1. A DNA Damage Response Pathway Controlled by Tel1 and the Mre11 Complex
Takehiko Usui, Hideyuki Ogawa, John H.J Petrini 
Molecular Cell 
Volume 7, Issue 6, Pages (June 2001)
DOI: /S (01)

2. Figure 1
Suppression of mec1 MMS Sensitivity in rad50S and sae2Δ Mutants
MMS sensitivity was assessed in the indicated strains (see Experimental Procedures). To facilitate comparison, the survival curves of wild-type, mec1 rad50S, and mec1 strains are included in each panel ([B]–[F]), represented by solid, broken, and dotted lines, respectively
Molecular Cell 2001 7, DOI: ( /S (01) )

3. Figure 2
Suppression of mec1 S/M Checkpoint Defects in rad50S and sae2Δ Mutants
(A) HU sensitivity and mitotic index in the indicated strains was assessed. Closed and open bars represent viability (left y axis) and the percentage of cells having elongated spindle (right y axis), respectively.
(B and C) The indicated strains were HU treated as above, and nuclear division was scored at the indicated times.
(D–G) Nuclear division in the indicated strains was scored by DAPI staining at the indicated times
Molecular Cell 2001 7, DOI: ( /S (01) )

4. Figure 3
Rad53 Kinase Activation in mec1 sae2Δ Mutants Requires Tel1, the Mre11 Complex, and Rad9
Rad53 activity was measured in FLAG-RAD53-expressing strains with or without ([A]) MMS or ([B]) HU treatments. FLAG-Rad53 immunoprecipitates were obtained from ([A]) asynchronous cells (Asn) and at 2.5 hr after MMS treatment (MMS). In (B), α factor-arrested cells (G1) and cells 1.5 hr after release arrest (HU) were assayed for Rad53 activity. 32P incorporation (middle panel) and FLAG-Rad53 abundance (lower panel) were quantified to determine the relative kinase activity (bar graph). Lanes 11 and 12 in (A) were from a separate gel with similar control values
Molecular Cell 2001 7, DOI: ( /S (01) )

5. Figure 4
The TM Pathway Influences Rad9 Phosphorylation and Interaction with Rad53
Western blot analyses of FLAG ([A]) and Rad9 ([B]) immunoprecipitates from extracts of MMS-treated FLAG-RAD53-expressing strains. Cell extracts were made from asynchronous cells treated without (Asn) or with 0.02% MMS for 2.5 hr (MMS). Immunoprecipitates were blotted with Rad9 antiserum (upper panel) and FLAG mAb (lower panel). The phosphorylated form of Rad9 is indicated by the asterisk
Molecular Cell 2001 7, DOI: ( /S (01) )

6. Figure 5 MMS-Induced Phosphorylation of Mre11 and Xrs2
(A) Western blot analysis of Mre11 (lanes 1–10) and Xrs2 (lanes 11 and 12) immunoprecipitates from the indicated strains. Rad50, Xrs2, and Mre11 were detected by sequentially blotting the membrane with Rad50, Xrs2, and Mre11 antisera.
(B) Lambda phosphatase treatment of Mre11 (left panel) and Xrs2 (right panel) immunoprecipitates of mec1 sae2Δ extracts with (+) and without (−) phosphatase. Extracts from asynchronous cells without (Asn) or with MMS (MMS) were analyzed. The phosphorylated forms of Mre11 or Xrs2 are indicated by asterisks. As previously observed, Xrs2 was present in multiple bands irrespective of prior treatment (Usui et al., 1998)
Molecular Cell 2001 7, DOI: ( /S (01) )

7. Figure 6 The TM Pathway Regulates the rad50S Checkpoint
(A–D) Meiotic cell cycle progression was inferred from the numbers of M I or M II cells at designated times after entering meiosis. At least 200 cells were scored for each time point.
(E) Immunolocalization of Mre11 on chromosome spreads prepared from nuclei of the indicated diploid strains at 4 hr after entering meiosis. Upper and lower panels of each strain are images from the same region with DAPI and FITC (Mre11) staining. Bar equals 5 μm. Exposure of the FITC image of the mre11-58 rad50S was about four times longer than those of the other two strains
Molecular Cell 2001 7, DOI: ( /S (01) )

8. Figure 7 Parallel Pathways in the DNA Damage Response
A model for the TM pathway is presented, relying in part on the assumption that meiotic and mitotic functions are similar. Given its role in the rad50S checkpoint, we propose that the TM pathway primarily responds to unprocessed DSBs via the sensing functions of the Mre11 complex, whereas the Rad24–Mec1 pathway, presented here in a simplified form, can be activated by either lesion. The suppression of mec1 defects suggests that the TM pathway is activated by a lesion accumulating in rad50S or sae2Δ, as indicated. Once activated, both pathways can converge on Rad53 and Rad9 in mitotic cells, and Mre4/Mek1 and Rad9 in meiosis. Phosphorylation of Rad9 is required for its interaction with the FHA domain of Rad53, which, like Rad9, is phosphorylated in a Mec1-Tel1-dependent manner (Pellicioli et al., 1999; Sun et al., 1998). Rad9 Rad53 interaction leads to the activation of downstream checkpoint functions, including cell cycle arrest and the activation of transcriptional programs. Given the requirement for Rad9 in the rad50S checkpoint, we speculate here that Mre4, like Rad53 in mitotic cells, interacts with Rad9 in the course of TM checkpoint activation. A Rad53-independent outcome of the TM checkpoint is its influence on the repair functions of the Mre11 complex via Tel1-dependent phosphorylation. An analogous effect on recombinational DNA repair appears to be affected by Mec1-dependent, Rad53-dependent phosphorylation of Rad55 (Bashkirov et al., 2000)
Molecular Cell 2001 7, DOI: ( /S (01) )