期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 101, 期 43, 页码 15392-15397出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0403748101
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资金
- NCI NIH HHS [T32 CA09503, T32 CA009503] Funding Source: Medline
- NIGMS NIH HHS [R01 GM054099-08S1, GM54099, T32 GM08224, R01 GM054099-09A1, T32 GM008224, R01 GM054099] Funding Source: Medline
To study the mechanism of gene targeting, we examined hetero-duplex 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.
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