Rif1 phosphorylation site analysis in telomere length regulation and the response to damaged telomeres

Abstract

Telomeres, the ends of eukaryotic chromosomes, consist of repetitive DNA sequences and their bound proteins that protect the end from the DNA damage response. Short telomeres with fewer repeats are preferentially elongated by telomerase. Tel1, the yeast homolog of human ATM kinase, is preferentially recruited to short telomeres and Tel1 kinase activity is required for telomere elongation. Rif1, a telomere-binding protein, negatively regulates telomere length by forming a complex with two other telomere binding proteins, Rap1 and Rif2, to block telomerase recruitment. Rif1 has 14 SQ/TQ consensus phosphorylation sites for ATM kinases, including 6 in a SQ/TQ Cluster Domain (SCD) similar to other DNA damage response proteins. These 14 sites were analyzed as N-terminal, SCD and C-terminal domains. Mutating some sites to non-phosphorylatable residues increased telomere length in cells lacking Tel1 while a different set of phosphomimetic mutants increased telomere length in cells lacking Rif2, suggesting that Rif1 phosphorylation has both positive and negative effects on length regulation. While these mutations did not alter the sensitivity to DNA damaging agents, inducing telomere-specific damage by growing cells lacking YKU70 at high temperature revealed a role for the SCD. Mass spectrometry of Rif1 from wild type cells or those induced for telomere-specific DNA damage revealed increased phosphorylation in cells with telomere damage at an ATM consensus site in the SCD, S1351, and non-ATM sites S181 and S1637. A phosphomimetic rif1-S1351E mutation caused an increase in telomere length at synthetic telomeres but not natural telomeres. These results indicate that the Rif1 SCD can modulate Rif1 function. As all Rif1 orthologs have one or more SCD domains, these results for yeast Rif1 have implications for the regulation of Rif1 function in humans and other organisms.

Keywords: Phosphorylation; Rif1; Saccharomyces cerevisiae; Telomere length regulation.

Figure 1. Mutation of Rif1 ATM consensus sites alters telomere length

A. SQ/TQ motifs and clustered domains are conserved from yeasts to humans. S. cerevisiae Rif1 has 14 SQ/TQ motifs, with 6 of them in a 108 a. a. SCD (SQ/TQ motif clustered domain, defined as 3 or more sites with 100 amino acids [12]). The presence of SCDs in Rif1 is evolutionarily conserved in Schizosaccharomyces pombe and humans. These S. cerevisiae motifs were divided into three groups for mutant analysis, N-terminus, SCD and C-terminus. The locations of the S and T residues in the SQ/TQ motifs are shown. The SQ/TQ motifs in the Rif1 SCD are S1308, T1316, S1330, S1351, T1386, and T1417. B. The telomere on Chromosome VIIL was replaced by a synthetic telomere with a marker gene URA3 After. digestion with Stu I, the terminal restriction fragments were measured on Southern blot by a probe against URA3 gene to calculate telomere length. C. Telomere length is increased for rif1-SCD-6E in rif2Δ cells. Telomere lengths of the Rif1 SCD mutations with or without the TEL1 or RIF2 genes were measured by Southern blotting using URA3 probe. The cells were grown for 100 generations after isolation of the mutant strains by serial dilution. The bar graph shows the averaged telomere size from 2 independent isolates, but only one representative Southern blot from one isolate was shown. The control bands are from internal loci and used as internal size standards for accurate measurement of the telomere band. M: DNA marker lane. * p<0.05 by two-way ANOVA analysis. D. Telomere length is increased for rif1-N6A in tel1Δ cells. Similar as in panel C but for Rif1 mutations at N-terminus. E. Telomere length is increased for rif1-C2A in tel1Δrif2Δ cells. Similar as in panel C but for Rif1 mutations at C-terminus.

Figure 2. The Rif1 SQ/TQ mutants have little effect on the length of chromosomal telomeres

A. Schematic illustration of telomeric regions in S. cerevisiae. The relative location of the TG_1–3/C_1–3A repeats between the middle repetitive elements X and Y′ and adjacent Y′ elements is indicated. Xho I digestion generates telomeric fragments in the 1.2–1.6 kb range in wild type strains from telomeres that end in Y′. B–D. The length of Y′ type telomeres in different RIF1 mutants with TEL1 RIF2, tel1Δ RIF2, TEL1 rif2Δ, or tel1Δ rif2Δ were analyzed with standard Southern blotting. All strains were first grown for over 100 generations to establish steady-state telomere lengths. Genomic DNA was prepared and digested with Xho I and telomeres were detected using a TG_1–3/C_1–3A as probe. These DNA samples are the same as those analyzed in Figure 1C–E.

Figure 3. Rif1 SCD mutations have no growth alterations in cells with damaged telomeres or cells treated with DNA damaging agents

A. cdc13-1 cells have unprotected telomeres and accumulate telomeric ssDNA at the non-permissive temperature. The checkpoint activation can be inhibited by overexpression of Rif1. B. Rif1 SCD mutants have similar temperature sensitivity as RIF1 in cdc13-1 cells. Serial five-fold dilutions of cells were plated onto YEPD plates and incubated at different temperatures. C. Rif1 SCD mutants have no growth defect upon MMS or HU treatment with or without TEL1 gene. Serial five-fold dilutions of cells were plated onto YEPD plates with different concentrations of MMS or HU. mre11Δ cells serve as a control for increased MMS or HU sensitivity. (P), petite phenotype (a mitochondrial DNA defect that may be induced by 5-FOA during strain construction [59]).

Figure 4. Three Rif1 phosphorylation sites are enriched in cells with short damaged cells

A. yku70Δ cells have inducible telomere damage at the non-permissive temperature. Extended growth at the restrictive temperature induces end resection and checkpoint activation at telomeres. B. Rif1 bands are stained by Coomassie blue after purification. Rif1 bands are indicated by arrow and protein marker bands are labeled. WCE: whole cell extract. IP: immunoprecipitation. M: protein marker. C. The summary of Rif1 phosphorylation sites and the alteration of their levels in mass spectrometry analysis. The phosphorylation level is calculated by the ratio of phosphopeptides to unmodified peptides (Table S1). The sites with more than 2-fold increase in wild type (YKU70) or yku70Δ cells are labeled in red or blue, respectively. SQ/TQ motifs are labeled as grey circles and S1351 in green. The black horizontal line represents Rif1 protein with a total of 1916 aa.

Figure 5. The rif1-SCD-6Eyku70Δ cells show increased sensitivity to high temperature and to the DNA damaging agents HU and MMS, while the rif1-SCD-6A yku70Δ cells show MMS resistance

A. The Rif N-terminal or C-terminal domain mutations did not change the resistance of yku70Δ cells to HU and MMS. Five-fold serial dilutions of cells growing exponentially at 25°C were spotted onto YEPD plates with indicated concentration of HU or MMS and cultured at 30 °C for 64 hours. The xrs2Δ and mre11Δ mutants were used as controls for strains that are highly sensitive to both drugs. B. The rif1-SCD-6E mutations impair the viability of yku70Δ cells at high temperature. Spot test analysis of viability of yku70Δ cells bearing the RIF1, rif1-SCD-6E or rif1-SCD-6A alleles at different temperatures. Five-fold serial dilutions of cells growing exponentially at 25°C were spotted onto room temperature YEPD plates and cultured at indicated temperatures for 93 hours. To monitor cell growth, plates were moved to room temperature for 1 hr every 24 to 27 hr to record cell growth, and then returned to the incubator. The (P) indicates that the rif1-SCD-6A and rif1-SCD-6E strains are petite.