Reduced Rif2 and lack of Mec1 target short telomeres for elongation rather than double-strand break repair

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...

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“…7D), although RIF1 deletion had a more modest effect than RIF2 deletion in allowing telomere addition (Fig. 7D) (26,37).…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2012

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“…We concluded that telomeres can be maintained by telomerase alone in sae2D sgs1D cells, but recombination also contributes to elongate telomeres in these cells. We next asked whether telomerase activity is sensitive to the negative regulation by Rif2, which competes with Tel1 binding at telomeres 27,44 , in the combined absence of Sae2 and Sgs1. We monitored the telomere length of sae2D sgs1D rif2D clones as described in Fig.…”

Section: Resultsmentioning

confidence: 99%

Sgs1 and Sae2 promote telomere replication by limiting accumulation of ssDNA

et al. 2014

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In budding yeast, DNA ends are processed by the consecutive action of MRX/Sae2 and two redundant pathways dependent on Sgs1/Dna2 and Exo1, and this processing is counteracted by Ku heterodimer. Here we show that DNA end resection by Sae2 and Sgs1 is dispensable for normal telomere maintenance by telomerase. Instead, these proteins facilitate telomere replication and limit the accumulation of single-strand DNA (ssDNA) at replication fork pause sites. Loss of Sae2 and Sgs1 drives selection for compensatory mutations, notably in Ku, which are responsible for abrupt telomere shortening in cells lacking Sae2 and Sgs1. In telomerase-negative cells, Sae2 and Sgs1 play non-overlapping roles in generating ssDNA at eroded telomeres and are required for the formation of type II survivors. Thus, although their primary function in telomerase-positive cells is to sustain DNA replication over the sites that are prone to fork pausing, Sae2 and Sgs1 contribute to telomere resection in telomerase-deficient cells.

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“…29,37,38 Like RPA, ATR and its yeast homolog Mec1 were both found to bind telomeres transiently during the cell cycle. 29,31 These findings suggest that the competition between RPA and POT1 on telomeric ssDNA may not be a simple one. Furthermore, the interplay between RPA and POT1 may be important for the coordination of telomere replication and telomere capping during the cell cycle.…”

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confidence: 87%

“…Indeed, in both yeast and humans, RPA was found to transiently associate with telomeres during S phase. [28][29][30][31] Furthermore, genetic studies in yeast have clearly implicated RPA in telomere maintenance. [32][33][34][35][36] While persistent activation of ATR at telomeres would likely trigger cell cycle arrest, senescence or apoptosis, ATR is also known to play a positive role in telomere maintenance.…”

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confidence: 99%