A tolerance of DNA heterology in the mammalian targeted gene repair reaction.
Targeted gene repair consists of at least two major steps, the pairing of an oligonucleotide to a site bearing DNA sequence complementarity followed by a nucleotide exchange reaction directed by the oligonucleotide. In this study, oligonucleotides with different structures were designed to target a stably integrated (mutant) enhanced green fluorescent protein (EGFP) gene and used to direct the repair of a single base mutation. We show that the efficiency of correction is influenced by the degree of DNA sequence homology existing between the oligonucleotide and target gene. Correction is reduced when a heterologous stretch of DNA sequence is placed in the center of the oligonucleotide and the mismatched base pair is then formed near the terminus. The negative impact of heterology is dependent on the type of DNA sequence inserted and on the size of the heterologous region. If the heterologous sequence is palindromic and adopts a secondary structure, the negative impact on the correction frequency is removed, and wild-type levels of repair are restored. Although differences in the efficiency of correction are observed in various cell types, the effect of structural changes on gene repair is consistent. These results reveal the existence of a directional-specific repair pathway that relies on the pairing stability of a bilateral complex and emphasize the importance of sequence homology between pairing partners for efficient catalysis of gene repair.
Drury
Miya D
MD
Department of Chemistry & Biochemistry, University of Delaware, Newark, DE 19716, USA.
Skogen
Michael J
MJ
Kmiec
Eric B
EB
D000818:Animals; D020137:Base Pair Mismatch; D020029:Base Pairing; D001483:Base Sequence; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D004247:DNA / Q000737:chemistry* / Q000235:genetics; D004260:DNA Repair / Q000235:genetics*; D005347:Fibroblasts / Q000378:metabolism; D005434:Flow Cytometry; D018390:Gene Targeting; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism*; D012689:Sequence Homology, Nucleic Acid
D009841:Oligonucleotides; D049452:Green Fluorescent Proteins; D004247:DNA