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Nucleic Acids Research, 2002, Vol. 30, No. 13 2742-2750
© 2002 Oxford University Press

Rad51p and Rad54p, but not Rad52p, elevate gene repair in Saccharomyces cerevisiae directed by modified single-stranded oligonucleotide vectors

Li Liu, Shuqiu Cheng1, Anja J. van Brabant1 and Eric B. Kmiec*

Department of Biology, Delaware Biotechnology Institute, University of Delaware, 15 Innovation Way, Room 270, Newark, DE 19711, USA and 1 NaPro Genomics, 15 Innovation Way, Newark, DE 19711, USA

Synthetic single-stranded DNA vectors have been used to correct point and frameshift mutations in episomal or chromosomal targets in the yeast Saccharomyces cerevisiae. Certain parameters, such as the length of the vector and the genetic background of the organism, have a significant impact on the process of targeted gene repair, and point mutations are corrected at a higher frequency than frameshift mutations. Genetic analyses reveal that expression levels of the recombination/repair genes RAD51, RAD52 and RAD54 can affect the frequency of gene repair. Overexpression of RAD51 enhances the frequency 4-fold for correction of an episomal target and 5-fold for correction of a chromosomal target; overexpression of RAD54 is also effective in stimulating gene repair, to the same extent as RAD51 in the chromosomal target. In sharp contrast, RAD52 gene expression serves to reduce gene repair activity in rescue experiments and in experiments where RAD52 is overexpressed in a wild-type strain. This may suggest an antagonist role for Rad52p. Consistent with this notion, the highest level of targeted repair occurs when the RAD51 gene is overexpressed in a strain of yeast deficient in RAD52 gene function.

* To whom correspondence should be addressed. Tel: +1 302 831 3420; Fax: +1 302 831 3427; Email: ekmiec{at}udel.edu


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