Chimera‐directed Gene Repair

Abstract

The information gained from the Human Genome Project must be formulated such that the function of newly discovered genes can be elucidated. A tool to aid in defining gene function is targeted gene repair, a technique that may also be valuable in human gene therapy.

Keywords: chimera; gene repair; chimeric RNA–DNA oligonucleotide; transfection; RAD51; yeast

Figure 1.

Generalized design features of the chimeric ribonucleic acid–deoxyribonucleic acid (RNA–DNA) oligonucleotide. The upper strand consists of RNA residues (1). Both ends of the molecule are configured into a hairpin structure (2). Each RNA residue has a 2′‐O‐methyl modification (3). The lower strand, which is all DNA residues, directs the nucleotide exchange during gene repair (4). A single, unligated phosphodiester bond exists to allow intertwining of the chimera with the target site (5).

Figure 2.

Current working hypothesis of the mechanism of chimera‐directed repair. The chimera (2) reaches the target site either through enzymatic or nonenzymatic activities. The double D‐loop structure, reaction intermediate, may be matured and/or stabilized by Rad59p (3). Protein Rad52p acts at an early stage to retard the reaction intermediate. The double D‐loop (4) containing a mismatched base pair G/T is recognized by a group of proteins, including those from the mismatch repair and nucleotide excision repair pathways (5). The excised base is replaced with a C residue, presumably by the action of a DNA polymerase (6). The chimera dissociates (between 7 and 8) and Msh2p and other mismatch repair activities correct the mismatch A/C left in the target sequence (8).

Figure 3.

Progress in targeted gene repair. References are shown for each seminal event in the development of chimera‐directed gene repair.

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Further Reading

Andersen MS, Sorensen CB, Bolund L and Jensen TG (2002) Mechanism underlying targeted gene correction using chimeric RNA/DNA and single‐stranded DNA oligonucleotides. Journal of Molecular Medicine 80: 770–781.

Liu C‐M, Liu D‐P and Liang C‐C (2002) Oligonucleotide‐mediated gene repair at DNA level: The potential applications for gene therapy. Journal of Molecular Medicine 80: 620–628.

Rando TA (2002) Oligonucleotide‐mediated gene therapy for muscular dystrophies. Neuromuscular Disorders 12: S55–S60.

Thorpe PH, Stevenson BJ and Porteous DJ (2002) Functional correction of episomal mutations with short DNA fragments and RNA–DNA oligonucleotides. Journal of Gene Medicine 4: 195–204.

Yanez RJ and Porter ACG (1999) Gene targeting is enhanced in human cells overexpressing hRAD51. Gene Therapy 6: 1282–1290.

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How to Cite close
Kmiec, Eric B(Jan 2006) Chimera‐directed Gene Repair. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1038/npg.els.0005763]