The SIR2/3/4 complex and SIR2 alone promote longevity in Saccharomyces cerevisiae by two different mechanisms

Abstract

The SIR genes are determinants of life span in yeast mother cells. Here we show that life span regulation by the Sir proteins is independent of their role in nonhomologous end joining. The short life span of a sir3 or sir4 mutant is due to the simultaneous expression of a and alpha mating-type information, which indirectly causes an increase in rDNA recombination and likely increases the production of extrachromosomal rDNA circles. The short life span of a sir2 mutant also reveals a direct failure to repress recombination generated by the Fob1p-mediated replication block in the rDNA. Sir2p is a limiting component in promoting yeast longevity, and increasing the gene dosage extends the life span in wild-type cells. A possible role of the conserved SIR2 in mammalian aging is discussed.

Figure 1

SIR2, SIR3, and SIR4, but not NHEJ, are required for longevity of haploid mother cells. (A) Mutation of either sir3 or sir4 results in a 20% reduction in mean life span. Mutation of sir2 reduces mean life span by ∼50%. Life spans were determined for W303R, and isogenic sir4Δ, sir3Δ, and sir2Δ mutants. Mean life spans and the number of mothers examined were as follows: W303R 21.4 (n = 50), sir4Δ 18.8 (n = 50), sir3Δ 18.0 (n = 50), and sir2Δ 11.6 (n = 50), (♦) W303R; (█) sir4; (▴) sir3; (x) sir2. (B) Mutation of hdf1 or hdf2 shortens life span. Life spans were determined for haploid W303R, hdf1Δ, and hdf2Δ strains. Mean life spans and the number of mothers examined were as follows: W303R 21.2 (n = 50), hdf1Δ 17.3 (n = 50), and hdf2Δ 14.3 (n = 50), (♦) W303R; (█) hdf1; (▴) hdf2. (C) Mutation of dnl4 does not affect life span. Life spans were determined for haploid W303R and dnl4Δ strains. Mean life spans and the number of mothers examined were as follows: W303R 23.7 (n = 50) and dnl4Δ 23.4 (n = 50), (♦) W303R; (█) dnl4.

Figure 2

The Sir2/3/4 complex extends life span by silencing the HM loci and preventing a/α coexpression. (A) The life span defect of a W303R MATa sir4Δ mutant is suppressed by deletion of hmlα. Life spans were determined for wild-type W303R, and isogenic derivatives, sir4Δ and sir4Δ hmlΔ. Mean life spans and the number of mothers examined were as follows: W303R 21.6 (n = 37), sir4Δ 18.2 (n = 49), and sir4Δ hmlΔ 22.4 (n = 48). (♦) W303R; (█) sir4; (▴) sir4 hml. (B) The life span defect of a W303R MATa sir3Δ mutant is suppressed by deletion of hmlα. Life spans were determined for W303R, and isogenic derivatives, sir3Δ and sir3Δ hmlΔ. Mean life spans and the number of mothers examined were as follows: W303R 20.3 (n = 48), sir3Δ 15.2 (n = 47), and sir3Δ hmlΔ 21.3 (n = 46). (♦) W303R; (█) sir3; (▴) sir3 hml. (C) The short life span of a W303R MATa sir2Δ mutant is not suppressed by deletion of hmlα. Life spans were determined for wild-type W303R, and isogenic derivatives, sir2Δ, and sir2Δ hmlΔ. Mean life spans and the number of mothers examined were as follows: W303R 21.4 (n = 58), sir2Δ 11.8 (n = 48), and sir2Δ hmlΔ 11.9 (n = 43). (♦) W303R; (█) sir2; (▴) sir2 hml. (D) Mating-type heterozygosity is sufficient to shorten haploid life span to the level of a sir3Δ or sir4Δ mutant. Life spans were determined for wild-type W303R, and isogenic derivatives, sir3Δ, sir4Δ, and W303R URA3/MATα. Mean life spans and the number of mothers examined were as follows: W303R 20.4 (n = 48), sir3Δ 15.2 (n = 47), sir4Δ 15.4 (n = 49), and W303R URA3/MATα 15.2 (n = 48). (♦) W303R; (█) sir4; (▴) sir3; (×) W303a/α.

Figure 3

SIR2, but not SIR4, is required for longevity in diploid cells. (A) Diploid life span is not regulated by SIR4. Life spans were determined for W303R/W303R, the isogenic derivative, sir4Δ/sir4Δ, W303R MATα, and W303R MATa. Mean life spans and the number of mothers examined were as follows: W303R/W303R 18.2 (n = 49), sir4Δ/sir4Δ 18.1 (n = 50), W303R MATα 21.1 (n = 50), and W303R MATa 20.5 (n = 50). (♦) W303R/W303R; (█) sir4/sir4; (▴) W303R MATα; (×) W303R MATa. (B) Homozygous mutation of sir2 causes a significant reduction in diploid life span. The sir2Δ/SIR2 heterozygote has a life span that is intermediate between the wild-type diploid and the sir2Δ/sir2Δ homozygote. Life spans were determined for W303R/W303R, and isogenic derivatives, sir2Δ/SIR2, and sir2Δ/sir2Δ. Mean life spans and the number of mothers examined were as follows: W303R/W303R 18.8 (n = 50), sir2Δ/SIR2 14.0 (n = 47), and sir2Δ/sir2Δ 12.1 (n = 48). (♦) W303R/W303R; (█) sir2/SIR2; (▴) sir2/sir2.

Figure 4

FOB1-dependent accumulation of ERCs occurs more rapidly in a sir2 mutant than in wild-type mother cells. DNA from young (unsorted) and old (mean age = 7 generations) mother cells was isolated and electrophoresed as described (Defossez et al. 1999). The gel was transferred and probed with rDNA. Chromosomal DNA is denoted by an arrowhead and ERC species by arrows. Both sorted and unsorted W303R sir2Δ mother cells show a greater accumulation of higher oligomeric species of ERCs than wild type. Mutation of fob1 in sir2Δ hmlΔ mother cells decreases ERC accumulation below levels observed in wild-type cells. Intensity of bands was quantitated on a PhosophorImager. The ratio of ERC DNA to genomic rDNA in young wild-type and sir2Δ hmlΔ cells is 0.2 and 0.4, respectively. In old wild-type and sir2Δ hmlΔ cells, this ratio is 2.1 and 3.1, respectively.

Figure 5

Sir4p does not function to extend life span after release of an ERC. ERCs were released in virgin cells as described (Sinclair and Guarente 1997). Life spans were determined for W303R, W303R induced to release an ERC (W303R + ERC), the isogenic derivative, sir4, and sir4 induced to release an ERC (sir4 + ERC). Mean life spans and the number of mothers examined were as follows: W303R 17.6 (n = 58), W303R + ERC 13.5 (n = 68), sir4 13.5 (n = 55), and sir4 + ERC 13.0 (n = 51). (♦) W303R; (█) W303R + ERC; (▴) sir4; (×) sir4 + ERC.

Figure 6

Mutation of fob1 suppresses the life span defect of a sir2 mutant. Life spans were determined for W303R, and the isogenic derivatives, sir2Δ hmlΔ, and sir2Δ hmlΔ fob1Δ. Mean life spans and the number of mothers examined were as follows: W303R 20.7 (n = 38), fob1Δ 27.0 (n = 37), sir2Δ hmlΔ 13.0 (n = 47) and sir2Δ hmlΔ fob1Δ 21.9 (n = 48). (♦) W303R; (█) fob1; (▴) sir2 hml; (×) sir2 hml fob1.

Figure 7

Sir2p is a limiting component of yeast longevity. (A) Overexpression of SIR2 extends the life span of W303R by ∼30%. A second copy of SIR2 was integrated at the URA3 locus (denoted SIR2/URA3) of strains W303R and sir2Δ. Life spans were determined for W303R, and the isogenic derivatives sir2Δ, W303R SIR2/URA3, and sir2Δ SIR2/URA3. Mean life spans and the number of mothers examined were as follows: W303R 23.0 (n = 48), W303R SIR2/URA3 28.8 (n = 50), sir2Δ 11.0 (49), and sir2Δ SIR2/URA3 21.3 (49). (♦) W303R; (█) W303R SIR2/URA3; (▴) sir2; (×) sir2 SIR2/URA3. (B) Overexpression of SIR2 does not affect the life span of a fob1 mutant. A second copy of SIR2 was integrated at the URA3 locus of fob1Δ. Life spans were determined for W303R, and the isogenic derivatives W303R SIR2/URA3, fob1Δ, and fob1Δ SIR2/URA3. Mean life spans and the number of mothers examined were as follows: W303R 22.2 (n = 50), W303R SIR2/URA3 29.9 (n = 50), fob1Δ 29.0 (n = 50), and fob1ΔSIR2/URA3 29.4 (n = 49). (♦) W303R; (█) W303R SIR2/URA3; (▴) fob1; (×) fob1 SIR2/URA3.

Figure 8

The Sir proteins regulate longevity by reducing the rate of ERC formation by two mechanisms. The Sir2/3/4p complex acts indirectly to inhibit ERC formation by preventing the simultaneous expression of a and α mating-type genes. When expressed, the a/α cell type increases recombination in the rDNA and, presumably, the formation of ERCs. Sir2p acts by a second mechanism in the rDNA to inhibit Fob1p-mediated formation of ERCs, presumably at blocked replication forks.