Rif2 protects Rap1-depleted telomeres from MRX-mediated degradation in Saccharomyces cerevisiae

Abstract

Rap1 is the main protein that binds double-stranded telomeric DNA in Saccharomyces cerevisiae. Examination of the telomere functions of Rap1 is complicated by the fact that it also acts as a transcriptional regulator of hundreds of genes and is encoded by an essential gene. In this study, we disrupt Rap1 telomere association by expressing a mutant telomerase RNA subunit (tlc1-tm) that introduces mutant telomeric repeats. tlc1-tm cells grow similar to wild-type cells, although depletion of Rap1 at telomeres causes defects in telomere length regulation and telomere capping. Rif2 is a protein normally recruited to telomeres by Rap1, but we show that Rif2 can still associate with Rap1-depleted tlc1-tm telomeres, and that this association is required to inhibit telomere degradation by the MRX complex. Rif2 and the Ku complex work in parallel to prevent tlc1-tm telomere degradation; tlc1-tm cells lacking Rif2 and the Ku complex are inviable. The partially redundant mechanisms may explain the rapid evolution of telomere components in budding yeast species.

Investigating the role of G-quadruplexes at Saccharomyces cerevisiae telomeres

“…Our findings suggest that G-quadruplex formation at telomeres is not essential for telomere capping nor cell viability in S. cerevisiae . In addition, our findings are not consistent with a previously proposed model whereby Est1-mediated G-quadruplex formation is required for telomerase activity [ 10 ], since tlc1-tm telomeres are efficiently extended by telomerase [ 17 ]. While we cannot exclude the possibility that less stable G-quadruplex structures (e.g.…”

“…While our findings are consistent with a previously proposed model in which G-quadruplexes protect cdc13-1 telomeres [ 11 ], the effect of tlc1-tm on cdc13-1 cells may instead be due to reduced levels of Rap1 at tlc1-tm telomeres [ 17 ] rather than a disruption in G-quadruplex formation. However, we do not favor this possibility because telomeres in tlc1-tm cells still retain wild-type telomeric sequence in their centromere-proximal regions, so that telomere-bound Rap1 is only reduced by 40% [ 17 ].…”

“…elongated, very short, or extensively degraded), and aberrant chromosome separation and segregation [ 18 , 19 ]. The tlc1-tm mutant introduces [(TG) 0–4 TGG] x ATTTGG mutant telomeric repeats instead of wild-type (TG) 0-6 TGGGTGTG(G) 0–1 repeats, and grows similar to a wild-type strain, even when one telomere consists entirely of mutant sequence [ 15 – 17 ]. Telomeres in the tlc1-tm mutant are on average longer and more heterogeneous in length than in wild-type strains [ 17 ], but the telomere profile of tlc1-tm is much less dramatically altered compared to most other mutants of TLC1 with altered template sequences [ 18 , 19 ].…”

“…Furthermore, we have previously reported several telomeric defects (e.g. disruption of telomere length homeostasis) in tlc1-tm cells [ 17 ]. While we believe that most of these defects can be largely attributed to depletion of telomere-bound Rap1, it is formally possible that impairment in the formation of telomeric G-quadruplexes could contribute to some of these defects.…”

“…In this study, we examined the function of G-quadruplexes at S. cerevisiae telomeres by expressing a mutant telomerase RNA subunit (tlc1-tm) that introduces [(TG) 0–4 TGG] x ATTTGG mutant telomeric repeats instead of wild-type (TG) 0-6 TGGGTGTG(G) 0-1 repeats [ 15 , 16 ]. The mutant repeats are impaired in the formation of G-quadruplexes, and we have previously shown that tlc1-tm repeats are poorly bound by Rap1 [ 17 ]. Despite being deficient in telomeric G-quadruplex formation, tlc1-tm cells are viable and grow as well as wild-type cells, suggesting that the ability to form telomeric G-quadruplexes is not essential for telomere capping and cell viability in S. cerevisiae .…”