The involvement of replication in single stranded oligonucleotide-mediated gene repair

• Published in: Nucleic acids research Vol. 34; no. 21; pp. 6183 - 6194
• Main Authors: Huen, Michael S Y, Li, Xin-tian, Lu, Lin-Yu, Watt, Rory M, Liu, De-Pei, Huang, Jian-Dong
• Format: Journal Article
• Language: English
• Published: England Oxford Publishing Limited (England) 01-12-2006; Oxford University Press
• Subjects: DNA Mismatch Repair; DNA Polymerase III - genetics; DNA Primers - chemistry; DNA Repair; DNA Replication; DNA, Single-Stranded - chemistry; Escherichia coli; Escherichia coli - genetics; Escherichia coli Proteins - genetics; Genes, Bacterial; Models, Genetic; Molecular Biology; Mutation; Oligonucleotides - chemistry; Recombination, Genetic; Replication Origin
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkl852

Targeted gene repair mediated by single-stranded oligonucleotides (SSOs) has great potential for use in functional genomic studies and gene therapy. Genetic changes have been created using this approach in a number of prokaryotic and eukaryotic systems, including mouse embryonic stem cells. However, the underlying mechanisms remain to be fully established. In one of the current models, the 'annealing-integration' model, the SSO anneals to its target locus at the replication fork, serving as a primer for subsequent DNA synthesis mediated by the host replication machinery. Using a lambda-Red recombination-based system in the bacterium Escherichia coli, we systematically examined several fundamental premises that form the mechanistic basis of this model. Our results provide direct evidence strongly suggesting that SSO-mediated gene repair is mechanistically linked to the process of DNA replication, and most likely involves a replication intermediate. These findings will help guide future experiments involving SSO-mediated gene repair in mammalian and prokaryotic cells, and suggest several mechanisms by which the efficiencies may be reliably and substantially increased.