Genetics, Vol. 163, 527-538, February 2003, Copyright © 2003

Targeted Nucleotide Repair of cyc1 Mutations in Saccharomyces cerevisiae Directed by Modified Single-Stranded DNA Oligonucleotides

Erin E. Brachmana and Eric B. Kmieca
a Department of Biology, Delaware Biotechnology Institute, University of Delaware, Newark, Delaware 19716

Corresponding author: Eric B. Kmiec, University of Delaware, Delaware Biotechnology Institute, 15 Innovation Way, Newark, DE 19711., ekmiec{at}udel.edu (E-mail)

Communicating editor: M. HAMPSEY

Modified single-stranded DNA oligonucleotides have been used to direct base changes in the CYC1 gene of Saccharomyces cerevisiae. In this process, the oligonucleotide is believed to hybridize to the target site through the action of a DNA recombinase and, once bound, DNA repair enzymes act to excise the nucleotide, replace it, and revert the gene to wild-type status. Nucleotide exchange exhibits a strand bias as, in most cases, a higher level of base reversal appears in cells in which the oligonucleotide is designed to hybridize to the nontemplate strand. But, in one case, a higher level was observed when an oligonucleotide complementary to the transcribed strand was used. Mutant haploid and diploid strains are reverted to wild type at this locus with approximately the same frequency and all strains take up the oligonucleotide with approximately equal efficiency. Some repair preference for certain base mismatches was observed; for example, T/T and C/C mispairs exhibited the highest degree of reactivity. Finally, we demonstrate that proteins involved in DNA pairing can enhance the repair activity up to 22-fold, while others affect the reaction minimally. Taken together, these results confirm the importance and versatility of yeast as a model system to elucidate the factors regulating the frequency of nucleotide exchange directed by oligonucleotides.




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