At short telomeres Tel1 directs early replication and phosphorylates Rif1

Abstract

The replication time of Saccharomyces cerevisiae telomeres responds to TG1-3 repeat length, with telomeres of normal length replicating late during S phase and short telomeres replicating early. Here we show that Tel1 kinase, which is recruited to short telomeres, specifies their early replication, because we find a tel1Δ mutant has short telomeres that nonetheless replicate late. Consistent with a role for Tel1 in driving early telomere replication, initiation at a replication origin close to an induced short telomere was reduced in tel1Δ cells, in an S phase blocked by hydroxyurea. The telomeric chromatin component Rif1 mediates late replication of normal telomeres and is a potential substrate of Tel1 phosphorylation, so we tested whether Tel1 directs early replication of short telomeres by inactivating Rif1. A strain lacking both Rif1 and Tel1 behaves like a rif1Δ mutant by replicating its telomeres early, implying that Tel1 can counteract the delaying effect of Rif1 to control telomere replication time. Proteomic analyses reveals that in yku70Δ cells that have short telomeres, Rif1 is phosphorylated at Tel1 consensus sequences (S/TQ sites), with phosphorylation of Serine-1308 being completely dependent on Tel1. Replication timing analysis of a strain mutated at these phosphorylation sites, however, suggested that Tel1-mediated phosphorylation of Rif1 is not the sole mechanism of replication timing control at telomeres. Overall, our results reveal two new functions of Tel1 at shortened telomeres: phosphorylation of Rif1, and specification of early replication by counteracting the Rif1-mediated delay in initiation at nearby replication origins.

Figure 1. Tel1 is required for early replication of short telomeres.

(A) Telomere length analysis in wild-type (YKU70 TEL1), tel1Δ, yku70Δ, and yku70Δ tel1Δ strains. Terminal chromosome fragments were detected by probing a Southern blot of XhoI-digested genomic DNA for TG_1–3 sequence. Smear represents average length of Y′ telomeres. (B) Replication kinetics of various genomic sequences in wild-type and short telomere mutants yku70Δ, tel1Δ and yku70Δ tel1Δ. Telomere-proximal sequences shown are Y′ (solid line with filled circles), ARS522 (solid line with filled diamonds), and proARS1202 (solid line with filled triangles). Non-telomeric marker sequences (dashed lines) are early origins ARS305 (open squares), late origin ARS1412 (open circles), and Chr XIV-internal sequences (open diamonds). Strains were released from α-factor block at 30°C. (C) Replication indices (RI) values from experiments in B, where replication times are normalized to early origin ARS305 (RI = 0) and Chr.XIV-int (RI = 1). Strains are BB14-3a (wild-type), ASY5 (tel1Δ), AW99 (yku70Δ) and ASY13 (yku70Δ tel1Δ; corresponding to second isolate in part A); all are in A364a background as listed in Table S1.

Figure 2. Tel1 stimulates activation of an origin neighboring an induced short telomere upon release into hydroxyurea.

(A) Cartoon showing HO endonuclease-inducible short telomere construct on the left arm of Chr. VII, with positions of HindIII (H) and XmnI (X) restriction sites. Triangles represent TG repeat sequences and the filled circle, ARS700.5. Not to scale. (B) HO endonuclease cutting efficiency in the hydroxyurea-arrested cultures used for 2D gel analysis in C. Cells were arrested with α-factor then galactose added to induce HO cutting, followed by release into hydroxyurea. (C) 2D gel analysis of replication intermediates present at early origin ARS305 (upper panels), ARS700.5 (middle panels), and late origin ARS1412 (lower panels), in TEL1 (left) and tel1Δ (right) strains. The same blot of HindIII-digested DNA was probed sequentially for the three origins. Strains used are YAB1410, SMKY10 (TEL1) and SMKY13 (tel1Δ).

Figure 3. Rif1 acts downstream of Tel1 in regulating telomere replication time.

(A) Telomere length analysis in wild-type, tel1Δ, rif1Δ and rif1Δ tel1Δ strains. Southern blot analysis carried out as in Fig. 1A. (B) Replication kinetics of various genomic sequences in rif1Δ and rif1Δ tel1Δ strains. Plots and symbols as in Fig. 1B. (C) Replication indices from experiments in B, along with values from wild-type and tel1Δ experiments from Fig. 1. Strains are HYLS44 (rif1Δ) and ASY14 (rif1Δ tel1Δ; corresponding to first isolate in part A).

Figure 4. Rif1 is phosphorylated at Tel1 consensus sites in the short telomere mutant yku70Δ.

(A) Telomere length gel confirms Myc-tagged Rif1 protein is functional. (B) Upper panel (i): Western blot analyzing Rif1-Myc protein in Whole Cell Extract (WCE), immunoprecipitated sample (IP) and supernatant (Unbound). All lanes show equivalent cell loading. Lower panel (ii): SyproRUBY-stained gel showing Rif1-Myc isolated from YKU70 and yku70Δ strains, and mock IP from untagged control sample. Rif1 was quantified based on SyproRUBY gel bands and equivalent quantities mixed for SILAC mass spectrometry analysis. 260 kD marker position is indicated. The predicted size of Rif1-13Myc is 232 kD; Rif1-13Myc migration is slightly retarded relative to its predicted mass. (C) Cartoon of Rif1p sequence, illustrating the position of the 14 S/TQ sites. In enlarged sequence below S/TQ sequences are bold, and colored green are the two sites identified as phosphorylated in an initial mass spectrometry run (carried out using Rif1-Myc from YKU70 and yku70Δ strains). Blue arrowheads indicate trypsin digestion sites. (D) Plot of SILAC ratio for phosphorylated peptide containing S-1308 in yku70Δ relative to YKU70 (16.4× increased). In this and similar plots relative values are normalized during processing to the median H/L ratio of all Rif1 peptides. (E) MS spectrum showing raw results for the same S-1308 phosphorylated peptide [KVDS(ph)QDIQVPATQGM(ox)K], with light (R0K0) peptide from YKU70 on left and heavy (R10K8) peptide from yku70Δ on right. (F) & (G) show equivalent SILAC analysis for the S-1351 phosphorylated peptide NTAIM(ox)NSS(ph)QQESHANR (4.1× increased in Δyku70 relative to YKU70). Strains (BY4741 strain background) are SHY201 (untagged wild-type), ASY25 (YKU70 RIF1-13Myc), ASY30 (yku70Δ RIF1-13Myc), Y00870 (untagged yku70Δ) and HYLS44 (rif1Δ*; asterisk indicating A364a strain background). An initial, non-SILAC, mass spectrometry analysis depicted in C used W303 Rad5+ strains YSM20 (YKU70 RIF1-13Myc) and ASY17 (yku70Δ RIF1-13Myc).

Figure 5. Phosphorylation of Rif1 Serine-1308 depends on Tel1.

(A) Plots shows relative levels of the S-1308 phosphorylated peptide [KVDS(ph)QDIQVPATQGM(ox)K] in yku70Δ (Light-labeled R0K0) and yku70Δ tel1Δ (Heavy-labeled R10K8) strains. H/L ratio is 0.10. (B) Equivalent plot for S-1351 phosphorylated peptide NTAIM(ox)NSSQQESHANR. H/L ratio is 0.97048. Strains used are ASY30 (yku70Δ RIF1-13Myc), and ASY46 (yku70Δ tel1Δ RIF1-13Myc).

Figure 6. Non-phosphorylatable rif1-7S→A does not delay early replication of short yku70Δ telomeres.

(A) Telomere length analysis of Rif1 phospho-site mutants. Genomic DNA was extracted from the indicated strains and telomere length analyses performed as described. Smear indicates average length of Y′ telomeres. rif1Δscd represents an internal deletion within the RIF1 C-terminal region made as a strain construction intermediate (see Supplementary Materials & Methods). RIF1-7S→S represents a RIF1 reconstruction, where wild-type sequence was re-inserted into rif1Δscd to control for telomere length recovery. (B) Replication program of yku70Δ rif1-7S→A, released from an α-factor block at 30°C. Sequences analyzed are as in Fig. 1. (C) Replication indices from yku70Δ rif1-7S→A experiment shown in B, along with values from wild-type and yku70Δ experiments from Fig. 1B&C. Strains in part A are BB14-3a (wild-type), HYLS44 (rif1Δ), ASY5 (tel1Δ), AW99 (yku70Δ), ASY51 (rif1Δscd); ASY81 (RIF1-7S→S). For rif1-7S→A asterisk indicates ASY69, used for replication timing in Fig. S11; for rif1-7S→E asterisk indicates ASY73, used for replication timing in Fig. S12; for yku70Δ rif1-7S→A asterisk indicates ASY76, used for replication timing in Fig. 6 B&C and S10; for yku70Δ rif1-7S→E asterisk indicates ASY78.

Figure 7. Model of replication timing control by Tel1 and Rif1.

(A) In wild-type cells, terminal TG_1–3 tract is bound by Rap1 (open triangles) which recruits Rif1 (grey hexagons) and Rif2 (small grey triangles). If the telomere is normal in length, Rif1 signals to nearby origins (such as telomere-proximal Y′ or ARS522 origins) specifying their late replication time (filled circle). (B) If telomeres are short (as in a yku70Δ mutant) Tel1 kinase is recruited and neutralizes the Rif1 delaying signal, so that nearby origins initiate early (white circle). (C) In tel1Δ mutant cells, the delaying effect of Rif1 cannot be neutralized so that nearby origins initiate late despite the short telomeres. (D) A rif1Δ mutant lacks the delaying signal, with the result that nearby origins initiate replication early despite their extended TG_1–3 repeat length.