mcm5/cdc46-bob1 bypasses the requirement for the S phase activator Cdc7p - PubMed (original) (raw)

mcm5/cdc46-bob1 bypasses the requirement for the S phase activator Cdc7p

C F Hardy et al. Proc Natl Acad Sci U S A. 1997.

Abstract

Cdc7p is a protein kinase that is required for G1/S transition and initiation of DNA replication in Saccharomyces cerevisiae. The mechanisms whereby Cdc7p and its substrates exerts their effects are unknown. We report here the characterization in S. cerevisiae of a recessive mutation in a member of the MCM family, MCM5/CDC46, which bypasses the requirement for Cdc7p and its interacting factor Dbf4p. Because the MCM family of evolutionarily conserved proteins have been implicated in restricting DNA replication to once per cell cycle, our studies suggest that Cdc7p is required late in G1 because in its absence the Mcm5p/Cdc46p blocks the initiation of DNA replication. Moreover, Mcm5p/Cdc46p may have both positive and negative effects on the ability of cell to initiate replication.

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Figures

Figure 3

Figure 3

FACS analysis of wild-type and cdc46-bob1 strains. Cells of wild-type strain 311 (dark solid line) and cdc46-bob1 strain 728 (light dotted line) were synchronized by α-factor treatment at 22°C in rich medium and samples were analyzed by FACS as described (30). Cell number is plotted versus the DNA content of the cells. Depicted are samples taken at 0, 80, 90, and 100 min after release from α-factor. 1C and 2C indicates the position of G1 and G2 phase cells, respectively. Yeast strains 311 and 728 are isogenic with A364a.

Figure 1

Figure 1

The bob1 mutation results in a Pro → Leu substitution at residue 83 of Mcm5p/Cdc46p. (A) bob1::URA3 dbf4-1 strains expressing wild-type Cdc46p, mutant cdc46-bob1p, or mutant/wild-type chimeras were tested for growth at 23°C and 37°C. +, Growth; −, no growth after 5 days on rich media. (B) Pro is conserved among Mcm2p, Mcm4p, and Mcm5p family members. The conserved residues are shown in boldface type.

Figure 2

Figure 2

Cell cycle distribution of cdc7 and bob1 strains. Cell number is plotted versus the DNA content of the cells. 1C and 2C indicate the amount of DNA in a G1 and G2 phase haploid cell, respectively. Cells of yeast strains 421 BOB1 CDC7, P180 bob1 CDC7, P208 BOB1 cdc7-4, P171 cdc7-4 bob1, and P190 cdc7::HIS3 bob1 are isogenic with A364a, grown in rich media at 22°C and processed for FACS analysis as described (30).

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