Genetic re-engineering of Saccharomyces cerevisiae RAD51 leads to a significant increase in the frequency of gene repair in vivo.
Oligonucleotides can be used to direct the alteration of single nucleotides in chromosomal genes in yeast. Rad51 protein appears to play a central role in catalyzing the reaction, most likely through its DNA pairing function. Here, we re-engineer the RAD51 gene in order to produce proteins bearing altered levels of known activities. Overexpression of wild-type ScRAD51 elevates the correction of an integrated, mutant hygromycin resistance gene approximately 3-fold. Overexpression of an altered RAD51 gene, which encodes a protein that has a higher affinity for ScRad54, enhances the targeting frequency nearly 100-fold. Another mutation which increases the affinity of Rad51 for DNA was also found to increase gene repair when overexpressed in the cell. Other mutations in the Rad51 protein, such as one that reduces interaction with Rad52, has little or no effect on the frequency of gene repair. These data provide the first evidence that the Rad51 protein can be modified so as to increase the frequency of gene repair in yeast.
Liu
Li
L
Department of Biological Sciences, University of Delaware, Delaware Biotechnology Institute, 15 Innovation Way, Room 270, Newark, DE 19711, USA.
Maguire
Katie K
KK
Kmiec
Eric B
EB
D001483:Base Sequence; D015153:Blotting, Western; D002934:Cinnamates / Q000494:pharmacology; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D025141:Drug Resistance, Fungal / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D005818:Genetic Engineering / Q000379:methods*; D049452:Green Fluorescent Proteins; D006921:Hygromycin B / Q000031:analogs & derivatives* / Q000494:pharmacology; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D012331:RNA, Fungal / Q000235:genetics / Q000378:metabolism; D051135:Rad51 Recombinase; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D014170:Transformation, Genetic
D002934:Cinnamates; D004268:DNA-Binding Proteins; D008164:Luminescent Proteins; D012331:RNA, Fungal; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; D006921:Hygromycin B; C026273:hygromycin A; C495151:RAD51 protein, S cerevisiae; D051135:Rad51 Recombinase