Protection of telomeres by the Ku protein in fission yeast

Abstract

Schizosaccharomyces pombe cells survive loss of telomeres by a unique pathway of chromosome circularization. Factors potentially involved in this survival mechanism include the heterodimeric Ku protein and ligase IV, both of which are involved in the repair of DNA double-strand breaks in mammalian cells. Furthermore, Ku plays a role in telomere maintenance as well as in DNA double-strand break repair in Saccharomyces cerevisiae. We have identified Ku and ligase IV homologues in S. pombe and analyzed their functions during normal growth and in cells undergoing senescence. In the absence of either a Ku subunit (pku70(+)) or ligase IV (lig4(+)), nonhomologous DNA end-joining was severely reduced. Lack of functional Ku led to shorter but stable telomeres and caused striking rearrangements of telomere-associated sequences, indicating a function for Ku in inhibiting recombinational activities near chromosome ends. In contrast to S. cerevisiae, concurrent deletion of pku70(+) and the gene for the catalytic subunit of telomerase (trt1(+)) was not lethal, allowing for the first time the dissection of the roles of Ku during senescence. Our results support a model in which Ku protects chromosome termini from nucleolytic and recombinational activities but is not involved in the formation of chromosome end fusions during senescence. The conclusion that nonhomologous end-joining is not required for chromosome circularization was further supported by analysis of survivors in strains lacking the genes for both trt1(+) and lig4(+).

Figure 1

(A) Double-strand break repair defect in pku70⁻ and lig4⁻ strains. For each strain, the efficiency of repair is expressed as the number of transformants obtained with linear plasmid divided by the number of transformants obtained with circular plasmid. Numbers of colonies for a typical experiment were as follows: 1012 wild-type circular; 127 wild-type linear; 1160 pku70⁻ circular; 12 pku70⁻ linear; 1560 lig4⁻ circular; and 22 lig4⁻ linear. (B) Senescence and generation of survivors in the absence of telomerase and pku70⁺. A diploid strain heterozygous for deletions of pku70⁺ and trt1⁺ was sporulated, and the tetrads were dissected and germinated on YES plates. The resulting colonies were used to inoculate precultures in YES liquid medium. Growth curves were recorded as described in MATERIALS AND METHODS. Day 1 on the x axis corresponds to ∼30 generations after germination. The cell density on each day is plotted for wild type (▴), pku70⁻ trt1⁺ (▪ and □), pku70⁺ trt1⁻ (♦), and pku70⁻ trt1⁻ (● and ○). (C) As in B except that the diploid starter strain was heterozygous for deletions of lig4⁺ and trt1⁺. The cell density on each day is plotted for wild type (▴), lig4⁻ trt1⁺ (▪ and □), lig4⁺ trt1⁻ (♦), and lig4⁻ trt1⁻ (● and ○).

Figure 2

Telomere length in wild-type and pku70⁻ strains. Genomic DNA of pku70⁺ trt1⁺ and pku70⁻ trt1⁺ strains was prepared on the indicated days of monitored growth in liquid culture (lanes a–e) or after three and seven restreaks on plates (lanes f–h). A total of 15 μg of DNA from each sample was digested with EcoRI and subjected to agarose gel electrophoresis and Southern transfer onto a nylon membrane. The blot was subsequently probed with a ³²P-labeled telomeric fragment. As a loading control, a ³²P-labeled fragment of the single-copy pol1⁺ gene was included in the hybridization mix. Size markers were 100-bp and 1-kb ladders from New England Biolabs.

Figure 3

Stability of TAS. (A) Restriction enzyme sites in the telomeric and telomere-associated sequences of one chromosome arm cloned in the plasmid pNSU70 (Sugawara, 1988). Locations of the probes used for Southern blotting are indicated by the bottom bars. Because of extensive homology between the TAS on different chromosomes, these probes hybridize to subtelomeric fragments on all six chromosome arms. (B and C) Genomic DNA (15 μg) was prepared from cells grown in liquid culture for the indicated number of days (see Figure 1), digested with NsiI, and fractionated on agarose gels. DNA was transferred onto a nylon membrane and hybridized to a ³²P-labeled TAS1 probe. Size markers are as in Figure 2.

Figure 4

Telomere-shortening phenotype of trt1⁻ and pku70⁻ trt1⁻ strains. Sister strains obtained by tetrad dissection of a trt1⁺/trt1⁻ pku70⁺/pku70⁻ strain were grown in liquid culture. Genomic DNA was prepared on the indicated days, and 20 μg of each sample was digested with EcoRI. DNA was transferred onto a nylon membrane and hybridized with a ³²P-labeled telomeric fragment.

Figure 5

Dynamic rearrangements of TAS in survivors of senescence. (A) Genomic DNA (15 μg) was prepared from trt1⁻ and pku70⁻ trt1⁻ cultures on the indicated days, digested with NsiI, and fractionated on agarose gels. DNA was transferred onto a nylon membrane and hybridized to a ³²P-labeled TAS1 probe. Size markers are as in Figure 2. (B) The blot shown in A was stripped as described in MATERIALS AND METHODS and hybridized to a TAS3 probe.

Figure 6

Chromosome circularization in trt1⁻ and pku70⁻ trt1⁻ strains. (A) Scheme of NotI restriction sites in S. pombe chromosomes. The terminal fragments on chromosomes I and II are shown in gray. Chromosome III lacks a NotI restriction site. (B and C) Pulsed-field gel analysis of NotI-digested genomic DNA from the same trt1⁻ and pku70⁻ trt1⁻ strains used in Figure 5. The gel was run and processed as described in MATERIALS AND METHODS. DNA was transferred onto a nylon membrane and hybridized to internal probes on the I, L, M, and C fragments. The terminal fragments of linear chromosomes I and II are indicated on the left, and the fragments resulting from chromosome circularization (I+L and C+M) are indicated on the right.

Figure 7

Chromosome dynamics in survivors. (A) Intact S. pombe chromosomal DNA was prepared from wild-type and trt1⁻ strains and fractionated by pulsed-field gel electrophoresis. DNA was stained with ethidium bromide. (B) Viability of survivors. Cells were grown in liquid culture and counted with the use of a hemacytometer, and the equivalent of 500 cells were plated in triplicate for each strain. Colonies were counted after 2.5 d. (C) NotI-digested chromosomal DNA was fractionated by pulsed-field gel electrophoresis and stained with ethidium bromide (lanes a–f). The DNA was then transferred onto a nylon membrane that was sequentially hybridized with a probe specific for the K fragment on chromosome I (lanes g–l) and probes specific for the I and L fragments on chromosome I (lanes m–r). The K fragment is an internal restriction fragment on chromosome I and is indicated by an open circle next to lane g.