1. Volume 28, Issue 1, Pages 134-146 (October 2007)
Ctp1 Is a Cell-Cycle-Regulated Protein that Functions with Mre11 Complex to Control Double-Strand Break Repair by Homologous Recombination 
Oliver Limbo, Charly Chahwan, Yoshiki Yamada, Robertus A.M. de Bruin, Curt Wittenberg, Paul Russell 
Molecular Cell 
Volume 28, Issue 1, Pages (October 2007)
DOI: /j.molcel
Copyright © 2007 Elsevier Inc. Terms and Conditions

2. Figure 1 Ctp1 Is Required for Survival of IR and CPT
(A) ctp1Δ cells are sensitive to IR. Their sensitivity is equivalent to nbs1Δ cells, and combining the mutations has no additive effect.
(B) ctp1Δ cells arrest division in response to IR, with the septation index dropping to <2%, showing that the DNA damage checkpoint is intact. The rad3Δ strain is checkpoint defective.
(C) Chk1 undergoes activating phosphorylation in response to IR in ctp1Δ cells as indicated by the appearance of a slow-mobility species of Chk1 that is hyperphosphorylated. The crb2Δ strain is checkpoint defective.
(D) ctp1Δ cells are sensitive to CPT. Their sensitivity is equivalent to nbs1Δ cells, and combining the mutations has no additive effect.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

3. Figure 2
Ctp1 Is Essential for Meiosis and for HR Repair of DSBs in Mitotic Cells
(A) Asci from a ctp1Δ × ctp1Δ mating are abnormal. DNA is stained with DAPI.
(B) Spore viability in a ctp1Δ × ctp1Δ mating is very low. This defect is partially suppressed in a rec12Δ background that is unable to form programmed meiotic DSBs.
(C) Ctp1 is required for HR, but not NHEJ. HR was measured by integration of a transformed linearized plasmid into homologous sequences. NHEJ was measured by circularization of a transformed linearized plasmid. Values are calculated with respect to transformants obtained with an uncut plasmid and are normalized to wild-type. Error bars show standard deviation values derived from at least five transformation assays.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

4. Figure 3
Exo1 Can Substitute for Ctp1 in Repair of DSBs, and Recruitment of RPA to a DSB Is Defective in ctp1Δ Cells
(A) The IR and CPT survival defects of ctp1Δ cells are suppressed by eliminating Ku80. This rescue depends on Exo1.
(B) Recruitment of RPA to a DSB is reduced in ctp1Δ and mre11Δ strains relative to wild-type. ChIP analysis of RPA (rad11-TAP) around an HO-induced DSB. Expression of HO endonuclease was controlled by using the thiamine-repressible nmt1 promoter. Sites located 0.2, 2, 9, and 16 kb from the DSB were assayed for enrichment of RPA (see Figure 4 for map of probes). The act1 probe is included as a negative control. Microscopic analyses confirmed that >90% of the cells in all strains arrested division as a result of HO expression, confirming highly efficient cutting by HO endonuclease.
(C) Recruitment of RPA to a DSB is reduced in ctp1Δ and mre11Δ strains relative to wild-type. Quantitative real-time PCR was used to measure enrichment of RPA at sites located 0.2 or 9 kb from the HO-induced DSB.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

5. Figure 4 Ctp1 Localizes at a DSB by an Mre11-Dependent Mechanism
(A) ChIP analysis of Ctp1 and phospho-H2A around an HO-induced DSB. Expression of HO endonuclease was controlled by using the thiamine-repressible nmt1 promoter. Assays were performed in a ctp1+ background or a strain in which the endogenous copy of ctp1+ was modified to encode Ctp1-TAP. Ctp1-TAP was enriched 0.2 kb from the DSB (lane 8), whereas phospho-H2A was enriched 2, 9, and 16 kb from the DSB (lane 10). Specific enrichment of Ctp1-TAP at 0.2 kb from the DSB was confirmed with four independent strains. WCE, whole-cell extract; IP, TAP, immunoprecipitated Ctp1-TAP; and P-H2A, immunoprecipitated C-terminally phosphorylated histone H2A.
(B) ChIP analysis of Ctp1 and Mre11 around an HO-induced DSB. Ctp1-TAP is specifically detected 0.2 kb from the DSB in mre11+, but not mre11Δ cells. In contrast, Mre11-TAP is specifically detected 0.2 kb from the DSB in both ctp1+ and ctp1Δ cells. Genotoxin survival studies confirmed that Mre11-TAP is functional. The absence of Ctp1-TAP from the DSB in mre11Δ cells was confirmed with four independent strains. The 9 kb and act1 products are not shown. Microscopic analyses of the 24 hr samples confirmed that >90% of the cells in all strains arrested division as a result of HO expression, indicating highly efficient cutting by HO endonuclease. The delayed detection of Mre11-TAP at the HO break site in ctp1Δ cells may reflect delayed cutting at HO.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

6. Figure 5
The Telomere Maintenance Function of MRN Is Independent of Ctp1
Southern blot analysis of EcoRI-digested genomic DNA from the indicated strains probed with the telomere associated-1 (TAS1) probe.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

7. Figure 6 Cell-Cycle Control of Ctp1 Abundance
(A) Transcription of ctp1+ is regulated by MBF. Wild-type and nrm1Δ strains were synchronized in G2 by centrifugal elutriation. Samples were taken as cells underwent mitosis and septation. The septation index approximately coincides with S phase. Transcript levels from ctp1+ and cdc22+ were determined by real-time PCR, normalized to act1+ transcript levels, and shown as relative transcript levels (%) to maximum wild-type levels during the cell cycle (wild-type maximum is 100%).
(B) MBF localizes at the ctp1+ promoter region. Association of Cdc10-HA, Res2-HA, and Nrm1-HA with the ctp1+, cdc22+, and act1+ promoters was determined by ChIP using log-phase cultures. Tagged constructs were expressed from the endogenous loci. Whole-cell extract (WCE) from the “no tag” strain was used as a control. Immunoblots confirmed that Nrm1 protein is expressed in res2Δ cells (data not shown).
(C) Immunoblot of Ctp1-TAP in asynchronous, irradiated, and HU-treated cells. Immunoblot of Cdc2 with PSTAIR antisera is the loading control.
(D) Immunoblot of Ctp1-TAP in asynchronous, nitrogen-starved, or carbon-starved cells. Immunoblot of tubulin is the loading control.
(E) Immunoblot of Ctp1-TAP in cells released from an HU arrest. Septation index indicates completion of the cell cycle.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

8. Figure 7 Conserved C-Terminal Domain of Ctp1
(A) The domain structures of S. pombe Ctp1, A. thaliania AtGR1, and H. sapiens CtIP/RBBP8 are shown. These proteins share C-terminal core homology domains of ∼70 amino acids that include the CxxC and RHR motifs. Alignments of the C-terminal core homology domains of S. pombe Ctp1 (Sp) and its homologs in A. thaliana, At (4e-21); Oryza sativa, Os (4e-19); H. sapiens, Hs (5e-17); Dictyostelium discoideum, Dd (9e-15); Gallus gallus, Gg (1e-17); Danio rerio, Dr (6e-16); Xenopus laevis, Xl (3e-16); and Caenorhabditis elegans, Ce (6e-10). The PSI-BLAST expect values are shown in parentheses.
(B) The conserved CxxC motif is required for Ctp1 activity. The CxxC motif was mutated to GxxG or SxxS. These mutations and a C-terminal TAP tag were introduced into the genomic copy of ctp1+. Two wild-type constructs with the C-terminal TAP tag serve as controls. Immunoblotting confirmed that the mutant proteins were expressed at levels at or above the wild-type amount (O.L. and P.R., unpublished data).
(C) Yeast two-hybrid assays show that Ctp1 self-associates but does not interact with Mre11, Rad50, or Nbs1. Mre11-Mre11, Mre11-Rad50, and Mre11-Nbs1 interactions are also detected. No interactions were detected involving Rad50-Rad50, Nbs1-Nbs1, or Rad50-Nbs1 (data not shown).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions