A Balance between Tel1 and Rif2 Activities Regulates Nucleolytic Processing and Elongation at Telomeres

Abstract

Generation of G-strand overhangs at Saccharomyces cerevisiae yeast telomeres depends primarily on the MRX (Mre11-Rad50-Xrs2) complex, which is also necessary to maintain telomere length by recruiting the Tel1 kinase. MRX physically interacts with Rif2, which inhibits both resection and elongation of telomeres. We provide evidence that regulation of telomere processing and elongation relies on a balance between Tel1 and Rif2 activities. Tel1 regulates telomere nucleolytic processing by promoting MRX activity. In fact, the lack of Tel1 impairs MRX-dependent telomere resection, which is instead enhanced by the Tel1-hy909 mutant variant, which causes telomerase-dependent telomere overelongation. The Tel1-hy909 variant is more robustly associated than wild-type Tel1 to double-strand-break (DSB) ends carrying telomeric repeat sequences. Furthermore, it increases the persistence at a DSB adjacent to telomeric repeats of both MRX and Est1, which in turn likely account for the increased telomere resection and elongation in TEL1-hy909 cells. Strikingly, Rif2 is unable to negatively regulate processing and lengthening at TEL1-hy909 telomeres, indicating that the Tel1-hy909 variant overcomes the inhibitory activity exerted by Rif2 on MRX. Altogether, these findings highlight a primary role of Tel1 in overcoming Rif2-dependent negative regulation of MRX activity in telomere resection and elongation.A highly ordered nucleoprotein complex called the telomere prevents the ends of linear chromosomes from being recognized as DNA double-strand breaks (DSBs) (reviewed in reference 30). Another key function of telomeres is to compensate for the incomplete replication of chromosome ends caused by discontinuous DNA synthesis. In most eukaryotes, telomeric DNA comprises tandemly repeated G-rich sequences (TG 1-3 repeats in Saccharomyces cerevisiae yeast and T 2 AG 3 in vertebrates), ending in a single-stranded 3= overhang (G tail) (reviewed in reference 20). The addition of telomeric repeats depends on the action of telomerase, a specialized reverse transcriptase that extends the TGrich strand of chromosome ends (23). The yeast telomerase complex consists of a reverse transcriptase subunit (Est2), a template RNA (TLC1), and two accessory proteins (Est1 and Est3), which are required for telomerase activity in vivo but not in vitro.The single-stranded G tails of budding yeast telomeres are short (about 10 to 15 nucleotides [nt]) for most of the cell cycle, but their length increases transiently to 50 to 100 nucleotides in late S phase (15, 50). While telomeric G tails can be generated during lagging-strand replication by removal of the last RNA primer, the 5= C strand of the telomere generated by leadingstrand synthesis must be nucleolytically processed (resected) to generate 3= overhangs (32, 51). Cyclin-dependent kinase (Cdk1 in S. cerevisiae) activity is required for this telomeric C-strand resection (17, 49), which occurs only during S and G 2 cell cycle phases, when Cdk1 activity is high and telomeres are elongated by telomerase (36...

Sgs1 and Sae2 promote telomere replication by limiting accumulation of ssDNA

“…9a). Although we cannot rule out that SAE2 and RIF2 deletions have additive independent effects, this result is in line with the role of Rif2 in regulating the function of MRX/Sae2 in resection 60 . In this case, premature senescence of the rif2D sae2D est2D triple mutant as compared with the sae2D est2D double mutant would suggest that Sae2 is not the only activity inhibited by Rif2.…”

Functions and regulation of the MRX complex at DNA double-strand breaks

“…In S. cerevisiae , MRX is known to interact with Rif2, which is recruited to telomeric DNA ends by Rap1 and negatively regulates telomerase action 39404142. Rif2 was recently found to be recruited also to intrachromosomal DSBs in a manner partially dependent on MRX 43.…”

To trim or not to trim: Progression and control of DSB end resection

“…MRX/ MRN is the major responsible for the generation of the G-tail at telomeres, where its activity is stimulated by the PIKK kinase Tel1. 88 Indeed, mrx mutants are defective in G-tail formation in a de novo telomere addition reaction; 89,90 moreover, both mrx mutants and MRN-depleted cells exhibit shorter G-tails. 91,92 Further investigations indicated that MRX and Sae2 act together in the processing the C-strand, even though MRX may give a chromatin remodeler belonging to the Etl1 Snf2 family.…”