Suppression of cdc13-2-associated senescence by pif1-m2 requires Ku-mediated telomerase recruitment

Abstract

In Saccharomyces cerevisiae, recruitment of telomerase to telomeres requires an interaction between Cdc13, which binds single-stranded telomeric DNA, and the Est1 subunit of telomerase. A second pathway involving an interaction between the yKu complex and telomerase RNA (TLC1) contributes to telomerase recruitment, but cannot sufficiently recruit telomerase on its own to prevent replicative senescence when the primary Cdc13-Est1 pathway is abolished—for example, in the cdc13-2 mutant. In this study, we find that mutation of PIF1, which encodes a helicase that inhibits telomerase, suppresses the replicative senescence of cdc13-2 by increasing reliance on the yKu-TLC1 pathway for telomerase recruitment. Our findings reveal new insight into telomerase-mediated telomere maintenance.

Deletion ofMEC1suppresses replicative senescence of thecdc13-2mutant inSaccharomyces cerevisiae

“…We also previously reported that the pif1-m2 can suppress the senescence of a cdc13-F237A tlc1Δ48 double mutant (Fekete-Szücs et al 2022), in which the Cdc13 EBM-N -Est1 and yKu-TLC1 interactions are both disrupted (Chen et al 2018). Unlike pif1-m2 , we find that mec1Δ cannot suppress cdc13-F237A tlc1Δ48 senescence, although it does delay senescence (Fig 1F).…”

“…We previously reported that the pif1-m2 mutant, which is depleted for nuclear Pif1 (Schulz and Zakian 1994), can suppress the cdc13-2 est phenotype, but not if the Sir4-yKu-TLC1 pathway is disrupted (Fekete-Szücs et al 2022). To test whether this was also true for mec1Δ suppression of cdc13-2 , we monitored the growth of haploid meiotic progeny derived from the sporulation of a CDC13/cdc13-2 MEC1/mec1Δ SML1/sml1Δ TLC1/tlc1Δ48 diploid strain (Fig 1E).…”

“…The mutations were confirmed by DNA sequencing. Plasmid pEFS4 (pRS415- cdc13-F237A ) was generated in the same manner from a previous study (Fekete-Szücs et al 2022).…”

“…Standard yeast genetic and molecular methods were used (Sherman 2002;Amberg et al 2005). Plasmids pFR95 (pRS415-cdc13- E252K), pYY1 (pRS415-cdc13-S306A), and pYY2 (pRS415-cdc13-E252K, Plasmid pEFS4 (pRS415-cdc13-F237A) was generated in the same manner from a previous study (Fekete-Szücs et al 2022).…”

“…We recently discovered that mutation of PIF1 can suppress the est phenotype of the cdc13-2 mutant (Fekete-Szücs et al 2022). In this study, we show that deletion of MEC1 can also suppress the cdc13-2 est phenotype.…”