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 Tyr 522 , Arg 635 , and Ile 633 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 Lys 568 was isolated that did not affect the essential function of Cdc13p. The Lys 568 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 .