Requirement of the Mre11 complex and exonuclease 1 for activation of the Mec1 signaling pathway

Abstract

The large protein kinases, ataxia-telangiectasia mutated (ATM) and ATM-Rad3-related (ATR), orchestrate DNA damage checkpoint pathways. In budding yeast, ATM and ATR homologs are encoded by TEL1 and MEC1, respectively. The Mre11 complex consists of two highly related proteins, Mre11 and Rad50, and a third protein, Xrs2 in budding yeast or Nbs1 in mammals. The Mre11 complex controls the ATM/Tel1 signaling pathway in response to double-strand break (DSB) induction. We show here that the Mre11 complex functions together with exonuclease 1 (Exo1) in activation of the Mec1 signaling pathway after DNA damage and replication block. Mec1 controls the checkpoint responses following UV irradiation as well as DSB induction. Correspondingly, the Mre11 complex and Exo1 play an overlapping role in activation of DSB- and UV-induced checkpoints. The Mre11 complex and Exo1 collaborate in producing long single-stranded DNA (ssDNA) tails at DSB ends and promote Mec1 association with the DSBs. The Ddc1-Mec3-Rad17 complex associates with sites of DNA damage and modulates the Mec1 signaling pathway. However, Ddc1 association with DSBs does not require the function of the Mre11 complex and Exo1. Mec1 controls checkpoint responses to stalled DNA replication as well. Accordingly, the Mre11 complex and Exo1 contribute to activation of the replication checkpoint pathway. Our results provide a model in which the Mre11 complex and Exo1 cooperate in generating long ssDNA tracts and thereby facilitate Mec1 association with sites of DNA damage or replication block.

FIG. 1.

Effect of mec1Δ, tel1Δ, or xrs2 mutations on phleomycin-induced Rad53 phosphorylation. Cells carrying YCpT-RAD53-HA were arrested at G₂/M with nocodazole, and untreated (−) or treated (+) with phleomycin, maintaining the G₂/M arrest for 60 min. Cells were then collected and subjected to immunoblotting analysis with anti-HA antibodies. Strains used are wild type (KSC1560), mec1Δ (KSC1561), tel1Δ (KSC1661), xrs2Δ (KSC1562), and xrs2-11 (KSC1563). All the strains contain an sml1Δ mutation that suppresses the lethality of mec1Δ mutants (63).

FIG. 2.

Overlapping function of the Mre11 complex and Exo1 in response to phleomycin-induced DNA damage. (A) Effect of mre11Δ, xrs2Δ, and exo1Δ mutations on cell viability after exposure to phleomycin. Cells were grown in log phase and incubated with phleomycin. At the indicated times, aliquots of cells were collected and the viability was estimated. Strains used are wild type (KSC1516), mre11Δ (KSC1933), xrs2Δ (KSC1562), exo1Δ (KSC1846), mre11Δ exo1Δ (KSC1980), and xrs2Δ exo1Δ (KSC1979). (B) Effect of mre11Δ, xrs2Δ, and exo1Δ mutations on phleomycin-induced Rad53 phosphorylation at G₂/M. Cells carrying YCpT-RAD53-HA were arrested with nocodazole and incubated with phleomycin maintaining the arrest. Cells were then collected at the indicated time and analyzed as in Fig. 1 to detect Rad53 phosphorylation. Strains used are the same as in panel A. (C) Effect of mre11Δ, xrs2Δ, and exo1Δ mutations on phleomycin-inducedChk1 phosphorylation at G₂/M. Cells carrying YCpT-CHK1-HA were analyzed as in panel B to detect Chk1 phosphorylation. Strains used are wild type (KSC1516), xrs2Δ (KSC1562), exo1Δ (KSC1846), and xrs2Δ exo1Δ (KSC1979). (D) Cell cycle progression delay after phleomycin treatment in G₂/M phase. Cells were synchronized with nocodazole at G₂/M and then treated with phleomycin (+PM) or untreated (−PM). At the indicated times after release from nocodazole, the percentage of uninucleate large-budded cells was scored after DAPI staining. Strains used are wild type (KSC1516), xrs2Δ (KSC1562), exo1Δ (KSC1846), xrs2Δ exo1Δ (KSC1979), sml1Δ (KSC1560), and mec1Δ sml1Δ (KSC1561). (E) Effect of mre11Δ, xrs2Δ, and exo1Δ mutations on phleomycin-induced Rad53 phosphorylation at G₁. Cells carrying YCpT-RAD53-HA were arrested with α-factor and incubated with phleomycin maintaining the arrest. Cells were then collected at the indicated time and analyzed as in panel B. Strains used are the same as in panel C.

FIG. 3.

Effect of xrs2Δ and exo1Δ mutations on cellular responses to DSBs. (A) Schematic of the HO cleavage site at the ADH4 locus (ADH4cs). An HO cleavage site, marked with HIS2, was introduced at the ADH4 locus on chromosome VII. The primer pairs were designed to amplify regions 1 and 2 kb apart from the HO cleavage site. An arrow represents the telomere. The black and gray bars indicate probes to examine the rate of degradation of the DSB ends (38). (B) Rad53 phosphorylation in response to HO-induced DSBs. 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. Aliquots were harvested at the indicated time points and analyzed as in Fig. 1. Strains used are wild type (KSC1516), exo1Δ (KSC1846), xrs2Δ (KSC1562), and xrs2Δ exo1Δ (KSC1979). (C) Degradation of the HO-induced DSB ends. Wild-type (KSC1516), exo1Δ (KSC1846), xrs2Δ (KSC1562), and xrs2Δ exo1Δ (KSC1979) cells carrying YCpA-GAL-HO were treated as in panel B. Purified DNAs were fixed to a membrane and hybridized with RNA probes, each complementary to the 5′- to 3′- or 3′- to 5′-degrading strand.

FIG. 4.

Effect of xrs2Δ and exo1Δ mutations on association of Mec1 and Ddc1 with the HO-induced DSB. (A) Association of Mec1 with DSBs. Cells expressing HA-tagged Mec1 were transformed with 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 carried out with the primers for the HO cleavage site at the ADH4 locus and for the control SMC2 locus (see Fig. 3A). PCR products from the respective input extracts are shown below. Strains used here are wild type (KSC1512), exo1Δ (KSC1633), xrs2Δ (KSC1632), and xrs2Δ exo1Δ (KSC1634). (B) Association of Ddc1 with the HO-induced DSB. Cells expressing Ddc1-HA were analyzed as in panel A. Strains used here are wild type (KSC1637), xrs2Δ (KSC1638), exo1Δ (KSC1639), and xrs2Δ exo1Δ (KSC1640).

FIG. 5.

Effect of rfa1-t11 mutation on DSB association of Mec1 and Ddc1 in xrs2Δ mutants. (A) Association of Mec1 with DSBs. Cells expressing Mec1-HA were analyzed as in Fig. 4A. Strains used here are wild type (KSC1512), rfa1-t11 (KSC1984), and rfa1-t11 xrs2Δ (KSC1985). (B) Association of Ddc1 with the HO-induced DSB. Cells expressing Ddc1-HA were analyzed as in panel A. Strains used here are wild type (KSC1637), rfa1-t11 (KSC1986), and rfa1-t11 xrs2Δ (KSC1987). (C) Degradation of the HO-induced DSB ends. Wild-type (KSC1516), rfa1-t11 (KSC1982), and rfa1-t11 xrs2Δ (KSC1983) cells carrying YCpA-GAL-HO were analyzed as in Fig. 3C.

FIG. 6.

Redundant role of Xrs2 and Exo1 in cellular responses to UV irradiation. (A) Cell viability after exposure to UV light. Viability was determined after UV irradiation at the indicated dosages. Strains used are the same as in Fig. 2C. (B) Rad53 phosphorylation after UV irradiation at G₂/M. The same cells as in panel A were transformed with YCpT-RAD53-HA. Transformed cells were grown in log phaseand treated with nocodazole. After arrest at G₂/M, the culture was irradiated with UV light. Cells were harvested at the indicated times and analyzed as in Fig. 1. (C) Chk1 phosphorylation after UV irradiation at G₂/M. The same cells as in panel A were transformed with YCpT-CHK1-HA and analyzed as in panel B. (D) G₂/M-phase cell cycle progression after exposure to UV light. Cells were grown in YEPD and arrested with nocodazole. After synchronization at G₂/M, cells were irradiated with UV (+UV) or mock-treated (−UV). At the indicated times after release of cultures from nocodazole, the percentage of uninucleate large-budded cells was scored after DAPI staining. Strains used are the same as in Fig. 2D. (E) Rad53 phosphorylation after UV irradiation at G₁. Cells carrying YCpT-RAD53-HA were treated with α-factor. After arrest at G₁, the culture was irradiated with UV light. Cells were harvested at the indicated times and analyzed as in panel B. Strains used are the same as in panel A.

FIG. 7.

Effects of xrs2Δ and exo1Δ mutations on checkpoint responses to DNA replication block. (A) Rad53 phosphorylation after HU treatment. Cells were transformed with YCpT-RAD53-HA. Transformed cells were grown in log phase and treated with α-factor. After arrest at G₁, cells were released into medium with HU (+) or without HU (−). Cells were harvested at the indicated times and analyzed as in Fig. 1. Strains used are the same as in Fig. 2C. (B) Mitotic entry after exposure to HU. Cells were grown in YEPD and arrested with α-factor at G₁. Cells were then released into medium with or without HU. At the indicated times after release, the percentage of cells with elongated spindles was scored after staining with DAPI and antitubulin antibodies. Strains used are the same as in Fig. 2D.