The Dbf4-Cdc7 kinase promotes S phase by alleviating an inhibitory activity in Mcm4

Abstract

Eukaryotic DNA replication uses kinase regulatory pathways to facilitate coordination with other processes during cell division cycles and response to environmental cues. At least two cell cycle-regulated protein kinase systems, the S-phase-specific cyclin-dependent protein kinases (S-CDKs) and the Dbf4-Cdc7 kinase (DDK, Dbf4-dependent protein kinase) are essential activators for initiation of DNA replication. Although the essential mechanism of CDK activation of DNA replication in Saccharomyces cerevisiae has been established, exactly how DDK acts has been unclear. Here we show that the amino terminal serine/threonine-rich domain (NSD) of Mcm4 has both inhibitory and facilitating roles in DNA replication control and that the sole essential function of DDK is to relieve an inhibitory activity residing within the NSD. By combining an mcm4 mutant lacking the inhibitory activity with mutations that bypass the requirement for CDKs for initiation of DNA replication, we show that DNA synthesis can occur in G1 phase when CDKs and DDK are limited. However, DDK is still required for efficient S phase progression. In the absence of DDK, CDK phosphorylation at the distal part of the Mcm4 NSD becomes crucial. Moreover, DDK-null cells fail to activate the intra-S-phase checkpoint in the presence of hydroxyurea-induced DNA damage and are unable to survive this challenge. Our studies establish that the eukaryote-specific NSD of Mcm4 has evolved to integrate several protein kinase regulatory signals for progression through S phase.

Figure 1. An inhibitory activity within the Mcm4 NSD is responsible for the dependency of cells on DDK for viability

a, Yeast strains were grown on YPD plates at permissive and non-permissive temperatures (30°C and above for cdc7-4 and 35°C and above for dbf4-1). The mcm4^Δ2-174 allele was introduced to cdc7-4 and dbf4-1 cells by two-step gene replacement. Shown are parental strains (top sectors) and three isolates of the second-step homologous recombination products. b, The dbf4-1 mcm4^Δ2-174 cells transformed with empty vector (V) or vector carrying MCM4 were streaked on selective media and allowed to grow at 37°C or 22°C for 5 days. c, diagram of Mcm4 and summary of transformation assay and complementation of mcm4Δ by the same plasmid constructs (Fig. S2c). However, mcm4^{Δ2-145,5A+2A} does exhibit growth defect even in the presence of DDK .

Figure 2. The status of the Mcm4 N-terminus determines the efficiency of DDK-independent cell proliferation

a-c, a modified plasmid shuffle assay was used to identify MCM4 alleles that allow cell growth in the absence of CDC7 and MCM4 when expressed from single-origin vectors. The tester yeast strain was constructed with both MCM4 and CDC7 genes deleted from its chromosomes while carrying both essential genes on plasmids with the URA3 gene that can be counter-selected in the presence of 5-FOA. The tester cells were transformed with indicated plasmids and assayed for growth on YPD and 5-FOA media. All mcm4 alleles used in the assay complement mcm4Δ. a, DDK-independent cell growth by the mcm4 mutants described in figure 1. b, Top panel, diagram of mcm4^Δ74-174 derivatives with mutations at CDK phosphorylation sites and their preceding phospho-acceptor residues within the distal NSD (residues 2-73). Bottom panel, the ability of mcm4 alleles described above to support DDK-independent growth. c, DDK-independent cell growth supported by mcm2^1-200-mcm4^Δ2-174.

Figure 3. Removal of the N-terminal inhibitory domain of Mcm4 allows DDK-independent initiation of DNA replication and S phase progression

a, Cell cycle progression of WT, mcm4^Δ74-174 and cdc7Δ mcm4^Δ74-174 cells. Cells were synchronized by G1 arrest and released into fresh YPD media at 25°C, collected at indicated times, and analyzed for DNA content by flow cytometry. b, Kinetics of Cdc45-MCM complex formation. Cell extracts were prepared from samples collected in a and subjected to immuno-precipitatation (IP) using monoclonal antibody against Mcm2. Cell extracts and IP were analyzed by immunoblotting. c, Flow cytometry analysis for DNA content in G1. CDK bypass cells containing SD fusion (sld3-600,609,622A-DBP11(253-764) and GAL-sld2-T84D, with or without additional modification (GAL-DBF4 or mcm4^Δ74-174) were synchronized and held in G1 using α-factor (α-F) and galactose (gal) was added to induce expression of sld2-T84D and Dbf4.

Figure 4. Deletion of the N-terminal inhibitory domain of Mcm4 does not bypass the requirement for DDK for cell survival and checkpoint activation in the presence of HU

a, Yeast strains of indicated genotypes at endogenous loci were grown on YPD media with or without 100 mM HU. b and c, Immunoblot analysis for Rad53 and Orc6 phosphorylation status in WT, mcm4^Δ74-174 and cdc7Δ mcm4^Δ74-174 cells. b, Protein samples were prepared from cells synchronized in G1 and released into 200 mM HU for indicated time. c, Protein samples were prepared from log-phase cells treated with HU for indicated time.