medicine, Winston-Salem, North Carolina, 27157, United States of America.
authors.forename: Jung H
authors.initials: JH
authors.lastname: Kim
authors.affiliation: Department of Chemistry, Furman University, Greenville, South Carolina, 29613, United States of America.
authors.forename: Banabihari
authors.initials: B
authors.lastname: Giri
authors.affiliation: Department of Cancer Biology, Wake Forest School of Medicine, Winston-Salem, North Carolina, 27157, United States of America; Harmonyx Diagnostics, Cordova, Tennessee, 38016, United States of America.
authors.forename: Steven D
authors.initials: SD
authors.lastname: Creacy
authors.affiliation: Department of Biochemistry, Wake Forest School of Medicine, Winston-Salem, North Carolina, 27157, United States of America.
authors.forename: Roy R
authors.initials: RR
authors.lastname: Hantgan
authors.affiliation: Department of Hematology and Oncology, Roper St. Francis Hospital, Charleston, South Carolina, 29401, United States of America.
authors.forename: Steven A
authors.initials: SA
authors.lastname: Akman
authors.affiliation: Department of Cancer Biology, Wake Forest School of Medicine, Winston-Salem, North Carolina, 27157, United States of America.
authors.forename: James P
authors.initials: JP
authors.lastname: Vaughn
mesh_terms: D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002942:Circular Dichroism; D053487:DEAD-box RNA Helicases / Q000378:metabolism*; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D054856:G-Quadruplexes; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D011188:Potassium / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D012964:Sodium / Q000378:metabolism; D016615:Telomere / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D004247:DNA; D012964:Sodium; C466988:DHX36 protein, human; D053487:DEAD-box RNA Helicases; D011188:Potassium