Telomere-binding and Stn1p-interacting activities are required for the essential function of Saccharomyces cerevisiae Cdc13p.
Yeast Saccharomyces cerevisiae Cdc13p is the telomere-binding protein that protects telomeres and regulates telomere length. It is documented that Cdc13p binds specifically to single-stranded TG(1-3) telomeric DNA sequences and interacts with Stn1p. To localize the region for single-stranded TG(1-3) DNA binding, Cdc13p mutants were constructed by deletion mutagenesis and assayed for their binding activity. Based on in vitro electrophoretic mobility shift assay, a 243-amino-acid fragment of Cdc13p (amino acids 451-693) was sufficient to bind single-stranded TG(1-3) with specificity similar to that of the native protein. Consistent with the in vitro observation, in vivo one-hybrid analysis also indicated that this region of Cdc13p was sufficient to localize itself to telomeres. However, the telomere-binding region of Cdc13p (amino acids 451-693) was not capable of complementing the growth defects of cdc13 mutants. Instead, a region comprising the Stn1p-interacting and telomere-binding region of Cdc13p (amino acids 252-924) complemented the growth defects of cdc13 mutants. These results suggest that binding to telomeres by Cdc13p is not sufficient to account for the cell viability, interaction with Stn1p is also required. Taken together, we have defined the telomere-binding domain of Cdc13p and showed that both binding to telomeres and Stn1p by Cdc13p are required to maintain cell growth.
Wang
M J
MJ
Institute of Biopharmaceutical Science, National Yang-Ming University, Shih-Pai, 112, Taipei, Taiwan, Republic of China.
Lin
Y C
YC
Pang
T L
TL
Lee
J M
JM
Chou
C C
CC
Lin
J J
JJ
D019926:Cyclin B / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D005816:Genetic Complementation Test; D009154:Mutation; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D016615:Telomere / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D019926:Cyclin B; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D010446:Peptide Fragments