The fission yeast Rad32 (Mre11)-Rad50-Nbs1 complex is required for the S-phase DNA damage checkpoint

Abstract

Mre11, Rad50, and Nbs1 form a conserved heterotrimeric complex that is involved in recombination and DNA damage checkpoints. Mutations in this complex disrupt the S-phase DNA damage checkpoint, the checkpoint which slows replication in response to DNA damage, and cause chromosome instability and cancer in humans. However, how these proteins function and specifically where they act in the checkpoint signaling pathway remain crucial questions. We identified fission yeast Nbs1 by using a comparative genomic approach and showed that the genes for human Nbs1 and fission yeast Nbs1 and that for their budding yeast counterpart, Xrs2, are members of an evolutionarily related but rapidly diverging gene family. Fission yeast Nbs1, Rad32 (the homolog of Mre11), and Rad50 are involved in DNA damage repair, telomere regulation, and the S-phase DNA damage checkpoint. However, they are not required for G(2) DNA damage checkpoint. Our results suggest that a complex of Rad32, Rad50, and Nbs1 acts specifically in the S-phase branch of the DNA damage checkpoint and is not involved in general DNA damage recognition or signaling.

FIG. 1.

Alignment of the Nbs1/Xrs2 homologs. (A) Graphic representation of the sequence similarity between the Nbs1/Xrs2 homologs. Percent identity is shown for regions identified as significantly similar by BLASTP. The FHA and BRCT domains recognized by CDD are depicted. The putative S. pombe BRCT domain is in gray because although it is similar to the human BRCT domain, it is not recognized by either the CDD or ProfileScan algorithms. (B) Phylogenetic tree of the Nbs1/Xrs2 homologs. The branch lengths represent relative phylogenetic distances, as determined by ClustalX (28). (C) Alignment of the FHA domain, the BRCT domain, and a conserved carboxy-terminal motif. Residues identical in any two sequences are boxed; residues similar a majority of the sequences are shaded. The signature motifs of a BRCT domain are underlined. The conserved carboxy-terminal motif is contained within a region that has been implicated in binding to Mre11 (65).

FIG. 2.

Nbs1 is required for DNA damage resistance. (A) Sensitivity of nbs1Δ (CC3223), rad32Δ (TMN2799), rad50Δ (NR2841), rad32Δ nbs1Δ (CC3227), rad50Δ nbs1Δ (CC3228), and rad50Δ rad32Δ nbs1Δ (CC3229) cells to ionizing radiation. (B) Sensitivity of nbs1Δ (CC3223), rad32Δ (TMN2799), rad50Δ (NR2841), rad32Δ nbs1Δ (CC3227), rad50Δ nbs1Δ (CC3228), and rad50Δ rad32Δ nbs1Δ (CC3229) cells to MMS.

FIG. 3.

Nbs1 is involved in telomere-length maintenance. (A) Telomere length in wild-type (wt) (TMN2665), rad32Δ (TMN2799), rad50Δ (NR2840), nbs1Δ (TMN3224), rad32Δ nbs1Δ (CC3227), rad32Δ rad50Δ nbs1Δ (CC3229), rad3Δ (TMN2937), rad3Δ rad32Δ (TMN2994), rad3Δ rad50Δ (CC3233), rad3Δ nbs1Δ (CC3230), tel1Δ (TMN2967), rad3Δ tel1Δ (TMN3052) and tel1Δ nbs1Δ (CC3231) cells. A Southern blot of ApaI-digested S. pombe chromosomal DNA was hybridized to telomere-specific probes (44). The ApaI site is located in the telomere-associated sequence (TAS) 30 to 40 bp away from telomeric repeat sequences in both ends of chromosomes I and II and at least one end of chromosome III, giving rise to a broad ∼300-bp telomere hybridization signal in the wt strain (Telomeres). Hybridization signals (TAS & rDNA adjacent telomeres) come from cross-hybridization to TAS or hybridization to telomere(s) of chromosome III which contain rDNA repeats directly adjacent to the telomeric repeat sequence and therefore lack the TAS-associated ApaI site directly adjacent to the telomeric repeat sequence. (B) NotI restriction enzyme map of S. pombe chromosomes (vertical lines). The telomeric fragments C, I, L, and M are filled black. Chromosome III lacks a NotI site. (C) Chromosome structure in wt (TMN2665), trt1 (TMN2669), rad32 (TMN2799), tel1 (TMN2967), nbs1 (TMN3224), rad3 tel1 (TMN3052), rad3 rad32 (TMN2994), rad3 rad50 (CC3233), and rad3 nbs1 (CC3230) cells. A pulsed-field gel Southern blot of NotI-digested S. pombe chromosomal DNA was hybridized to C-, I-, L-, and M-specific probes (43). Four telomeric fragments (C, I, L, and M) and fusion products (C+M and I+L) are marked.

FIG. 4.

The Nbs1 and Rad32 associate in vivo. Protein A affinity (TAP)-tagged Nbs1 was precipitated with IgG Sepharose from a soluble nbs1-TAP rad32-13Myc (CC3235) cell lysate. The IgG beads were then washed four times with binding buffer. Protein from the cell lysate, protein from the first and last washes, and protein remaining on the IgG beads was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and probed with anti-Myc antibodies to visualize Rad32-13Myc. Rad32-13Myc coprecipitated with the IgG-bound Nbs1-TAP; insignificant Rad32-13Myc precipitated in the absence of Nbs1-TAP.

FIG. 5.

The MRN proteins are required for the S-phase DNA damage checkpoint but not the G₂ DNA damage checkpoint. (A) Fluorescence-activated cell sorter (FACS) analysis of S-phase DNA damage checkpoint in cdc10-M17ts (EN2682), rad3Δ cdc10-M17ts (yFS260), and rad32Δ cdc10-M17ts (yFS263) cells. Synchronous G₁ cultures were released from a G₁ block in the presence or absence of 0.03% MMS. Progression through the S phase was determined by FACS. (B) Quantitation of the data in shown in panel A and data from rad50Δ cdc10-M17ts (yFS265) and nbs1Δ cdc10-M17ts (yFS267) cells. The percentage of progress through the S phase was calculated as the position of the mean of the FACS signal between the means of the 1C and 2C controls. (C) FACS analysis of S-phase DNA damage checkpoint in wild-type (PR109) and rad32Δ(TMN2799) cells. Elutriation-synchronized G₂ cultures were treated with 0.01% MMS, a dose that does not activate the G₂ DNA damage checkpoint but does activate the S-phase DNA damage checkpoint, and followed through mitosis and the S phase by FACS. For each time point, a separate set of FACS results is shown: the bold line represents the DNA content of the MMS-treated cells, the right-hand gray peak is a control consisting of the same time point of a parallel untreated culture, and the left-hand gray peak is a 1C control consisting of the 140-min sample of a parallel hydroxyurea-treated culture. (D) Division kinetics of the cells shown in panel C as determined by microscopically monitoring cell septation. (E) The response of wild-type (PR109), rad3Δ (NR1826), rad32Δ (TMN2799), rad50Δ (NR2841), and nbs1Δ (CC3223) cells to ionizing radiation during G₂. Elutriation-synchronized G₂ cultures were X-ray irradiated, and cells completing mitosis were identified by microscopically monitoring cell septation.