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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 In Vivo CMG Ubiquitylation at the End of S Phase(A) cdc48-aid (YMM228) and cdc48-aid dia2-ΔTPR (YPNK334) were synchronized in G1 phase at 30°C and then released into S phase for 60 min in the presence of 0.2 M hydroxyurea. For depletion of Cdc48-aid, 0.5 mM auxin was added for 60 min, before release into fresh medium containing auxin but lacking hydroxyurea. DNA content was monitored by flow cytometry, and samples were taken at the indicated times (“1” and “2”) to prepare pH 9 cell extracts containing 700 mM salt.(B) CMG helicase was isolated as above by immunoprecipitation of TAP-tagged Sld5 subunit.
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fig3: Tethering of SCFDia2 to the Replisome Progression Complex Increases the Efficiency of In Vivo CMG Ubiquitylation at the End of S Phase(A) cdc48-aid (YMM228) and cdc48-aid dia2-ΔTPR (YPNK334) were synchronized in G1 phase at 30°C and then released into S phase for 60 min in the presence of 0.2 M hydroxyurea. For depletion of Cdc48-aid, 0.5 mM auxin was added for 60 min, before release into fresh medium containing auxin but lacking hydroxyurea. DNA content was monitored by flow cytometry, and samples were taken at the indicated times (“1” and “2”) to prepare pH 9 cell extracts containing 700 mM salt.(B) CMG helicase was isolated as above by immunoprecipitation of TAP-tagged Sld5 subunit.

Mentions: Ubiquitylation of the CMG helicase is restricted to the end of chromosome replication in vivo, when it is coupled rapidly to Cdc48-dependent disassembly [3]. For visualization of ubiquitylated CMG in vivo, it is necessary to inactivate Cdc48 before cells terminate DNA replication and then prepare “high salt” extracts that block the in vitro ubiquitylation of the CMG helicase. In order to assess the contribution of replisome tethering of SCFDia2 to CMG ubiquitylation in vivo, we synchronized cdc48-aid and dia2-ΔTPR cdc48-aid cells (aid [auxin inducible degron]) in early S phase and then depleted Cdc48-aid, before allowing cells to proceed with chromosome replication (Figure 3A). As shown in Figure 3B, in vivo ubiquitylation of the Mcm7 subunit of CMG could still be detected at the end of S phase in dia2-ΔTPR cdc48-aid cells but was markedly reduced. These data indicate that the tethering of SCFDia2 to the RPC increases the efficiency of CMG ubiquitylation at the end of chromosome replication in budding yeast.


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 In Vivo CMG Ubiquitylation at the End of S Phase(A) cdc48-aid (YMM228) and cdc48-aid dia2-ΔTPR (YPNK334) were synchronized in G1 phase at 30°C and then released into S phase for 60 min in the presence of 0.2 M hydroxyurea. For depletion of Cdc48-aid, 0.5 mM auxin was added for 60 min, before release into fresh medium containing auxin but lacking hydroxyurea. DNA content was monitored by flow cytometry, and samples were taken at the indicated times (“1” and “2”) to prepare pH 9 cell extracts containing 700 mM salt.(B) CMG helicase was isolated as above by immunoprecipitation of TAP-tagged Sld5 subunit.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4562905&req=5

fig3: Tethering of SCFDia2 to the Replisome Progression Complex Increases the Efficiency of In Vivo CMG Ubiquitylation at the End of S Phase(A) cdc48-aid (YMM228) and cdc48-aid dia2-ΔTPR (YPNK334) were synchronized in G1 phase at 30°C and then released into S phase for 60 min in the presence of 0.2 M hydroxyurea. For depletion of Cdc48-aid, 0.5 mM auxin was added for 60 min, before release into fresh medium containing auxin but lacking hydroxyurea. DNA content was monitored by flow cytometry, and samples were taken at the indicated times (“1” and “2”) to prepare pH 9 cell extracts containing 700 mM salt.(B) CMG helicase was isolated as above by immunoprecipitation of TAP-tagged Sld5 subunit.
Mentions: Ubiquitylation of the CMG helicase is restricted to the end of chromosome replication in vivo, when it is coupled rapidly to Cdc48-dependent disassembly [3]. For visualization of ubiquitylated CMG in vivo, it is necessary to inactivate Cdc48 before cells terminate DNA replication and then prepare “high salt” extracts that block the in vitro ubiquitylation of the CMG helicase. In order to assess the contribution of replisome tethering of SCFDia2 to CMG ubiquitylation in vivo, we synchronized cdc48-aid and dia2-ΔTPR cdc48-aid cells (aid [auxin inducible degron]) in early S phase and then depleted Cdc48-aid, before allowing cells to proceed with chromosome replication (Figure 3A). As shown in Figure 3B, in vivo ubiquitylation of the Mcm7 subunit of CMG could still be detected at the end of S phase in dia2-ΔTPR cdc48-aid cells but was markedly reduced. These data indicate that the tethering of SCFDia2 to the RPC increases the efficiency of CMG ubiquitylation at the end of chromosome replication in budding yeast.

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