Ten1p promotes the telomeric DNA-binding activity of Cdc13p: implication for its function in telomere length regulation

Abstract

In Saccharomyces cerevisiae, the essential gene CDC13 encodes a telomeric single-stranded DNA-binding protein that interacts with Stn1p and Ten1p genetically and physically, and is required for telomere end protection and telomere length control. The molecular mechanism by which Ten1 participates in telomere length regulation and chromosome end protection remains elusive. In this work, we observed a weak interaction of Cdc13p and Ten1p in a gelfiltration analysis using purified recombinant Cdc13p and Ten1p. Ten1p itself exhibits a weak DNA-binding activity, but enhances the telomeric TG 1-3 DNA-binding ability of Cdc13p. Cdc13p is co-immunoprecipitated with Ten1p. In the mutant ten1-55 or ten1-66 cells, the impaired interaction between Ten1p and Cdc13p results in much longer telomeres, as well as a decreased association of Cdc13p with telomeric DNA. Consistently, the Ten1-55 and Ten1-66 mutant proteins fail to stimulate the telomeric DNA-binding activity of Cdc13p in vitro. These results suggest that Ten1p enhances the telomeric DNA-binding activity of Cdc13p to negatively regulate telomere length.

TEN1Is Essential forCDC13-Mediated Telomere Capping

“…Consistent with the increased Cdc13 telomere binding observed in ykuD strains (Fisher et al 2004) and in late S/G2 when telomeres transiently have longer single-stranded termini (Taggart et al 2002), an increase in the single-stranded nature of the elongated ten1-ts telomeres would be expected to promote the overall extent of Cdc13 association in these strains. These data are consistent with the interpretation that Ten1 is not necessary for Cdc13 to bind telomeres in vivo, in agreement with Qian et al (2009).…”

“…Wild-type Ten1 protein expression is not as robust; it is not clear if this is a factor in the apparently weaker two-hybrid interactions of wild-type Ten1 at 23°( Figure 1D). The Ten1-103 protein reproducibly shows altered mobility on SDS-PAGE; it was recently shown that mutating the adjacent residue (R47E) also impacts Ten1 gel migration (Qian et al 2009). Next, Ten1 interaction was tested with Stn1 and Cdc13 across a range of temperatures.…”

“…These results show that in vivo Cdc13 can bind to telomere repeats independent of Ten1, and demonstrate that the binding of Cdc13 to telomeres is not sufficient to provide a functional telomere cap. It was recently reported that Cdc13 association with a subtelomeric region is reduced in two ten1 strains (Qian et al 2009). The lesions in these mutants perturb the Ten1-Cdc13 contact and the strains show significant telomere elongation, but no impairment of viability (Qian et al 2009).…”

“…It was recently reported that Cdc13 association with a subtelomeric region is reduced in two ten1 strains (Qian et al 2009). The lesions in these mutants perturb the Ten1-Cdc13 contact and the strains show significant telomere elongation, but no impairment of viability (Qian et al 2009). Since these sets of ten1 alleles are all deficient for Cdc13 interaction, how Cdc13 telomere association is affected in ten1 strains may depend more on the overall structure of the chromosome ends than specifically upon the robustness of the interaction between Cdc13 with Ten1.…”

Cdk1 Regulates the Temporal Recruitment of Telomerase and Cdc13-Stn1-Ten1 Complex for Telomere Replication

“…In particular, Stn1 contains binding domains for both Cdc13 and Ten1, which are essential for the formation of the heterotrimeric Cdc13-Stn1-Ten1 (CST) complex at the chromosome ends. In the absence of Stn1, the interaction between Cdc13 and Ten1 is unstable (22). Recent bioinformatic analysis and protein structure modeling have indicated that Stn1 and Ten1 share several structural similarities with Rpa2 and Rpa3, the subunits of the replication protein A (RPA) complex (5,23,24).…”