Abstract
Full Text
Uninterrupted MCM2-7 Function Required for DNA Replication Fork Progression
Karim Labib, José Antonio Tercero, and John F. X. Diffley

Supplementary Material

Construction and use of degron mutantsmcm-td ubr1Δ::GAL-UBR1 strains were constructed in the following way. PCR products representing the first 400 to 500 base pairs of each MCM open reading frame were used to replace the Hind III fragment of the plasmid pPW66R [R.J.Dohmen, P.Wu, A.Varshavsky, Science263, 1273 (1994)]. The resulting six plasmids were linearized within the MCM sequence and integrated at the corresponding locus in the strain W303-1a. This generated six strains in which the only functional copy of a particular MCM gene was expressed from the copper-inducible CUP1 promoter, with the t.s. degron cassette in frame at the 5' end. The UBR1 gene was then placed under the control of the GAL1,10 promoter. A PCR product containing 1.4 kb from the 5' end of the UBR1 open reading frame (20% of the total) was subcloned downstream of the GAL1,10 promoter, in the plasmid pRS303 [R. S. Sikorski and P. Hieter, Genetics122, 19 (1989)]. The resulting plasmid was then integrated at the UBR1 locus of each strain containing an mcm degron mutant. This produced strains in which the only functional copy of the UBR1 gene was expressed under the control of the GAL1,10 promoter. In all experiments described in this paper, mcm-td ubr1Δ::GAL-UBR1 strains were grown in medium containing 0.1 mM CuSO4 at 24°C, and CuSO4 was subsequently omitted from the medium when cells were shifted to 37°C, to induce degradation of the degron-fusion protein.

Antibodies used in immunoblot analysis
The degron-fusion proteins contain the influenza HA epitope, and so can be detected in immunoblots with the monoclonal antibody 12CA5. In Fig. 1C, Mcm2 and Mcm7 were detected with the Santa Cruz polyclonal antibodies sc-6680 and sc6688, respectively, while Mcm4 was fused to green fluorescent protein (GFP) so that it could be detected with an antibody to GFP. For the experiment shown in Fig. 4, Ubr1 was tagged with the c-myc epitope at the NH2-terminus and detected with the monoclonal antibody 9E10.

Dense isotope substitution
MCM4 ubr1Δ::GAL-UBR1 ADE2+ ARS305Δ and mcm4-td ubr1Δ::GAL-UBR1 ADE2+ ARS305Δ strains were grown for six generations in YNB lacking (NH4)2SO4 (Difco 0335-15-9), supplemented with 0.1 mM CuSO4, 0.1% 13C glucose and 0.01% 15N (NH4)2SO4 (CK Gas Products Ltd, UK). This medium caused the DNA to be labeled with dense isotopes, but was not compatible with efficient induction of UBR1 expression from the GAL promoter. Therefore, to surmount this problem, cells were arrested in G2/M with 1.7 μg/ml Nocodazole after labeling with dense isotopes, and then washed twice with YP+Galactose containing 0.1 mM CuSO4 and 5 μg/ml Nocodazole. Incubation was continued in this medium at 24°C for 1 hour, to allow cells to adapt to the change of medium. Cells were then transferred to fresh YP+Galactose medium containing 0.1 mM CuSO4 and 5 μg/ml α-factor. After 4 hours at 24°C, cells had arrested in G1 phase, and they were subsequently released into fresh YP+Galactose medium containing 0.2 M hydroxyurea for 90 min, to allow the activation of early origins of replication. The cultures were then shifted to 37°C for 45 min to induce degradation of Mcm4-td, before being released into fresh medium at 37°C, that lacked hydroxyurea. Samples were taken throughout the experiment and used to prepare genomic DNA, which was then digested with Cla I. The samples were then fractionated on CsCl gradients as described previously [R. M. McCarroll and W. L. Fangman, Cell54, 505 (1988)]. The refractive index of the samples was measured, and every second fraction between the values of 1.407 and 1.403 was slot-blotted onto Hybond N+ membrane. The membranes were then hybridized with specific probes labeled with [α-32P] dCTP. The following probes were used (the numbers correspond to those in Fig. 3): probe 1, nucleotides 73001-73958 from chromosome III; probe 2, nucleotides 2062-3100 from chromosome III; probe 3, nucleotides 198945-199832 from chromosome VI; probe 4, nucleotides 260048-261088 from chromosome VI. Hybridization signals were quantitated using a Molecular Dynamics PhosphorImager, and the extent of replication of each fragment was calculated using the following equation: % replication = 100 [0.5 HL/HH + 0.5 HL]. HL and HH represent the proportion of each restriction fragment in the heavy-light and heavy-heavy peaks, respectively.


Supplemental Figure 1. Inactivation of Mcm degron mutants at 37°C is Ubr1 dependent. The indicated strains were streaked at 37°C either on YPD plates (GAL-UBR1OFF) or on YPGal plates (GAL-UBR1ON).


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