Distinct roles for Sld3 and GINS during establishment and progression of eukaryotic DNA replication forks

Abstract

The Cdc45 protein is crucial for the initiation of chromosome replication in eukaryotic cells, as it allows the activation of prereplication complexes (pre-RCs) that contain the MCM helicase. This causes the unwinding of origins and the establishment of DNA replication forks. The incorporation of Cdc45 at nascent forks is a highly regulated and poorly understood process that requires, in budding yeast, the Sld3 protein and the GINS complex. Previous studies suggested that Sld3 is also important for the progression of DNA replication forks after the initiation step, as are Cdc45 and GINS. In contrast, we show here that Sld3 does not move with DNA replication forks and only associates with MCM in an unstable manner before initiation. After the establishment of DNA replication forks from early origins, Sld3 is no longer essential for the completion of chromosome replication. Unlike Sld3, GINS is not required for the initial recruitment of Cdc45 to origins and instead is necessary for stable engagement of Cdc45 with the nascent replisome. Like Cdc45, GINS then associates stably with MCM during S-phase.

Figure 1

Sld3 does not move with DNA replication forks away from origins. (A, B) The indicated strains were grown at 24°C in YPRaff medium and then synchronised in G1-phase with mating phermone. The cells were then transferred to YPGal medium containing mating pheromone for 50′, washed twice in YPGal medium, and samples taken at the indicated times for (a) ChIP analysis and (b) flow cytometry. The strains in (B) expressed a stable form of the CDK inhibitor Sic1 (Sic1ΔNT) under the control of the GAL1,10 promoter.

Figure 2

Sld3 and Cdc45 interact unstably (or indirectly) with MCM before initiation; GINS and Cdc45 interact stably with MCM after initiation (also see Supplementary Figure 4). (A) (a) YMK456 was grown at 24°C in YPD medium and then synchronised in G1-phase with alpha factor (G1), before releasing into fresh medium containing 0.2 M hydroxyurea (HU) for 90′. At each stage of the experiment, aliquots of cells were incubated in the presence or absence of the crosslinking agent formaldehyde, and cell extracts were prepared and then processed as for ChIP. After immunoprecipitation of Mcm4-5FLAG, tagged proteins were detected by immunoblotting with specific monoclonal antibodies; and (b) a similar experiment was performed with YMK487. (B) (a) YMK301 and (b) YMK425 were synchronised in G1-phase at 24°C in YPRaff medium as above and a sample was then processed for ChIP (24°C). Cells were then washed twice with YPGal medium containing mating pheromone and incubated at 24°C for 40′ to induce expression of GAL-UBR1, before shifting the culture to 36°C for 60′ to deplete Mcm4-td protein in YMK425, and taking another ChIP sample (36°C). (c) Cell extracts were prepared and used to confirm by immunoblotting that Cdc45-18Myc was not affected by degradation of Mcm4-td in YMK425 at 36°C.

Figure 3

Sld3 is not required for completion of chromosome replication after release from HU arrest. (A) YMK302 (control), YBH42 (cdc45-td), YKL55 (cdc7-1) and YMK517 (sld3-7td) were grown as for the experiment in Figure 2B, and then released from G1 arrest at 37°C for the indicated times. After confirming that budding had occurred in all cultures, mating pheromone was added once again at the 45′ timepoint, so that any cells completing mitosis and cell division would arrest in the subsequent G1 phase. (B) An equivalent experiment was performed in which cells were first released from G1 arrest for 60′ into YPRaff medium containing 0.2 M HU at 24°C, before induction of GAL-UBR1 in YPGal+HU medium at 24°C for 35′, transfer to YPGal+HU medium at 37°C for 50′, and finally release into YPGal medium without HU for the indicated times. As before, mating pheromone was added again 45′ after release from HU arrest.

Figure 4

Recruitment of Cdc45 to origins of DNA replication without GINS. (A) YMK301 (CDC45-18MYC) and YMK292 (cdc102-td CDC45-18MYC) were grown in YPRaff medium at 24°C and then synchronised in G2/M phase with nocodazole, before inducing expression of GAL-UBR1 for 35′. Cells were shifted to 36°C for 50′ to deplete Psf2/Cdc102-td in YMK292 and a sample processed for ChIP (G2/M). Cells were then washed twice in fresh medium containing alpha factor and incubation was continued at 36°C for 90′ until cell division was complete and cells arrested in the next G1-phase, before processing another ChIP sample (G1). (B) DNA content was measured by flow cytometry throughout the experiment; cells were also released from G1-arrest at the end of the experiment for the indicated times, to confirm that chromosome replication did not occur after depletion of Psf2/Cdc102-td.

Figure 5

GINS is essential for incorporation of Cdc45 into an active replisome. (A) YMK301 (CDC45-18MYC) and YMK292 (cdc102-td CDC45-18MYC) were synchronised in G1 phase as above, before induction of GAL-UBR1 for 45′ and depletion of Psf2/Cdc102-td in YMK292 at 36°C. Cells were then released from G1-arrest at 36°C and samples processed for ChIP (a) and flow cytometry (b) at the indicated times. (B) A similar experiment was performed with shorter timepoints, to facilitate analysis of fork progression through the region adjacent to ARS305. In (a) and (b), the data corresponding to the ARS305 proximal region are duplicated in an enlarged form below the main graph for greater clarity.

Figure 6

Mcm4 remains at ARS305 when cells enter S-phase in the absence of GINS, and Sld3 is slowly lost from the origin. (A) Experiments equivalent to those shown in Figure 5A were performed with YMK423 (MCM4-FLAG) and YMK424 (MCM4-FLAG cdc102-td). ChIP and flow cytometry data are presented as before. Nocodazole was added to the medium 50′ after release from G1 arrest, to prevent any cells that had completed chromosome replication from passing through mitosis. (B) Identical experiments were performed with YMK297 (SLD3–MYC) and YMK296 (SLD3–MYC cdc102-td).

Figure 7

GINS is essential for activation of the pre-RC. (A) (a) Control cells (YMK302) were synchronised in G1-phase or G2/M phase as before and protein–DNA complexes at ARS305 (indicated by the black bar) were then examined by genomic footprinting. The asterisks mark three ORC-induced hypersensitive sites that are suppressed when pre-RC formation occurs during G1-phase. (b) Control (YMK302) and cdc102-td cells (YMK458) were synchronised in G1-phase as above and then Psf2/Cdc102-td was depleted in YMK458 for 45′ before both strains were released into fresh medium at 36°C. Samples were taken at the indicated times. (B) The region of the gel that corresponds to the three hypersensitive sites in (A) (b) was quantified for the 0′ and 60′ samples as described in Materials and methods.

Figure 8

A model for the loading of Cdc45 at origins by Sld3 and GINS during the initiation of chromosome replication. MCM is assembled into pre-RCs at origins during G1-phase (for simplicity other proteins such as the Origin Recognition Complex are not shown). Following activation of CDK and Cdc7, Sld3 recruits GINS and Cdc45 to MCM, converting the pre-RC into a pre-IC. The order of assembly may vary in different species or at different origins; thus, at early origins in budding yeast the initial recruitment of Cdc45 occurs during G1-phase. GINS then allows stable incorporation of Cdc45 into an active replisome, so that MCM–Cdc45–GINS move away with the newly formed DNA replication forks. Sld3 is displaced during the initiation reaction.