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

Abstract

Disassembly of the Cdc45-MCM-GINS (CMG) DNA helicase, which unwinds the parental DNA duplex at eukaryotic replication forks, is the key regulated step during replication termination but is poorly understood. In budding yeast, the F-box protein Dia2 drives ubiquitylation of the CMG helicase at the end of replication, leading to a disassembly pathway that requires the Cdc48 segregase. The substrate-binding domain of Dia2 comprises leucine-rich repeats, but Dia2 also has a TPR domain at its amino terminus that interacts with the Ctf4 and Mrc1 subunits of the replisome progression complex, which assembles around the CMG helicase at replication forks. Previous studies suggested two disparate roles for the TPR domain of Dia2, either mediating replisome-specific degradation of Mrc1 and Ctf4 or else tethering SCF(Dia2) (SCF [Skp1/cullin/F-box protein]) to the replisome to increase its local concentration at replication forks. 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. Instead, the tethering of SCF(Dia2) to the replisome progression complex increases the efficiency of ubiquitylation of the Mcm7 subunit of CMG, both in vitro and in vivo. 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.

Graphical abstract

Figure 1

Tethering of SCF^Dia2 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.

Figure 2

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.

Figure 3

Tethering of SCF^Dia2 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.

Figure 4

The CMG Disassembly Defect of dia2-ΔTPR Is Augmented by Mutations in CDC48 (A) dia2Δ (YHM130), dia2-ΔTPR (YTM265), cdc48-aid (YMM228), and dia2-ΔTPR cdc48-aid (YPNK334) were arrested in G1 phase at 30°C, in medium lacking auxin (permissive conditions for cdc48-aid), before isolation of TAP-Sld5 from “high-salt pH 9 cell extracts.” (B) Serial dilutions of the indicated strains were grown on rich medium (YPD) in the absence or presence of the DNA-damaging agent methyl methanesulfonate. (C) Asynchronous cultures of control (YASD375) and dia2-ΔTPR (YTM265) were grown at 30°C, before isolation of TAP-Sld5 from high-salt pH 9 cell extracts as above. The arrows denote the increased association of Cdc45 and Mcm2-7 proteins with GINS in extracts of asynchronous dia2-ΔTPR cells. (D) The diploid strain CDC48/cdc48-3 DIA2/dia2-ΔTPR was sporulated and subjected to tetrad analysis. The germinated spores were grown for 3 days at 24°C before imaging. See also Figures S3 and S4.