The involvement of replication in single stranded oligonucleotide-mediated gene repair.
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.
Huen
Michael S Y
MS
Department of Biochemistry, The University of Hong Kong 3/F Laboratory Block, Faculty of Medicine, Building, 21 Sassoon Road, Pokfulam, Hong Kong SAR, China.
Li
Xin-tian
XT
Lu
Lin-Yu
LY
Watt
Rory M
RM
Liu
De-Pei
DP
Huang
Jian-Dong
JD
D053843:DNA Mismatch Repair; D004258:DNA Polymerase III / Q000235:genetics; D017931:DNA Primers / Q000737:chemistry; D004260:DNA Repair; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics; D005798:Genes, Bacterial; D008957:Models, Genetic; D009154:Mutation; D009841:Oligonucleotides / Q000737:chemistry*; D011995:Recombination, Genetic; D018741:Replication Origin
D017931:DNA Primers; D004277:DNA, Single-Stranded; D029968:Escherichia coli Proteins; D009841:Oligonucleotides; D004258:DNA Polymerase III; C468316:dnaQ protein, E coli