title: Stimulation of oligonucleotide-directed gene correction by Redβ expression and MSH2 depletion in human HT1080 cells.
abstract: The correction of disease-causing mutations by single-strand oligonucleotide-templated DNA repair (ssOR) is an attractive approach to gene therapy, but major improvements in ssOR efficiency and consistency are needed. The mechanism of ssOR is poorly understood but may involve annealing of oligonucleotides to transiently exposed single-stranded regions in the target duplex. In bacteria and yeast it has been shown that ssOR is promoted by expression of Redβ, a single-strand DNA annealing protein from bacteriophage lambda. Here we show that Redβ expression is well tolerated in a human cell line where it consistently promotes ssOR. By use of short interfering RNA, we also show that ssOR is stimulated by the transient depletion of the endogenous DNA mismatch repair protein MSH2. Furthermore, we find that the effects of Redβ expression and MSH2 depletion on ssOR can be combined with a degree of cooperativity. These results suggest that oligonucleotide annealing and mismatch recognition are distinct but interdependent events in ssOR that can be usefully modulated in gene correction strategies.
authors.affiliation: Tianjin Key Laboratory of Lung Cancer Metastasis and Tumor Microenviroment, Tianjin Lung Cancer Institute, Tianjin Medical University General Hospital, Tianjin 300052, China ; Gene Targeting Group, Department of Hematology, Faculty of Medicine, Imperial College London, London W12 0NN, UK.
authors.forename: Ke
authors.initials: K
authors.lastname: Xu
authors.affiliation: Genomics, Bio Innovations Zentrum, Technische Universitaet Dresden, 01307 Dresden, Germany.
authors.forename: A Francis
authors.initials: AF
authors.lastname: Stewart
authors.affiliation: Gene Targeting Group, Department of Hematology, Faculty of Medicine, Imperial College London, London W12 0NN, UK.
authors.forename: Andrew C G
authors.initials: AC
authors.lastname: Porter
mesh_terms: D010582:Bacteriophage lambda / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000378:metabolism*; D006801:Humans; D051718:MutS Homolog 2 Protein / Q000037:antagonists & inhibitors*; D009841:Oligonucleotides / Q000737:chemistry / Q000235:genetics; D052416:Targeted Gene Repair / Q000379:methods*; D014162:Transfection; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D004277:DNA, Single-Stranded; D009841:Oligonucleotides; D014764:Viral Proteins; C497172:MSH2 protein, human; D051718:MutS Homolog 2 Protein
keywords: DNA repair; Redβ; gene correction; mismatch repair; single-strand oligonucleotide