Genetic analysis reveals essential and non-essential amino acids within the telomeric DNA-binding interface of Cdc13p.
Cdc13p is a specific single-stranded telomeric DNA-binding protein of Saccharomyces cerevisiae. It is involved in protecting telomeres and regulating telomere length. The telomere-binding domain of Cdc13p is located between residues 497 and 693, and its structure has been resolved by NMR spectroscopy. A series of aromatic, hydrophobic and basic residues located at the DNA-binding surface of Cdc13p are involved in binding to telomeres. Here we applied a genetic approach to analyse the involvements of these residues in telomere binding. A series of mutants within the telomere-binding domain of Cdc13p were identified that failed to complement cdc13 mutants in vivo. Among the amino acids that were isolated, the Tyr522, Arg635, and Ile633 residues were shown to locate at the DNA-binding surface. We further demonstrated that Y522C and R635A mutants failed to bind telomeric DNA in vitro, indicating that these residues are indeed required for telomere binding. We did not, however, isolate other mutant residues located at the DNA-binding surface of Cdc13p beyond these three residues. Instead, a mutant on Lys568 was isolated that did not affect the essential function of Cdc13p. The Lys568 is also located on the DNA-binding surface of Cdc13p. Thus these results suggested that other DNA-binding residues are not essential for telomere binding. In the present study, we have established a genetic test that enabled the identification of telomere-binding residues of Cdc13p in vivo. This type of analysis provides information on those residues that indeed contribute to telomere binding in vivo.
Lin
Yi-Chien
YC
Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Shih-Pai 112, Taipei, Taiwan, People's Republic of China.
Wu Lee
Yan-Hwa
YH
Lin
Jing-Jer
JJ
D000596:Amino Acids / Q000235:genetics* / Q000378:metabolism*; D001665:Binding Sites; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D010641:Phenotype; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D016615:Telomere / Q000235:genetics / Q000378:metabolism*; D034501:Telomere-Binding Proteins / Q000235:genetics / Q000378:metabolism*
D000596:Amino Acids; C452219:Cdc13 protein, S cerevisiae; D004271:DNA, Fungal; D029701:Saccharomyces cerevisiae Proteins; D034501:Telomere-Binding Proteins