Liu Li, Maguire Katie K, Kmiec Eric B
Department of Biological Sciences, University of Delaware, Delaware Biotechnology Institute, 15 Innovation Way, Room 270, Newark, DE 19711, USA.
Nucleic Acids Res. 2004 Apr 15;32(7):2093-101. doi: 10.1093/nar/gkh506. Print 2004.
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.
寡核苷酸可用于指导酵母染色体基因中单个核苷酸的改变。Rad51蛋白似乎在催化该反应中起核心作用，很可能是通过其DNA配对功能。在此，我们对RAD51基因进行重新设计，以产生具有已知活性水平改变的蛋白质。野生型ScRAD51的过表达使整合的突变潮霉素抗性基因的校正提高了约3倍。一个改变的RAD51基因的过表达，该基因编码一种对ScRad54具有更高亲和力的蛋白质，使靶向频率提高了近100倍。还发现另一个增加Rad51对DNA亲和力的突变在细胞中过表达时会增加基因修复。Rad51蛋白中的其他突变，例如减少与Rad52相互作用的突变，对基因修复频率几乎没有影响。这些数据提供了第一个证据，即Rad51蛋白可以被修饰以增加酵母中的基因修复频率。
