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Tethering of SCF(Dia2) to the Replisome Promotes Efficient Ubiquitylation and Disassembly of the CMG Helicase.

Maculins T, Nkosi PJ, Nishikawa H, Labib K - Curr. Biol. (2015)

Bottom Line: Here, we show that SCF(Dia2) does not mediate replisome-specific degradation of Mrc1 and Ctf4, either during normal S phase or in response to replication stress.Correspondingly, loss of tethering reduces the efficiency of CMG disassembly in vivo and is synthetic lethal in combination with a disassembly-defective allele of CDC48.Residual ubiquitylation of Mcm7 in dia2-ΔTPR cells is still CMG specific, highlighting the complex regulation of the final stages of chromosome replication, about which much still remains to be learned.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK Manchester Institute, University of Manchester, Wilmslow Road, Manchester M20 4BX, UK.

No MeSH data available.


Related in: MedlinePlus

Tethering of SCFDia2 to the Replisome Progression Complex Increases the Efficiency of CMG Ubiquitylation In Vitro(A) An asynchronous culture of MCM4-5FLAG MRC1-18MYC cells (YGDP219) was grown at 30°C, before addition of 500 μg/ml cycloheximide for the indicated times. Cell extracts were treated with DNase before immunoprecipitation of Mcm4-5FLAG and detection of the indicated proteins by immunoblotting.(B) (i) Flow cytometry analysis from the same experiment. (ii) The same strain as above was arrested in G1 phase and then released into S phase for 60 min in the presence of 0.2 M hydroxyurea. Cycloheximide was added for the indicated times and samples processed as before.(C) The samples from (Bii) were processed as in (A).(D) Control cells (YTM325) and MCM4-5FLAG (YTM326) were synchronized at 30°C in the G1 phase of the cell cycle by addition of mating pheromone, before release into S phase for 20 min. DNA content was monitored by flow cytometry (upper panels). “pH 9 cell extracts” were then prepared as described in the Supplemental Experimental Procedures and incubated with magnetic beads coupled to anti-FLAG monoclonal antibody. The immunoprecipitated proteins were then monitored by immunoblotting (lower panels).(E) Control (YASD375), ctf4Δ (YTM403), mrc1Δ (YLG31), and dia2-ΔTPR (YTM265) were synchronized in early S phase as above, before immunoprecipitation of TAP-Sld5 from pH 9 cell extracts on IgG beads.See also Figures S1 and S2.
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fig1: Tethering of SCFDia2 to the Replisome Progression Complex Increases the Efficiency of CMG Ubiquitylation In Vitro(A) An asynchronous culture of MCM4-5FLAG MRC1-18MYC cells (YGDP219) was grown at 30°C, before addition of 500 μg/ml cycloheximide for the indicated times. Cell extracts were treated with DNase before immunoprecipitation of Mcm4-5FLAG and detection of the indicated proteins by immunoblotting.(B) (i) Flow cytometry analysis from the same experiment. (ii) The same strain as above was arrested in G1 phase and then released into S phase for 60 min in the presence of 0.2 M hydroxyurea. Cycloheximide was added for the indicated times and samples processed as before.(C) The samples from (Bii) were processed as in (A).(D) Control cells (YTM325) and MCM4-5FLAG (YTM326) were synchronized at 30°C in the G1 phase of the cell cycle by addition of mating pheromone, before release into S phase for 20 min. DNA content was monitored by flow cytometry (upper panels). “pH 9 cell extracts” were then prepared as described in the Supplemental Experimental Procedures and incubated with magnetic beads coupled to anti-FLAG monoclonal antibody. The immunoprecipitated proteins were then monitored by immunoblotting (lower panels).(E) Control (YASD375), ctf4Δ (YTM403), mrc1Δ (YLG31), and dia2-ΔTPR (YTM265) were synchronized in early S phase as above, before immunoprecipitation of TAP-Sld5 from pH 9 cell extracts on IgG beads.See also Figures S1 and S2.

Mentions: Previous work showed that the association of budding yeast Mrc1 and Ctf4 with the Cdc45-MCM-GINS (CMG) helicase was lost in control cells, but not in dia2Δ, when cycloheximide was used to inhibit protein synthesis in asynchronous cell cultures [4]. This was taken as evidence that SCFDia2 specifically ubiquitylates the fraction of Mrc1 and Ctf4 that is incorporated into the replisome progression complex at replication forks. We repeated the same experiment with control cells expressing DIA2 by immunoprecipitating the Mcm4 helicase subunit from cell extracts after addition of cycloheximide. Whereas Mcm4 still associated with the remaining subunits of the Mcm2-7 complex in cycloheximide-treated cells, association with all other RPC subunits was lost (Figure 1A). Rather than reflecting the specific degradation of RPC-associated Mrc1 and Ctf4, these data thus indicated that the RPC is no longer present when control cells are treated with cycloheximide. A simple explanation for this is provided by the fact that protein synthesis is required for G1 phase cells to enter S phase, but S phase cells can complete DNA replication without ongoing protein synthesis [7–9]. Consistent with this view, flow cytometry data from the same experiment indicated that the S phase population of cells was lost upon addition of cycloheximide to the asynchronous cell culture (Figure 1Bi). Cycloheximide should thus block the assembly, but not the disassembly, of the RPC.


Tethering of SCF(Dia2) to the Replisome Promotes Efficient Ubiquitylation and Disassembly of the CMG Helicase.

Maculins T, Nkosi PJ, Nishikawa H, Labib K - Curr. Biol. (2015)

Tethering of SCFDia2 to the Replisome Progression Complex Increases the Efficiency of CMG Ubiquitylation In Vitro(A) An asynchronous culture of MCM4-5FLAG MRC1-18MYC cells (YGDP219) was grown at 30°C, before addition of 500 μg/ml cycloheximide for the indicated times. Cell extracts were treated with DNase before immunoprecipitation of Mcm4-5FLAG and detection of the indicated proteins by immunoblotting.(B) (i) Flow cytometry analysis from the same experiment. (ii) The same strain as above was arrested in G1 phase and then released into S phase for 60 min in the presence of 0.2 M hydroxyurea. Cycloheximide was added for the indicated times and samples processed as before.(C) The samples from (Bii) were processed as in (A).(D) Control cells (YTM325) and MCM4-5FLAG (YTM326) were synchronized at 30°C in the G1 phase of the cell cycle by addition of mating pheromone, before release into S phase for 20 min. DNA content was monitored by flow cytometry (upper panels). “pH 9 cell extracts” were then prepared as described in the Supplemental Experimental Procedures and incubated with magnetic beads coupled to anti-FLAG monoclonal antibody. The immunoprecipitated proteins were then monitored by immunoblotting (lower panels).(E) Control (YASD375), ctf4Δ (YTM403), mrc1Δ (YLG31), and dia2-ΔTPR (YTM265) were synchronized in early S phase as above, before immunoprecipitation of TAP-Sld5 from pH 9 cell extracts on IgG beads.See also Figures S1 and S2.
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Related In: Results  -  Collection

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fig1: Tethering of SCFDia2 to the Replisome Progression Complex Increases the Efficiency of CMG Ubiquitylation In Vitro(A) An asynchronous culture of MCM4-5FLAG MRC1-18MYC cells (YGDP219) was grown at 30°C, before addition of 500 μg/ml cycloheximide for the indicated times. Cell extracts were treated with DNase before immunoprecipitation of Mcm4-5FLAG and detection of the indicated proteins by immunoblotting.(B) (i) Flow cytometry analysis from the same experiment. (ii) The same strain as above was arrested in G1 phase and then released into S phase for 60 min in the presence of 0.2 M hydroxyurea. Cycloheximide was added for the indicated times and samples processed as before.(C) The samples from (Bii) were processed as in (A).(D) Control cells (YTM325) and MCM4-5FLAG (YTM326) were synchronized at 30°C in the G1 phase of the cell cycle by addition of mating pheromone, before release into S phase for 20 min. DNA content was monitored by flow cytometry (upper panels). “pH 9 cell extracts” were then prepared as described in the Supplemental Experimental Procedures and incubated with magnetic beads coupled to anti-FLAG monoclonal antibody. The immunoprecipitated proteins were then monitored by immunoblotting (lower panels).(E) Control (YASD375), ctf4Δ (YTM403), mrc1Δ (YLG31), and dia2-ΔTPR (YTM265) were synchronized in early S phase as above, before immunoprecipitation of TAP-Sld5 from pH 9 cell extracts on IgG beads.See also Figures S1 and S2.
Mentions: Previous work showed that the association of budding yeast Mrc1 and Ctf4 with the Cdc45-MCM-GINS (CMG) helicase was lost in control cells, but not in dia2Δ, when cycloheximide was used to inhibit protein synthesis in asynchronous cell cultures [4]. This was taken as evidence that SCFDia2 specifically ubiquitylates the fraction of Mrc1 and Ctf4 that is incorporated into the replisome progression complex at replication forks. We repeated the same experiment with control cells expressing DIA2 by immunoprecipitating the Mcm4 helicase subunit from cell extracts after addition of cycloheximide. Whereas Mcm4 still associated with the remaining subunits of the Mcm2-7 complex in cycloheximide-treated cells, association with all other RPC subunits was lost (Figure 1A). Rather than reflecting the specific degradation of RPC-associated Mrc1 and Ctf4, these data thus indicated that the RPC is no longer present when control cells are treated with cycloheximide. A simple explanation for this is provided by the fact that protein synthesis is required for G1 phase cells to enter S phase, but S phase cells can complete DNA replication without ongoing protein synthesis [7–9]. Consistent with this view, flow cytometry data from the same experiment indicated that the S phase population of cells was lost upon addition of cycloheximide to the asynchronous cell culture (Figure 1Bi). Cycloheximide should thus block the assembly, but not the disassembly, of the RPC.

Bottom Line: Here, we show that SCF(Dia2) does not mediate replisome-specific degradation of Mrc1 and Ctf4, either during normal S phase or in response to replication stress.Correspondingly, loss of tethering reduces the efficiency of CMG disassembly in vivo and is synthetic lethal in combination with a disassembly-defective allele of CDC48.Residual ubiquitylation of Mcm7 in dia2-ΔTPR cells is still CMG specific, highlighting the complex regulation of the final stages of chromosome replication, about which much still remains to be learned.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK Manchester Institute, University of Manchester, Wilmslow Road, Manchester M20 4BX, UK.

No MeSH data available.


Related in: MedlinePlus