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TRANSCRIPTIONAL REGULATION

Strand Bias in Targeted Gene Repair Is Influenced by Transcriptional Activity

Li Liu, Michael C. Rice, Miya Drury, Shuqiu Cheng, Howard Gamper, Eric B. Kmiec
Li Liu
1Department of Biology and Delaware Biotechnology Institute, University of Delaware, Newark, Delaware 19716
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Michael C. Rice
1Department of Biology and Delaware Biotechnology Institute, University of Delaware, Newark, Delaware 19716
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Miya Drury
1Department of Biology and Delaware Biotechnology Institute, University of Delaware, Newark, Delaware 19716
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Shuqiu Cheng
2Genomics Division, NaPro BioTherapeutics, Inc., Delaware Biotechnology Institute, Newark, Delaware 19711
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Howard Gamper
1Department of Biology and Delaware Biotechnology Institute, University of Delaware, Newark, Delaware 19716
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Eric B. Kmiec
1Department of Biology and Delaware Biotechnology Institute, University of Delaware, Newark, Delaware 19716
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  • For correspondence: ekmiec@udel.edu
DOI: 10.1128/MCB.22.11.3852-3863.2002
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  • Strand Bias in Targeted Gene Repair Is Influenced by Transcriptional Activity - August 01, 2003

ABSTRACT

Modified single-stranded DNA oligonucleotides can direct nucleotide exchange in Saccharomyces cerevisiae. Point and frameshift mutations are corrected in a reaction catalyzed by cellular enzymes involved in various DNA repair processes. The present model centers on the annealing of the vector to one strand of the helix, followed by the correction of the designated base. The choice of which strand to target is a reaction parameter that can be controlled, so here we investigate the properties of strand bias in targeted gene repair. An in vivo system has been established in which a plasmid containing an actively transcribed, but mutated, hygromycin-enhanced green fluorescent protein fusion gene is targeted for repair and upon conversion will confer hygromycin resistance on the cell. Overall transcriptional activity has a positive influence on the reaction, elevating the frequency. If the targeting vector is synthesized so that it directs nucleotide repair on the nontranscribed strand, the level of gene repair is higher than if the template strand is targeted. We provide data showing that the targeting vector can be displaced from the template strand by an active T7 phage RNA polymerase. The strand bias is not influenced by which strand serves as the leading or lagging strand during DNA synthesis. These results may provide an explanation for the enhancement of gene repair observed when the nontemplate strand is targeted.

  • Copyright © 2002 American Society for Microbiology
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Strand Bias in Targeted Gene Repair Is Influenced by Transcriptional Activity
Li Liu, Michael C. Rice, Miya Drury, Shuqiu Cheng, Howard Gamper, Eric B. Kmiec
Molecular and Cellular Biology Jun 2002, 22 (11) 3852-3863; DOI: 10.1128/MCB.22.11.3852-3863.2002

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Strand Bias in Targeted Gene Repair Is Influenced by Transcriptional Activity
Li Liu, Michael C. Rice, Miya Drury, Shuqiu Cheng, Howard Gamper, Eric B. Kmiec
Molecular and Cellular Biology Jun 2002, 22 (11) 3852-3863; DOI: 10.1128/MCB.22.11.3852-3863.2002
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KEYWORDS

DNA repair
Saccharomyces cerevisiae

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