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1.
FIG. 5.

FIG. 5. From: Mre11 Nuclease Activity and Ctp1 Regulate Chk1 Activation by Rad3ATR and Tel1ATM Checkpoint Kinases at Double-Strand Breaks .

Mre11 endonuclease activity is required for Tel1 signaling to Chk1. (A) Deletion of Mre11 abolishes Chk1 phosphorylation in the ctp1Δ rad3Δ background. (B) The nuclease dead allele of Mre11 (mre11-H134S) abolishes Chk1 phosphorylation in the ctp1Δ rad3Δ background. (C) Mre11 nuclease activity is required for the positive genetic interactions between ctp1Δ and rad3Δ. (D) IR triggers histone H2A phosphorylation in mre11-H134S rad3Δ cells, indicating that phosphorylation of histone H2A by Tel1 does not require Mre11 nuclease activity. (E) Nuclease dead mre11-D65N also abolishes Chk1 phosphorylation in the ctp1Δ rad3Δ background. (F) Tel1 phosphorylates histone H2A in a mre11-D65N background.

Oliver Limbo, et al. Mol Cell Biol. 2011 February;31(3):573-583.
2.
FIG. 4.

FIG. 4. From: Mre11 Nuclease Activity and Ctp1 Regulate Chk1 Activation by Rad3ATR and Tel1ATM Checkpoint Kinases at Double-Strand Breaks .

Tel1 activation of Chk1 requires the 9-1-1 checkpoint clamp and Crb2. (A) Hus1 is required for Chk1 phosphorylation in the ctp1Δ rad3Δ background. (B) Deletion of Hus1 abolishes rescue in the ctp1Δ rad3Δ background. (C) Crb2 is required for Chk1 phosphorylation in the ctp1Δ rad3Δ background. The crb2-T215A and H2A-AQ mutations reduce but do not eliminate Chk1 phosphorylation in the ctp1Δ rad3Δ background. (D) Quantitation of Chk1 phosphorylation. Error bars represent the standard errors from three independent experiments. (E) Both pathways of Crb2 recruitment contribute to the rescue in the ctp1Δ rad3Δ background. Deletion of Crb2 abolishes this rescue. (F) IR survival assays. (G) UV survival assays.

Oliver Limbo, et al. Mol Cell Biol. 2011 February;31(3):573-583.
3.
FIG. 6.

FIG. 6. From: Mre11 Nuclease Activity and Ctp1 Regulate Chk1 Activation by Rad3ATR and Tel1ATM Checkpoint Kinases at Double-Strand Breaks .

Biphasic DNA damage checkpoint. DNA damage checkpoint signaling at DSBs is depicted. The MRN complex recruits Tel1 to DSBs. Tel1 phosphorylates histone H2A. Tel1 can phosphorylate Chk1 in a pre-resection phase of the DNA damage checkpoint. Mre11 endonuclease activity is required for phosphorylation of Chk1 but not histone H2A, indicating that Mre11 endonuclease activity is required for recruitment of additional checkpoint factors needed for Chk1 phosphorylation. These factors are likely the 9-1-1 complex. Recruitment of Ctp1 through its interaction with Nbs1 leads to resection and disengagement of the MRN complex, Ctp1, and Tel1 from the resected DNA ends. RPA loading on ssDNA leads to Rad3-Rad26 recruitment and Rad3-dependent activation of Chk1.

Oliver Limbo, et al. Mol Cell Biol. 2011 February;31(3):573-583.
4.
FIG. 3.

FIG. 3. From: Mre11 Nuclease Activity and Ctp1 Regulate Chk1 Activation by Rad3ATR and Tel1ATM Checkpoint Kinases at Double-Strand Breaks .

Duration of Mre11 binding to DSBs is regulated by Ctp1. (A) Locations of the PCR primer pairs used for amplifying DNA regions adjacent to the HO cleavage site in chromosome I. (B) ChIP analysis of Mre11-TAP in ctp1+ and ctp1Δ backgrounds. Mre11 binding close to the DSB (0.2-kb probe) is weak and transient in ctp1+ cells, whereas it is strong and persistent in ctp1Δ cells. Expression of the HO endonuclease from the nmt41 promoter initiates ca. 16 to 20 h after the shift to thiamine-free (−B1) media.

Oliver Limbo, et al. Mol Cell Biol. 2011 February;31(3):573-583.
5.
FIG. 2.

FIG. 2. From: Mre11 Nuclease Activity and Ctp1 Regulate Chk1 Activation by Rad3ATR and Tel1ATM Checkpoint Kinases at Double-Strand Breaks .

Tel1 is required for the Chk1 activation in ctp1Δ rad3Δ cells. (A) Positive genetic interactions between ctp1Δ and rad3Δ mutations. Double-mutant ctp1Δ rad3Δ cells are more resistant to DNA damage than the most sensitive single mutant. These assays also show that ctp1Δ and nbs1Δ mutations have different effects in a rad3Δ background. Fivefold serial dilutions of exponentially growing cells were plated and exposed the indicated agents. (B) Chk1 is required for the positive genetic interactions between ctp1Δ and rad3Δ mutations that confer genotoxin resistance (IR, CPT, and UV). Cds1 is also required for CPT resistance. (C) Suppression of rad3Δ UV sensitivity by ctp1Δ requires Chk1. Exponentially growing cells were plated in triplicate and exposed to the indicated dose of UV. The resulting colonies were compared relative to an untreated sample. (D) Chk1 activation in ctp1Δ rad3Δ cells requires Tel1. (E) Tel1 expression from a plasmid restores Chk1 phosphorylation in ctp1Δ rad3Δ tel1Δ cells.

Oliver Limbo, et al. Mol Cell Biol. 2011 February;31(3):573-583.
6.
FIG. 1.

FIG. 1. From: Mre11 Nuclease Activity and Ctp1 Regulate Chk1 Activation by Rad3ATR and Tel1ATM Checkpoint Kinases at Double-Strand Breaks .

Deletion of Ctp1 restores the DNA damage checkpoint in rad3Δ cells. (A) Regulatory connections between ATM/ATR and Chk1/Chk2 orthologs in mammals, S. cerevisiae, and S. pombe. ATM phosphorylates Chk2 and ATR phosphorylates Chk1. CtIP mediates an ATM-to-ATR switch through DNA end resection in mammals (44, 53). ATM promotes Chk1 activation by stimulating CtIP-dependent resection through an unknown mechanism. In S. cerevisiae, Mec1 phosphorylates both Rad53 and Chk1. Deleting Sae2 uncovers a Tel1-to-Rad53 signaling pathway and enhances Rad53 activation (47). In S. pombe, Cds1 and Chk1 activation is dependent on Rad3. (B) Chk1 phosphorylation peaks in wild-type (wt) (top panel) and ctp1Δ cells (bottom panel) 30 min after exposure to 90 Gy of IR in log-phase cultures. Chk1 phosphorylation in ctp1Δ cells prior to IR exposure likely arises from an inability to repair spontaneous DNA damage (23). Immunoblots were probed for the HA epitope-tagged Chk1 or Cdc2 as a loading control. (C) Chk1 phosphorylation is reduced at least 2-fold in ctp1Δ cells relative to the wild type. Quantification of blots from panel B expressed as a ratio of phospho-Chk1 (upper band) versus nonphospho-Chk1 (lower band) was performed. The phospho-Chk1 signal in untreated ctp1Δ cells was subtracted from the IR-treated samples to more accurately measure the IR-induced phosphorylation. (D) The ctp1Δ mutation restores Chk1 phosphorylation in rad3Δ cells. Cells were harvested immediately after mock or 90-Gy IR treatment and blotted for HA epitope tag. Ponceau staining shows equal loading. (E) Quantitation of Chk1 phosphorylation. Error bars represent the standard errors from three independent experiments. (F) The checkpoint arrest is restored in ctp1Δ rad3Δ cells. Cells synchronized in G2 by elutriation were mock treated or exposed to 100 Gy of IR. Cell cycle progression was tracked by microscopic observation.

Oliver Limbo, et al. Mol Cell Biol. 2011 February;31(3):573-583.

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