Fission yeast rad12+ regulates cell cycle checkpoint control and is homologous to the Bloom's syndrome disease gene

Abstract

The human BLM gene is a member of the Escherichia coli recQ helicase family, which includes the Saccharomyces cerevisiae SGS1 and human WRN genes. Defects in BLM are responsible for the human disease Bloom's syndrome, which is characterized in part by genomic instability and a high incidence of cancer. Here we describe the cloning of rad12+, which is the fission yeast homolog of BLM and is identical to the recently reported rhq1+ gene. We showed that rad12 null cells are sensitive to DNA damage induced by UV light and gamma radiation, as well as to the DNA synthesis inhibitor hydroxyurea. Overexpression of the wild-type rad12+ gene also leads to sensitivity to these agents and to defects associated with the loss of the S-phase and G2-phase checkpoint control. We showed genetically and biochemically that rad12+ acts upstream from rad9+, one of the fission yeast G2 checkpoint control genes, in regulating exit from the S-phase checkpoint. The physical chromosome segregation defects seen in rad12 null cells combined with the checkpoint regulation defect seen in the rad12+ overproducer implicate rad12+ as a key coupler of chromosomal integrity with cell cycle progression.

FIG. 1

Schematic representation of rad12⁺. (a) Alignment of Rad12p and BLMp sequences, showing the conserved acidic (A), basic (B), RecQp-like helicase (H), and extended high-level-homology domains. The acidic and basic regions exhibit greater than 30% acidic or basic residues over a span of at least 20 amino acids. The highly conserved helicase domain has been previously defined (13). The extended region of high-level homology is a region of 37% identity (57% similarity) that extends for an additional 199 amino acid residues past the C terminus of the helicase domain. (b) Corresponding schematic representation of the rad12⁺ gene and the construct used to generate the gene disruption, rad12::ura4⁺ (Sp359).

FIG. 2

Sensitivity of the rad12::ura4⁺ mutant to DNA damage and transient inhibition of replication. Shown are UV light (a), γ radiation (b), and HU (c) survival curves of the wild-type (Sp30) (•), rad12-502 (Sp272) (▪), and rad12::ura4⁺ (Sp358) (▴) strains. (a and b) Strains were grown to mid-log phase, plated at appropriate dilutions, and treated with the indicated doses of radiation. Colonies were allowed to grow 4 days at 32°C, at which time the numbers of colonies were determined. Colony numbers were normalized to those of the nonirradiated samples. (c) Strains were grown to mid-log phase; then HU was added to 12 mM. Samples were removed from the HU at the indicated times and transferred to plates for viability testing. Colonies were allowed to grow 4 days at 32°C, at which time the numbers of colonies were determined. Colony numbers were normalized to those of the samples harvested at the time of addition of HU (time = 0). In all cases, error bars represent the standard deviations of the data. Where error bars are not visible, they are smaller than the symbols representing the data points.

FIG. 3

rad9⁺ acts downstream of rad12⁺ in the repression of basal levels of UVDE activity. (a) Cells were grown to late log phase and then treated with UV where indicated. Cells were allowed to recover for the indicated time periods and then harvested, and whole-cell extracts were prepared. The extracts were assayed for UVDE activity, as measured by cleavage of an oligonucleotide substrate with a single internal UV photoproduct from a 51mer to a 31mer. WT, wild type. (b) Basal levels of uve1⁺ mRNA were determined by Northern analysis in wild-type and rad9 null cells. Samples were subjected to electrophoresis, transfer, and hybridization against uve1⁺ and leu1⁺ probes simultaneously. (c) Time course of uve1⁺ mRNA induction in wild-type (972) and rad9 null (Sp325) cells after treatment with 50 J of UV light/m². Inductions were performed as described in Materials and Methods. Cells were harvested at the indicated times (in minutes) after induction. Poly(A)⁺ RNA was subjected to Northern analysis with probes directed against uve1⁺ and leu1⁺. (d) UVDE activity was assayed as for panel a, using strains rad12-502 (Sp275) (lane 3), rad9::ura4⁺ (Sp325) (lane 2), and rad12-502 rad9::ura4⁺ (Sp345) (lane 1) mutants.

FIG. 4

rad12⁺ and rad9⁺ are epistatic; the aberrant morphology of rad12 mutants is suppressed in a rad9::ura4⁺ background. (a) UV survival curves were constructed as described in Materials and Methods. Wild-type (972) (•) cells are highly resistant to UV light. rad12-502 cells (Sp275) (▪) are moderately sensitive to UV light. rad12-502 rad9::ura4⁺ double-mutant cells (Sp345) (✚) are no more sensitive to UV light than rad9::ura4⁺ single-mutant cells (Sp325) (⧫). (b) Late-log-phase cells which were either wild type (972), rad12-502 (Sp275), rad9::ura4⁺ (Sp325), or rad12-502 rad9::ura4⁺ (Sp345) were fixed with ethanol, stained with propidium iodide, and examined by fluorescence microscopy. rad12-502 cell populations exhibit many elongated cells, which are not present in rad12-502 rad9::ura4⁺ double mutants.

FIG. 5

rad12⁺ OP cells are defective in the S-phase checkpoint. Wild-type (Sp263), rad9::ura4⁺ (Sp325), rad12::ura4⁺ (Sp358), and rad12⁺ OP (Sp359) strains were grown to mid-log phase at 25°C, arrested with HU, and then released 5 h later. (a) Aliquots were fixed with ethanol each hour during the HU block and every 20 min after HU block release. Samples were stained with propidium iodide and analyzed by flow cytometry. (b) The indicated strains were prepared identically to those in panel a. The 0-h (log phase), 5-h post-HU addition (HU blocked), and 140-min post-HU removal (HU released) samples were stained with propidium iodide and examined by fluorescence microscopy.

FIG. 6

Sensitivity of the rad12⁺ OP to HU treatment. HU survival curves of the wild type (972) (•), rad12::ura4⁺ (Sp358) (▪), rad9::ura4⁺ (Sp325) (⧫), and rad12⁺ OP (Sp359) (▴). Strains were grown to mid-log phase; then 12 mM HU was added. Samples were removed from HU at the indicated times and plated. Colonies were allowed to grow for 4 days at 32°C. Colony numbers were normalized to the samples harvested at time zero, the time of addition of HU. In all cases, error bars represent the standard deviations of the data. Where error bars are not visible, they are smaller than the symbols representing the data points.