Gene targeting in yeast is initiated by two independent strand invasions.
To study the mechanism of gene targeting, we examined heteroduplex DNA (hDNA) formation during targeting of two separate chromosomal locations in Saccharomyces cerevisiae. We examined both replacement of the entire gene with a heterologous selectable marker and correction of a single base pair insertion mutation by gene targeting, and in all cases our results were consistent with separate strand invasion/resolution at the two ends of the targeting fragment as the dominant mechanism in wild-type cells. A small subset of transformants was consistent with assimilation of a single strand of targeting DNA encompassing both flanking homology regions and the marker into hDNA. hDNA formation during correction of a point mutation by targeted integration was conspicuously altered in a mismatch repair-deficient background and was consistent with single-strand invasion/assimilation without mismatch correction, confirming that gene targeting by this pathway is actively impeded in wild-type yeast. Finally, inversion of one targeted locus and mutation of an active origin of DNA replication at the other locus affected hDNA formation significantly, suggesting that formation of productive interactions between the targeting DNA and the targeted site in the chromosome is sensitive to local DNA dynamics.
Integrated Program in Cellular, Molecular, and Biophysical Studies and Department of Microbiology, Columbia University Medical Center, 701 West 168th Street, New York, NY 10032, USA.
Lance D
LD
Langston
Lorraine S
LS
Symington
D002352:Carrier Proteins / Q000502:physiology; D002874:Chromosome Mapping; D015825:Chromosomes, Fungal; D018390:Gene Targeting; D000070956:MutL Proteins; D009692:Nucleic Acid Heteroduplexes / Q000235:genetics; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000502:physiology
D002352:Carrier Proteins; D009692:Nucleic Acid Heteroduplexes; C088858:PMS1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D000070956:MutL Proteins