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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

The CMG Ubiquitylation Defects of ctf4Δ and mrc1Δ Can Be Rescued In Vitro(A) S phase cell extracts of control (YTM401), ctf4Δ (YTM438), and mrc1Δ (YTM440) were prepared at pH 9 as above and complemented with buffer or purified Ctf4 as indicated, before immunoprecipitation of Cdc45-ProteinA. The indicated proteins were then monitored by immunoblotting. Asterisks denote non-specific bands.(B) To test for in vitro rescue of the ubiquitylation defect of mrc1Δ cell extracts, we synchronized the indicated CDC45-ProteinA “recipient strains” (1, YTM401; 2–4, YTM440) and CDC45 “donor strains” (1–4, YSS3, YPNK314, YSS3, and YPNK342, respectively) in S phase at 30°C. Each of the indicated pairs of recipient and donor cultures were then mixed and used to prepare a single cell extract at pH 9 as above. After digestion of chromosomal DNA, the CMG helicase from recipient cells was isolated by immunoprecipitation of its ProteinA-tagged-Cdc45 subunit.
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fig2: The CMG Ubiquitylation Defects of ctf4Δ and mrc1Δ Can Be Rescued In Vitro(A) S phase cell extracts of control (YTM401), ctf4Δ (YTM438), and mrc1Δ (YTM440) were prepared at pH 9 as above and complemented with buffer or purified Ctf4 as indicated, before immunoprecipitation of Cdc45-ProteinA. The indicated proteins were then monitored by immunoblotting. Asterisks denote non-specific bands.(B) To test for in vitro rescue of the ubiquitylation defect of mrc1Δ cell extracts, we synchronized the indicated CDC45-ProteinA “recipient strains” (1, YTM401; 2–4, YTM440) and CDC45 “donor strains” (1–4, YSS3, YPNK314, YSS3, and YPNK342, respectively) in S phase at 30°C. Each of the indicated pairs of recipient and donor cultures were then mixed and used to prepare a single cell extract at pH 9 as above. After digestion of chromosomal DNA, the CMG helicase from recipient cells was isolated by immunoprecipitation of its ProteinA-tagged-Cdc45 subunit.

Mentions: To confirm that loss of tethering reduced the capacity of SCFDia2 to drive in vitro ubiquitylation of the CMG helicase, we repeated the above experiment with S phase extracts of control, ctf4Δ, or mrc1Δ and then complemented the extracts with buffer or with purified Ctf4 protein. Critically, addition of purified Ctf4 to the ctf4Δ extract restored the efficiency of ubiquitylation, producing a very similar pattern to the control extract (with di-ubiquitylated Mcm7 being the predominant form in the isolated CMG material), whereas addition of Ctf4 to an extract of mrc1Δ cells had no effect (Figure 2A). Similarly, we showed that the CMG ubiquitylation defect of mrc1Δ extracts could be rescued in vitro by mixing with extracts of cells that expressed Mrc1. We synchronized “recipient” cultures of CDC45-ProteinA in S phase alongside “donor cultures” expressing untagged CDC45 and then mixed cultures as indicated in Figure 2B, before making cell extracts and isolating “‘recipient CMG” by immunoprecipitation of Cdc45-ProteinA. A donor extract expressing Mrc1 was able to rescue the in vitro ubiquitylation defect of an mrc1Δ recipient extract (Figure 2B, sample 3), whereas an extract overexpressing Mrc1 further enhanced the ubiquitylation of CMG (Figure 2B, sample 4). These findings demonstrate that the TPR-dependent tethering of SCFDia2 to the RPC serves to increase the efficiency of CMG ubiquitylation in vitro.


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)

The CMG Ubiquitylation Defects of ctf4Δ and mrc1Δ Can Be Rescued In Vitro(A) S phase cell extracts of control (YTM401), ctf4Δ (YTM438), and mrc1Δ (YTM440) were prepared at pH 9 as above and complemented with buffer or purified Ctf4 as indicated, before immunoprecipitation of Cdc45-ProteinA. The indicated proteins were then monitored by immunoblotting. Asterisks denote non-specific bands.(B) To test for in vitro rescue of the ubiquitylation defect of mrc1Δ cell extracts, we synchronized the indicated CDC45-ProteinA “recipient strains” (1, YTM401; 2–4, YTM440) and CDC45 “donor strains” (1–4, YSS3, YPNK314, YSS3, and YPNK342, respectively) in S phase at 30°C. Each of the indicated pairs of recipient and donor cultures were then mixed and used to prepare a single cell extract at pH 9 as above. After digestion of chromosomal DNA, the CMG helicase from recipient cells was isolated by immunoprecipitation of its ProteinA-tagged-Cdc45 subunit.
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fig2: The CMG Ubiquitylation Defects of ctf4Δ and mrc1Δ Can Be Rescued In Vitro(A) S phase cell extracts of control (YTM401), ctf4Δ (YTM438), and mrc1Δ (YTM440) were prepared at pH 9 as above and complemented with buffer or purified Ctf4 as indicated, before immunoprecipitation of Cdc45-ProteinA. The indicated proteins were then monitored by immunoblotting. Asterisks denote non-specific bands.(B) To test for in vitro rescue of the ubiquitylation defect of mrc1Δ cell extracts, we synchronized the indicated CDC45-ProteinA “recipient strains” (1, YTM401; 2–4, YTM440) and CDC45 “donor strains” (1–4, YSS3, YPNK314, YSS3, and YPNK342, respectively) in S phase at 30°C. Each of the indicated pairs of recipient and donor cultures were then mixed and used to prepare a single cell extract at pH 9 as above. After digestion of chromosomal DNA, the CMG helicase from recipient cells was isolated by immunoprecipitation of its ProteinA-tagged-Cdc45 subunit.
Mentions: To confirm that loss of tethering reduced the capacity of SCFDia2 to drive in vitro ubiquitylation of the CMG helicase, we repeated the above experiment with S phase extracts of control, ctf4Δ, or mrc1Δ and then complemented the extracts with buffer or with purified Ctf4 protein. Critically, addition of purified Ctf4 to the ctf4Δ extract restored the efficiency of ubiquitylation, producing a very similar pattern to the control extract (with di-ubiquitylated Mcm7 being the predominant form in the isolated CMG material), whereas addition of Ctf4 to an extract of mrc1Δ cells had no effect (Figure 2A). Similarly, we showed that the CMG ubiquitylation defect of mrc1Δ extracts could be rescued in vitro by mixing with extracts of cells that expressed Mrc1. We synchronized “recipient” cultures of CDC45-ProteinA in S phase alongside “donor cultures” expressing untagged CDC45 and then mixed cultures as indicated in Figure 2B, before making cell extracts and isolating “‘recipient CMG” by immunoprecipitation of Cdc45-ProteinA. A donor extract expressing Mrc1 was able to rescue the in vitro ubiquitylation defect of an mrc1Δ recipient extract (Figure 2B, sample 3), whereas an extract overexpressing Mrc1 further enhanced the ubiquitylation of CMG (Figure 2B, sample 4). These findings demonstrate that the TPR-dependent tethering of SCFDia2 to the RPC serves to increase the efficiency of CMG ubiquitylation in vitro.

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