Evidence that the S.cerevisiae Sgs1 protein facilitates recombinational repair of telomeres during senescence

Abstract

RecQ DNA helicases, including yeast Sgs1p and the human Werner and Bloom syndrome proteins, participate in telomere biology, but the underlying mechanisms are not fully understood. Here, we explore the protein sequences and genetic interactors of Sgs1p that function to slow the senescence of telomerase (tlc1) mutants. We find that the S-phase checkpoint function of Sgs1p is dispensable for preventing rapid senescence, but that Sgs1p sequences required for homologous recombination, including the helicase domain and topoisomerase III interaction domain, are essential. sgs1 and rad52 mutations are epistatic during senescence, indicating that Sgs1p participates in a RAD52-dependent recombinational pathway of telomere maintenance. Several mutations that are synthetically lethal with sgs1 mutation and which individually lead to genome instability, including mus81, srs2, rrm3, slx1 and top1, do not speed the senescence of tlc1 mutants, indicating that the rapid senescence of sgs1 tlc1 mutants is not caused by generic genome instability. However, mutations in SLX5 or SLX8, which encode proteins that function together in a complex that is required for viability in sgs1 mutants, do speed the senescence of tlc1 mutants. These observations further define roles for RecQ helicases and related proteins in telomere maintenance.

Figure 1

Map of the 1447 amino acid Sgs1p protein. Indicated by shaded boxes are the helicase domain, the RQC domain (a C-terminal extension from the helicase domain of shared homology among RecQ-family helicases, which is involved in protein–protein interactions) and the HRCD domain (helicase/RNase D C-terminal domain, which appears to be involved in DNA binding). The locations of the N- and C-terminal deletion breakpoints used in this study are indicated by thick vertical lines and labels above the protein, as is the helicase-deficient alanine substitution mutant, K706A. Sequences containing amino acids that are essential for the binding of Sgs1p to Top3p or for the intra-S-phase checkpoint function of Sgs1p are indicated by horizontal bars below the protein.

Figure 2

Full-length and ΔC200 forms of Sgs1p rescue the fast senescence of tlc1 sgs1 mutants. Full-length SGS1, SGS1-ΔC200 or control vector sequences were integrated at the LEU2 locus of diploids heterozygous for sgs1 and tlc1 null mutations. tlc1 and tlc1 sgs1 spore products without or with the integrated SGS1 alleles (indicated by an asterisk) were compared in liquid senescence assays (see Materials and Methods). The cumulative population doubling since spore germination, and the density of cells after 22 h of growth, from cultures inoculated with 4 × 10⁵ cells/ml are shown. For each time point, the mean and standard errors for at least three independent spore products for each genotype are indicated. For each experiment, filled diamonds indicate tlc1, filled circles indicate tlc1 sgs1, open diamonds indicate tlc1 with an integrated * allele, and open circles indicate tlc1 sgs1 with an integrated * allele, as shown. (A) Rescue by full-length integrated SGS1* (B) Lack of rescue by the pRS405 vector used to integrate the SGS1* alleles. (C) Rescue by SGS1-ΔC200*, which encodes an Sgs1p derivative lacking the C-terminal 200 amino acids.

Figure 3

Evidence that Sgs1p functions via recombination to slow senescence. Senescence rates were measured in the same fashion as Figure 2. (A) rad52 and sgs1 mutations are epistatic during senescence. Haploid spore products were derived from diploids heterozygous for tlc1, sgs1 and rad52 mutations. Curves for tlc1 (filled diamonds), tlc1 rad52 (open diamonds), tlc1 sgs1 (filled circles) and tlc1 sgs1 rad52 (open circles) mutants are shown. This result was repeated two additional times with independently derived spore products (data not shown). (B) est2 and tlc1 mutations are equivalent in the context of sgs1 and rad52 mutations. The experiment was the same as in (A), but with telomerase inactivation via est2 rather than tlc1 deletion (C) Sgs1p helicase activity is required to slow senescence. Diploids heterozygous for tlc1 and sgs1 mutations and with an integrated SGS1-hd* allele, which encodes the helicase-defective K706A point mutant, were sporulated and the senesce rates of tlc1 (filled diamonds), tlc1 sgs1 (filled circles), tlc1 SGS1-hd* (open diamonds) and tlc1 sgs1 SGS1-hd* haploid products were measured. (D) Same as (C), except that the SGS1-ΔC795* allele, which encodes a derivative lacking the C-terminal 795 amino acids of Sgs1p was used instead of SGS1-hd*. (E) SGS1-hd* and SGS1-ΔC795* encode active proteins. The ability of the * alleles to rescue the synthetic slow growth of sgs1 top1 mutants was tested by generating top1 deletions in haploid cells with sgs1 deletion and the indicated * allele. Shown is growth of serial 10-fold dilutions of double mutants containing integrated vector, SGS1*, SGS1-ΔC795* or SGS1-hd* alleles, as indicated. (F) top1 deletion does not speed senescence. Diploids heterozygous for top1 and tlc1 deletions were sporulated and senescence rates of tlc1 (filled diamonds) and tlc1 top1 (open diamonds) haploid spore products were measured.

Figure 4

Sgs1p cooperates with Top3p to slow senescence. Strains were generated and senescence experiments were performed in the same fashion as in Figure 2. (A) SGS1-ΔN50*, which encodes a derivative lacking the N-terminal 50 amino acids of Sgs1p, does not rescue the fast senescence of tlc1 sgs1 mutants. Senescence curves for tlc1 (filled diamonds), tlc1 sgs1 (filled circles), tlc1 SGS1-ΔN50*(open diamonds) and tlc1 sgs1 SGS1-ΔN50* (open circles) haploid spore products are indicated. (B) TOP3-SGS1-ΔN106*, which encodes Top3p fused to an Sgs1p derivative lacking the N-terminal 106 amino acids, rescues rapid senescence. Senescence curves for tlc1 (filled diamonds), tlc1 sgs1 (filled circles) and tlc1 sgs1 TOP3-SGS1-ΔN106* (open circles) haploid spore products are indicated.

Figure 5

Effects of sgs1 synthetic lethal mutations on senescence. The indicated synthetic lethal mutations were introduced individually into diploids heterozygous for tlc1 deletion. Senescence rates of spore products were measured in the same fashion as Figure 2. In all cases, filled diamonds indicate tlc1 mutants, open diamonds indicate tlc1 plus the synthetic lethal mutation, and filled and open triangles indicate wild-type and the synthetic lethal mutant, respectively. Comparison of tlc1 and tlc1 with (A) slx1 deletion, (B) slx5 deletion, (C) slx8 deletion, (D) mus81 deletion, (E) srs2 deletion and (F) rrm3 deletion. Note the increased number of days (data points) spent in the nadir in tlc1 srs2 mutants.

Figure 6

Telomere shortening and survivor formation in tlc1 slx8 cells. (A) Y′-containing telomere lengths from wild type, slx8 and from two independent cultures each of tlc1 and tlc1 slx8 mutants during senescence were measured by digesting genomic DNA with XhoI and probing with Y′ sequences distal to the cut site. The number of population doublings (PD) since the start of spore growth is indicated. (B) Survivor type in four tlc1 (A–D) and four tlc1 slx8 (E–F) cultures was examined by Southern analysis of XhoI-digested genomic DNA visualized with a telomere repeat probe. Samples are from days 17 (PD 155–170) and 22 (PD 200–215) after sporulation, and samples were obtained from the cultures shown in Figure 5C. Y′ and Y′ term indicate the position of tandemly-repeated Y′ elements, and the terminal Y′ fragment, respectively, that are most prominent in type I survivors. On the right, samples from slx8, pure type I (tlc1 sgs1), pure type II (tlc1 rad51) and wild-type cultures are provided for comparison, and the position of DNA markers is indicated with sizes in kilobases (kb).