title: Correction of point mutations at the endogenous locus of the dihydrofolate reductase gene using repair-PolyPurine Reverse Hoogsteen hairpins in mammalian cells.
abstract: Correction of point mutations that lead to aberrant transcripts, often with pathological consequences, has been the focus of considerable research. In this work, repair-PPRHs are shown to be a new powerful tool for gene correction. A repair-PPRH consists of a PolyPurine Reverse Hoogsteen hairpin core bearing an extension sequence at one end, homologous to the DNA strand to be repaired but containing the wild type nucleotide instead of the mutation. Previously, we had corrected a single-point mutation with repair-PPRHs using a mutated version of a dihydrofolate reductase (dhfr) minigene. To further evaluate the utility of these molecules, different repair-PPRHs were designed to correct insertions, deletions, substitutions and a double substitution present in a collection of mutants at the endogenous locus of the dhfr gene, the product of which is the target of the chemotherapeutic agent methotrexate. We also describe an approach to use when the point mutation is far away from the homopyrimidine target domain. This strategy consists in designing Long-Distance- and Short-Distance-Repair-PPRHs where the PPRH core is bound to the repair tail by a five-thymidine linker. Surviving colonies in a DHFR selective medium, lacking glycine and sources of purines and thymidine, were analyzed by DNA sequencing, and by mRNA, protein and enzymatic measurements, confirming that all the dhfr mutants had been corrected. These results show that repair-PPRHs can be effective tools to accomplish a permanent correction of point mutations in the DNA sequence of mutant mammalian cells.
authors.affiliation: Department of Biochemistry and Molecular Biology, School of Pharmacy, IN2UB, University of Barcelona, 08028 Barcelona, Spain.
authors.forename: Anna
authors.initials: A
authors.lastname: Solé
authors.affiliation: Department of Biochemistry and Molecular Biology, School of Pharmacy, IN2UB, University of Barcelona, 08028 Barcelona, Spain. Electronic address: cciudad@ub.edu.
authors.forename: Carlos J
authors.initials: CJ
authors.lastname: Ciudad
authors.affiliation: Department of Biological Sciences, Columbia University, New York, NY 10027, United States.
authors.forename: Lawrence A
authors.initials: LA
authors.lastname: Chasin
authors.affiliation: Department of Biochemistry and Molecular Biology, School of Pharmacy, IN2UB, University of Barcelona, 08028 Barcelona, Spain.
authors.forename: Véronique
authors.initials: V
authors.lastname: Noé
mesh_terms: D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D003412:Cricetulus; D004260:DNA Repair; D005091:Exons; D015870:Gene Expression; D056426:Genetic Loci; D015316:Genetic Therapy / Q000379:methods; D007438:Introns; D055029:Inverted Repeat Sequences; D016254:Mutagenesis, Insertional; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000235:genetics; D017354:Point Mutation; D017384:Sequence Deletion; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
chemical_list: D009838:Oligodeoxyribonucleotides; D013762:Tetrahydrofolate Dehydrogenase
keywords: Gene correction; Mammalian cells; PPRH; Point mutation; dhfr