PMID: 9192655Jun 24, 1997Paper

Gene targeting by linear duplex DNA frequently occurs by assimilation of a single strand that is subject to preferential mismatch correction

Proceedings of the National Academy of Sciences of the United States of America
W LeungJ E Haber

Abstract

To study targeted recombination, a single linear 2-kb fragment of LEU2 DNA was liberated from a chromosomal site within the nucleus of Saccharomyces cerevisiae, by expression of the site-specific HO endonuclease. Gene targeting was scored by gene conversion of a chromosomal leu2 mutant allele by the liberated LEU2 fragment. This occurred at a frequency of only 2 x 10(-4), despite the fact that nearly all cells successfully repaired, by single-strand annealing, the chromosome break created by liberating the fragment. The frequency of Leu+ recombinants was 6- to 25-fold higher in pms1 strains lacking mismatch repair. In 70% of these cases, the colony was sectored for Leu+/Leu-. Similar results were obtained when a 4. 1-kb fragment containing adjacent LEU2 and ADE1 genes was liberated, to convert adjacent leu2 and ade1 mutations on the chromosome. These results suggest that a linear fragment is not assimilated into the recipient chromosome by two crossovers each close to the end of the fragment; rather, heteroduplex DNA between the fragment and the chromosome is apparently formed over the entire region, by the assimilation of one of the two strands of the linear duplex DNA. Moreover, the recovery of Leu+ transformants is frequentl...Continue Reading

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Citations

Aug 1, 2000·Mutation Research·J E Haber
Oct 5, 2001·Trends in Genetics : TIG·G B Gloor
Feb 18, 2003·Proceedings of the National Academy of Sciences of the United States of America·Wei J Gong, Kent G Golic
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Oct 19, 2004·Proceedings of the National Academy of Sciences of the United States of America·Lance D Langston, Lorraine S Symington
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Jun 25, 2015·Microbiology Spectrum·Cheng-Sheng Lee, James E Haber

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