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Nucleic Acids Research Advance Access published online on November 6, 2006
Nucleic Acids Research, doi:10.1093/nar/gkl852
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© 2006 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Molecular Biology

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

Michael S. Y. Huen1, Xin-tian Li1,2, Lin-Yu Lu1, Rory M. Watt3, De-Pei Liu2 and Jian-Dong Huang1,*

1 Department of Biochemistry, The University of Hong Kong 3/F Laboratory Block, Faculty of Medicine Building, 21 Sassoon Road, Pokfulam, Hong Kong SAR, China 2 National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (CAMS) and Peking Union Medical College (PUMC) Beijing 100005, P.R. China 3 Open Laboratory of Chemical Biology, The Institute of Molecular Technology for Drug Discovery and Synthesis, Department of Chemistry, The University of Hong Kong Pokfulam Road Hong Kong SAR, China

*To whom correspondence should be addressed. Tel: +852 2819 2810; Fax: +852 2855 1254; Email: jdhuang{at}hkucc.hku.hk

Received May 25, 2006. Revised October 4, 2006. Accepted October 10, 2006.

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.


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