Stimulation of Oligonucleotide-Directed Gene Correction by Redβ Expression and MSH2 Depletion in Human HT1080 Cells

  • Xu, Ke (Tianjin Key Laboratory of Lung Cancer Metastasis and Tumor Microenviroment, Tianjin Lung Cancer Institute, Tianjin Medical University General Hospital) ;
  • Stewart, A. Francis (Genomics, Bio Innovations Zentrum, Technische Universitaet Dresden) ;
  • Porter, Andrew C.G. (Gene Targeting Group, Department of Hematology, Faculty of Medicine, Imperial College London)
  • Received : 2014.06.10
  • Accepted : 2014.10.15
  • Published : 2015.01.31


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 $Red{\beta}$, a single-strand DNA annealing protein from bacteriophage lambda. Here we show that $Red{\beta}$ 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 $Red{\beta}$ 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.


DNA repair;gene correction;mismatch repair;$Red{\beta}$;single-strand oligonucleotide


Supported by : National Natural Science Foundation of China


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