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Results: 8

1.
Figure 5

Figure 5. Deletion of RAD9 gene accelerates DSB resection despites high Cdc5's levels.. From: Elevated Levels of the Polo Kinase Cdc5 Override the Mec1/ATR Checkpoint in Budding Yeast by Acting at Different Steps of the Signaling Pathway.

YEP+raffinose nocodazole-arrested cell cultures of wild type JKM MATa and isogenic rad9Δ strains, with or without GAL1::CDC5, were transferred to nocodazole-containing YEP + raffinose + galactose (time zero). (A,B) Analysis of ssDNA formation as described in Figure 4. (C) Ddc2 protein was analyzed by western blots using 12CA5 antibody; Rad53 protein was analysed by monoclonal antibody Mab.EL7.

Roberto Antonio Donnianni, et al. PLoS Genet. 2010 January;6(1):e1000763.
2.
Figure 2

Figure 2. Overproduction of Cdc5 affects DSB–induced Rad53 phosphorylation and activity.. From: Elevated Levels of the Polo Kinase Cdc5 Override the Mec1/ATR Checkpoint in Budding Yeast by Acting at Different Steps of the Signaling Pathway.

(A,B) YEP+raffinose nocodazole-arrested cell cultures of wild type JKM and isogenic GAL1::CDC5, GAL1::cdc5-kd (kinase dead, K110A mutation) and GAL1::cdc5-ad (adaptation defective, L251W mutation) strains were transferred to nocodazole-containing YEP + raffinose + galactose (time zero). (A) Samples were taken at the indicated time points and analyzed by FACS. (B) Overproduced Cdc5 proteins have an additional HA epitope and their accumulation in galactose was analyzed by western blots using 12CA5 antibody. Rad53 was analyzed by western blots with Mab.EL7 antibodies. Rad53 in situ auto-phosphorylation activity was analyzed by ISA assay.

Roberto Antonio Donnianni, et al. PLoS Genet. 2010 January;6(1):e1000763.
3.
Figure 3

Figure 3. Overproduction of Cdc5 overrides Mec1 checkpoint signaling.. From: Elevated Levels of the Polo Kinase Cdc5 Override the Mec1/ATR Checkpoint in Budding Yeast by Acting at Different Steps of the Signaling Pathway.

(A) YEP+raffinose nocodazole-arrested cell cultures of wild-type JKM and isogenic rad53-kd (kinase dead, K227A mutation) derivative strains, with or without GAL1::CDC5, were transferred to nocodazole-containing YEP + raffinose + galactose (time zero). Samples were taken at the indicated time points and Rad53 was analyzed by western blots using monoclonal antibodies Mab.EL7 or Mab.F9, which recognized, respectively, all the forms of Rad53 or only the auto-phosphorylated and active forms. (B) YEP+raffinose nocodazole-arrested cell cultures of wild type JKM and isogenic GAL1::CDC5 derivative strains, expressing DDC2-HA, were transferred to nocodazole-containing YEP + raffinose + galactose (time zero). Ddc2 protein was analyzed by western blots using 12CA5 antibody; Rad9 protein was analyzed by polyclonal antibodies. An arrow denotes the hyper-phosphorylation band of Rad9 accumulated specifically in response to DNA damage.

Roberto Antonio Donnianni, et al. PLoS Genet. 2010 January;6(1):e1000763.
4.
Figure 6

Figure 6. Recruitment to DSB of checkpoint factors in CDC5-overexpressing cells.. From: Elevated Levels of the Polo Kinase Cdc5 Override the Mec1/ATR Checkpoint in Budding Yeast by Acting at Different Steps of the Signaling Pathway.

(A) Schematic representation of the HO cleavage site with the positions of the primers used to amplify regions 1 kb (DSB) and 66 kb (CON) from the HO cut site. PCR analysis at the CON site is used as a control of background signal. (B–E) YEP+raffinose nocodazole-arrested cell cultures of wild-type JKM and isogenic GAL1::CDC5-MYC or GAL1-CDC5-HA strains, expressing DDC2-HA, DDC1-MYC, DPB11-MYC, and RAD9-MYC alleles, were transferred to nocodazole-containing YEP + raffinose + galactose (time zero). Cells were collected at the indicated times and then subjected to chromatin immunoprecipitation. Representative ChIP time-course analysis of protein-DSB association is shown for each protein tested before (Inputs) and after protein immunoprecipitation (IP).

Roberto Antonio Donnianni, et al. PLoS Genet. 2010 January;6(1):e1000763.
5.
Figure 1

Figure 1. Overproduction of Cdc5 overrides the DNA replication and DNA damage checkpoints.. From: Elevated Levels of the Polo Kinase Cdc5 Override the Mec1/ATR Checkpoint in Budding Yeast by Acting at Different Steps of the Signaling Pathway.

(A) Exponentially (L) growing culture of the strain Y114 (GAL1::CDC5) was grown in YEP+3%raffinose and treated for 3 hours with 0.02% MMS (time 0). Then the culture is split in two and 2% galactose was added to one half, while the other half was maintained in raffinose. Samples were taken at the indicated time and analysed by FACS. (B) Cultures of the strains Y79 (wild type), Y114 (GAL1::CDC5), exponentially (L) growing in YEP+3%raffinose were blocked in G2/M by nocodazole treatment (0). Zeocin (50 µg/ml) was then added to cause DSBs formation and after 30 minutes of treatment, 2% galactose was added. Samples were taken at the indicated time and Rad53 protein was analyzed by western blotting with Mab.EL7 antibody.

Roberto Antonio Donnianni, et al. PLoS Genet. 2010 January;6(1):e1000763.
6.
Figure 7

Figure 7. Analysis of Sae2 protein in CDC5 overexpressing cells.. From: Elevated Levels of the Polo Kinase Cdc5 Override the Mec1/ATR Checkpoint in Budding Yeast by Acting at Different Steps of the Signaling Pathway.

(A,B) YEP+raffinose nocodazole-arrested cell cultures of wild type JKM and isogenic GAL1::CDC5-MYC strain, expressing SAE2-3HA allele, were transferred to nocodazole-containing YEP + raffinose + galactose (time zero). Cells were collected at the indicated times and then subjected to chromatin immunoprecipitation (ChIP) as described in Figure 6. Representative ChIP time-course analysis of protein-DSB association is shown before (Inputs) and after protein immunoprecipitation (IP). (B) Western blot analysis of protein extracts. (C) Western blot analysis of protein extracts prepared 3 hrs after HO induction and treated with or without λ phosphatase before gel electrophoresis. (D) YEP-raffinose growing cells of wild type and of wild-type JKM MATa-inc and isogenic GAL1::CDC5-MYC strains, expressing SAE2-3HA allele, were split in two. One half was treated with nocodazole to block cells in G2. Galactose was then added to the cultures to induce overproduction of Cdc5. Cells were collected at the indicated times after galactose addition. (B–D) Sae2-HA protein was analyzed by western blots using 12CA5 antibody; Rad53 protein was analysed by monoclonal antibody Mab.EL7.

Roberto Antonio Donnianni, et al. PLoS Genet. 2010 January;6(1):e1000763.
7.
Figure 4

Figure 4. Overproduction of Cdc5 affects DSB processing.. From: Elevated Levels of the Polo Kinase Cdc5 Override the Mec1/ATR Checkpoint in Budding Yeast by Acting at Different Steps of the Signaling Pathway.

(A–D) YEP+raffinose nocodazole-arrested cell cultures of wild-type JKM MATα and isogenic GAL1::CDC5 strain were transferred to nocodazole-containing YEP + raffinose + galactose (time zero). (A) Schematic representation of the system used to detect DSB resection. Gel blots of SspI-digested genomic DNA separated on alkaline agarose gel were hybridized with a single-strand RNA probe specific for the un-resected strand at the MAT locus, which shows HO-cut and uncut fragments of 0.9 and 1.1 kb, respectively. 5′-to-3′ resection progressively eliminates SspI sites located 1.7, 3.5, 4.7, 5.9, 6.5, 8.9, and 15.8 kb centromere-distal from the HO-cut site, producing larger SspI fragments (r1–r7) detected by the probe. (B) Analysis of ssDNA formation as described in (A). (C) The time of the first appearance over the background of each undigested band in the blot shown in (B) was graphically represented for both the wild type and GAL1::CDC5 strains. (D) Western blot analysis of protein extracts with anti-Rad53 Mab.EL7 antibody.

Roberto Antonio Donnianni, et al. PLoS Genet. 2010 January;6(1):e1000763.
8.
Figure 8

Figure 8. Sae2 protein interacts with PBD of Cdc5.. From: Elevated Levels of the Polo Kinase Cdc5 Override the Mec1/ATR Checkpoint in Budding Yeast by Acting at Different Steps of the Signaling Pathway.

(A) Sae2 protein sequence. The putative Cdc5 phosphorylation sites and PBD binding sites are indicated. (B) Plasmid pEG202-PBD340–705, carrying the polo box domain of Cdc5 (PBD, aa 340 to 705), and pJG4-5-SAE2, carrying the full length SAE2 gene under the GAL1 promoter, were co-transformed with pSH18-34, a β-galactosidase reporter plasmid in the wild type yeast strain EGY48. To assess two-hybrid interaction, these strains were patched on to 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X Gal) plates containing either raffinose (RAF, prey repressed) or galactose (GAL, prey expressed). Accordingly to [50], the strain Y692 (PBD versus Swe1173–400 protein fragment) was used as positive control. (C) Cells of the strain Y202, expressing SAE2-3HA gene, were blocked in G2/M by nocodazole treatment. Whole cell protein extract was prepared and incubated with glutathione-Sepharose beads carrying GST or GST-PBD357–705. Input and pull-down samples were analyzed by western blotting with monoclonal antibody 12CA5 (αHA) or polyclonal antisera raised against GST (αGST). Asterisk denotes bands of GST-PBD degradation or expression of truncated proteins. (D) Schematic model to summarize the results presented in this work. (i) Sae2 transiently binds DSB, regulating ends resection and influencing Mec1-signaling. The checkpoint signal is amplified downstream, regulating several targets, including Cdc5. (ii) After a prolonged checkpoint response, adaptation to damage takes over and Cdc5 is re-activated, likely by an activating kinase (in human cells, it is aurora A [35]); Cdc5 then inhibits checkpoint signaling in a feedback regulatory loop, by likely targeting several factors, including Sae2 whose loading on the irreparable DSB increases, slowing down resection and contributing to counteract the checkpoint signaling (red circles denote phosphorylation). Alternatively, or in addition, Cdc5 function on several targets, including Sae2, is enhanced in the presence of elevated levels of Cdc5, a situation frequently found for Plks in tumor cells.

Roberto Antonio Donnianni, et al. PLoS Genet. 2010 January;6(1):e1000763.

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