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

Philip J. Smaldino, Eric D. Routh, Jung H. Kim, Banabihari Giri, Steven D. Creacy, Roy R. Hantgan, Steven A. Akman, James P. Vaughn

Research output: Contribution to journalArticle

6 Citations (Scopus)

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.

Original languageEnglish (US)
Article numbere0132668
JournalPloS one
Volume10
Issue number7
DOIs
StatePublished - Jul 14 2015

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Dissection
Recombinases
tail
DNA
chromosomes
mutation
telomerase
Chromosomes
guanine
adenine
topology
G-Quadruplexes
nucleotides
Holoenzymes
Mutation
Telomerase
DNA sequences
Guanine
nucleotide sequences
Human Chromosomes

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)

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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. / Smaldino, Philip J.; Routh, Eric D.; Kim, Jung H.; Giri, Banabihari; Creacy, Steven D.; Hantgan, Roy R.; Akman, Steven A.; Vaughn, James P.

In: PloS one, Vol. 10, No. 7, e0132668, 14.07.2015.

Research output: Contribution to journalArticle

Smaldino, Philip J. ; Routh, Eric D. ; Kim, Jung H. ; Giri, Banabihari ; Creacy, Steven D. ; Hantgan, Roy R. ; Akman, Steven A. ; Vaughn, James P. / 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. In: PloS one. 2015 ; Vol. 10, No. 7.
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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.",
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