ATM-related Tel1 associates with double-strand breaks through an Xrs2-dependent mechanism

Abstract

In budding yeast, TEL1 encodes a protein closely related to ATM. Xrs2 is an Nbs1 homolog and forms a complex with Mre11 and Rad50. We show here that Tel1 associates with double-strand breaks (DSBs) through a mechanism dependent on the C terminus of Xrs2. Although Xrs2 is required for the DNA degradation at DSBs, the C-terminal Xrs2 truncation does not affect the degradation. Tel1 and the C terminus of Xrs2 are similarly involved in cell survival and Rad53 phosphorylation after DNA damage. Our findings suggest that the Tel1 association with DNA lesions is required for the activation of DNA damage responses.

Figure 1.

Effect of the C-terminal deletion of Xrs2 on DNA sensitivity and the complex formation. (A,B) Sensitivity to phleomycin (A) and UV light (B). Serial dilutions of cultures were spotted on YEPD media without or with phleomycin. Cultures spotted on YEPD media were irradiated with UV light. All of the strains contain the sml1Δ mutation that suppresses the lethality of mec1 mutants (Zhao et al. 1998). Strains used were wild-type (KSC1560), xrs2Δ (KSC1562), xrs2-11 (KSC1563), tel1Δ (KSC1661), mec1-81 (KSC1662), mec1-81 tel1Δ (KSC1564), mec1-81 xrs2-11 (KSC1565), and mec1-81 tel1Δ xrs2-11 (KSC1566). (C) Interaction between Tel1and Xrs2. Cells were untreated (–) or treated with 50 μg/mL phleomycin for 1h (+). Extracts were prepared from cells and immunoprecipitated with anti-HA antibodies. Immunoprecipitates (IP) and whole extracts were subjected to immunoblotting analysis. Strains used were XRS2-myc (KSC1904), xrs2-11-myc (KSC1905), TEL1-HA (KSC1785), TEL1-HA XRS2-myc (KSC1906), and TEL1-HA xrs2-11-myc (KSC1907). (D) Interaction of Xrs2 with Mre11 and Rad50. Extracts were prepared from XRS2-HA (KSC1744), xrs2-11-HA (KSC1869), or untagged cells (KSC1516), and immunoprecipitated with anti-HA antibodies. IPs and whole extracts were subjected to immunoblotting analysis. Phosphatase treatment diminished the slower migrating forms of Xrs2-HA and Xrs2-11-HA (data not shown), suggesting that Xrs2 and Xrs2-11 are phosphorylated proteins.

Figure 2.

Effect of the C-terminal deletion of Xrs2 on the cellular response to DSBs. (A) A single HO restriction site at the ADH4 locus in MATa-inc cells. A solid box indicates a DNA fragment containing an HO cleavage site, which is marked with HIS2. The black and gray bars indicate probes to examine the rate of degradation of the DSB ends. The primer pairs HO1and HO2 were designed to amplify regions near the cleavage site on the ADH4 locus by PCR for the experiments described in Figures 3 and 4. An arrow represents telomere. The MATa locus was replaced with the MATa-inc allele in which the HO endonuclease does not generate a DSB. (B) Degradation of the HO-induced DSB ends. Wild-type (KSC1516), xrs2Δ (KSC1620), and xrs2-11 (KSC1621) cells carrying YCpA-GAL-HO were grown in sucrose and treated with nocodazole. After arrest at G2/M, the culture was incubated with galactose to induce HO expression. At the indicated time points, aliquots were harvested for DNA preparation. Purified DNAs were fixed to a membrane and probed with ³²P-labeled oligonucleotides, each complementary to a 5′-to-3′-degrading or 3′-to-5′-degrading strand as in A. (C) Rad53 phosphorylation after HO expression. Cells carrying YCpT-RAD53-HA and YCpA-GAL-HO were grown in sucrose and treated with nocodazole. After arrest at G2/M, the culture was incubated with galactose to induce HO expression, while part of the culture was maintained in sucrose to repress HO expression. At the indicated time points, aliquots were harvested for immunoblotting analysis. Cells used were wild-type (KSC1560), mec1Δ (KSC1561), tel1Δ (KSC1661), sae2Δ (KSC1567), mec1Δ sae2Δ (KSC1701), and tel1Δ sae2Δ (KSC1594). (D) Effect of the tel1Δ and xrs2-11 mutations on the HO-induced Rad53 phosphorylation. Cells were analyzed as in C to detect Rad53 phosphorylation. Tubulin was detected as a loading control. Cells used were sae2Δ (KSC1567), mec1-81 sae2Δ (KSC1775), tel1Δ sae2Δ (KSC1594), xrs2-11 sae2Δ (KSC1595), mec1-81 xrs2-11 sae2Δ (KSC1597), mec1-81 tel1Δ sae2Δ (KSC1596), and mec1Δ sae2Δ (KSC1701).

Figure 3.

Association of Tel1with sites near the HO-induced DSB. (A) The Tel1association with DSBs in SAE2 and sae2Δ cells. Cells expressing Tel1-HA (KSC1785 and KSC1786) were transformed with the YCpA-GAL-HO plasmid. Transformed cells were grown in sucrose and treated with nocodazole. After arrest at G2/M, the culture was incubated with galactose to induce HO expression, while part of the culture was maintained in sucrose to repress HO expression. Aliquots of cells were collected at the indicated times and subjected to chromatin immunoprecipitation. PCR was done with the primers for the ADH4 locus shown schematically in Figure 2A and for the control SMC2 locus. PCR products from the respective input extracts are shown in parallel. (B) Effect of the sae2Δ mutation on Tel1 association with DSBs in mec1Δ mutants. Cells expressing Tel1-HA (KSC1873, KSC1874 and KSC1875) were analyzed as in A. (C) Effect of xrs2Δ and xrs2-11 mutations on Tel1association with the DSBs. Cells expressing Tel1-HA (KSC1785, KSC1881 and KSC1882) were analyzed as in the top two panels of B. Immunoprecipitates were also subjected to immunoblotting analysis with anti-HA antibodies (bottom).

Figure 4.

Association of Xrs2 with sites near the HO-induced DSB. (A) Time course of the Xrs2 association with DSBs. Cells expressing Xrs2-HA (KSC1744) were analyzed as in Figure 3A. (B) Effect of mec1Δ, tel1Δ and sae2Δ mutations on the Xrs2 association with DSBs. Cells expressing Xrs2-HA were analyzed as in Figure 3B. Cells used were wild-type (KSC1744), tel1Δ (KSC1799), sae2Δ (KSC1798), sml1Δ (KSC1863), and mec1Δ sml1Δ (KSC1864). (C) Effect of the C-terminal truncation on the Xrs2 association with DSBs. XRS2-HA (KSC1744) and xrs2-11-HA (KSC1869) cells were analyzed as in B.