title: Repair of single-point mutations by polypurine reverse Hoogsteen hairpins.
abstract: Polypurine reverse Hoogsteen hairpins (PPRHs) are formed by two intramolecularly bound antiparallel homopurine domains linked by a five-thymidine loop. One of the homopurine strands binds with antiparallel orientation by Watson-Crick bonds to the polypyrimidine target sequence, forming a triplex. We had previously reported the ability of PPRHs to effectively bind dsDNA displacing the fourth strand away from the newly formed triplex. The main goal of this work was to explore the possibility of repairing a point mutation in mammalian cells using PPRHs as tools. These repair-PPRHs contain different combinations of extended sequences of DNA with the corrected nucleotide to repair the point mutation. As a model we used the dihydrofolate reductase gene. On the one hand, we demonstrate in vitro that PPRHs bind specifically to their polypyrimidine target sequence, opening the two strands of the dsDNA, and allowing the binding of a given repair oligonucleotide to the displaced strand of the DNA. Subsequently, we show at a cellular level (Chinese ovary hamster cells) that repair-PPRHs are able to correct a single-point mutation in a dihydrofolate reductase minigene bearing a nonsense mutation, both in an extrachromosomal location and when the mutated plasmid was stably transfected into the cells. Finally, this methodology was successfully applied to repair a single-point mutation at the endogenous locus, using the DA5 cell line with a deleted nucleotide in exon six of the dhfr gene.
authors.affiliation: Department of Biochemistry and Molecular Biology, School of Pharmacy, University of Barcelona , E08028 Barcelona, Spain .
authors.forename: Anna
authors.initials: A
authors.lastname: Solé
authors.forename: Xenia
authors.initials: X
authors.lastname: Villalobos
authors.forename: Carlos J
authors.initials: CJ
authors.lastname: Ciudad
authors.forename: Véronique
authors.initials: V
authors.lastname: Noé
mesh_terms: D000818:Animals; D020029:Base Pairing; D001665:Binding Sites; D016466:CHO Cells; D003412:Cricetulus; D004260:DNA Repair; D015316:Genetic Therapy / Q000379:methods*; D055029:Inverted Repeat Sequences; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000235:genetics; D017354:Point Mutation; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000235:genetics*; D014162:Transfection
chemical_list: D009838:Oligodeoxyribonucleotides; D013762:Tetrahydrofolate Dehydrogenase