Characterization of Saccharomyces cerevisiae dna2 mutants suggests a role for the helicase late in S phase

Abstract

The TOR proteins, originally identified as targets of the immunosuppressant rapamycin, contain an ATM-like "lipid kinase" domain and are required for early G1 progression in eukaryotes. Using a screen to identify Saccharomyces cerevisiae mutants requiring overexpression of Tor1p for viability, we have isolated mutations in a gene we call ROT1 (requires overexpression of Tor1p). This gene is identical to DNA2, encoding a helicase required for DNA replication. As with its role in cell cycle progression, both the N-terminal and C-terminal regions, as well as the kinase domain of Tor1p, are required for rescue of dna2 mutants. Dna2 mutants are also rescued by Tor2p and show synthetic lethality with tor1 deletion mutants under specific conditions. Temperature-sensitive (Ts) dna2 mutants arrest irreversibly at G2/M in a RAD9- and MEC1-dependent manner, suggesting that Dna2p has a role in S phase. Frequencies of mitotic recombination and chromosome loss are elevated in dna2 mutants, also supporting a role for the protein in DNA synthesis. Temperature-shift experiments indicate that Dna2p functions during late S phase, although dna2 mutants are not deficient in bulk DNA synthesis. These data suggest that Dna2p is not required for replication fork progression but may be needed for a later event such as Okazaki fragment maturation.

Figure 1

Identification of the mutations in dna2–20 and dna2–21 alleles. Schematic drawing of the DNA2 sequence with the location of the seven helicase domains shown hatched and drawn to scale. Regions that contain the mutations are enlarged. The yeast and human DNA2 sequences are shown on the top and bottom, respectively, with conserved residues connected by bars. The mutated residue is boxed and the resulting amino acid shown in parentheses.

Figure 2

Rescue of dna2–20 is TOR-specific. The dna2–20 mutant carrying pYDF88 (GAL1p→TOR1) was transformed with the following plasmids: pYDF18 (vector), pYDF66 (Tor1p), pJK3–3 (Tor2p) (Kunz et al., 1993), 324.34 (Vps34p) (Herman and Emr, 1990), YEp352-STT4 (Stt4p) (Yoshida et al., 1994), and YEp352-PIK1 (Pik1p) (Flanagan et al., 1993). All of these plasmids are 2μ-based and contain the genes under control of their endogenous promoters, except for the Tor1p expression construct that utilizes the GPD promoter (see MATERIALS AND METHODS). Transformants were selected on SCGal-leu minus the appropriate amino acid to also select for the various plasmids. Cells were scraped off plates (representing ∼100 colonies), resuspended in water, sonicated, and spotted onto YPD and YPGal plates. Ten thousand cells were spotted on the leftmost region with successive fivefold dilutions moving to the right. The plates were incubated for 3 d at 30°C and photographed.

Figure 3

Regions of Tor1p required for rescue of dna2–20 mutant. The dna2–20 mutant carrying pYDF88 (GAL1p→>TOR1) was transformed with various Tor1p deletion constructs (see MATERIALS AND METHODS). Cells were scraped off transformation plates (∼100 colonies), resuspended in water, sonicated, and spotted onto YPD plates. Spots represent 1200 and 150 cells applied. Plates were incubated at 30°C for 3 d. The apparently elevated activity seen in the Δ1–416 mutant is not reproducible and is thus represented as +. The absence of suppression by the Δ1–724 construct may be due to low protein expression, as this mutant protein is not detected by immunoblot analysis (our unpublished results).

Figure 4

dna2 mutants show synthetic interactions with the TOR pathway. (A). dna2 tor1 mutants are slow growing. Strains YMW1 (WT), YDF20 (Δtor1), 2–71 (dna2–20), 2–174 (dna2–21), YDF117 (dna2–20 Δtor1), and YDF121 (dna2–21 Δtor1) were streaked on YPD plates containing 0.5 M sorbitol at 37°C for 60 h before photographing. (B) dna2 mutants show increased rapamycin sensitivity. Strains YMW1 (WT) and YDF20 (Δtor1), and 2–174 (dna2–21) were streaked on YPD plates containing indicated amounts of rapamycin at 30°C and photographed after 72 h of growth.

Figure 5

A dna2-Δ20 deletion mutant is inviable and not suppressed by Tor1p. YDF27 (dna2-Δ20/DNA2⁺) (A), or YDF27 transformed with pYDF113 (DNA2) (B), or pYDF77 (GAL1p→TOR1) (C) were sporulated and tetrads dissected on either YPD (A), (B), or YPGal (C), and plates were incubated at 30°C for 4 d and photographed.

Figure 6

(A) Dna2p and Tor1p do not interact in vitro. Extracts from CB018 transformants were prepared and immunoprecipitations (labeled “IP”) performed with either affinity-purified anti-Dna2p antibodies (α-Dna2p) or 12CA5 (α-Tor1p) ascites. Washed immunoprecipitates were loaded onto 6% SDS-PAGE gels and immunoblotted with either anti-Dna2p (left) or anti-HA (right) antibodies. + and − indicate the presence and absence, respectively, of extract in the immunoprecipitate. (B). Effect of Tor1p overexpression on Dna2p levels. Left panel, strain CB018 was transformed with either PRS315 (vector) or pYDF125 (TOR1), and immunoblots were probed with either affinity-purified anti-Dna2p antibodies or anti-actin antisera. Right panel, strains YMW1(WT), 2–71(dna2–20), and 2–174(dna2–21) were transformed with pYDF77 (GAL1p→TOR1) and grown for 5 h in YPGal and either prepared for extracts (Gal) or shifted to YPD for 9 h (Dex) before preparation of extracts. Equal amounts of protein were loaded onto 8% gels and immunoblots probed with either anti-Dna2p (top) or anti-actin (bottom) antibodies. Protein levels were quantified by densitometry and normalized to actin levels.

Figure 7

Phenotype of the dna2–22 Ts mutant. Strains YDF100 (DNA2⁺) and YDF102 (dna2–22) were grown at 30°C in YPD to a density of 1.5 × 10⁶ cells/ml, and cultures were split and incubated at either 30°C or 37°C for 4 h. Top panel, DAPI staining. Shown are DIC images with DAPI staining overlaid using Adobe Photoshop 3.0. Bottom panel, cells were processed for propidium iodide (PI) staining and flow cytometric analysis.

Figure 8

dna2 mutants are rescued by DNA checkpoint mutations. (A) Strains YDF92 (mec1–1), YDF94 (dna2–22 mec1–1), YDF96 (rad9), YDF98 (dna2–22 rad9), YDF100 (WT), and YDF102 (dna2–22) were streaked on YPD plates containing 1 M sorbitol at 37°C for 3 d before photographing. (B) Strain 2–71 (dna2–20) was mated to strains YDF100 (WT), YDF92 (mec1–1), and YDF96 (rad9). After sporulation of diploids, tetrads were dissected on YPD. Plates were incubated at 30°C for 3 d before photographing.

Figure 9

Dna2p executes its function at around the time of late S phase. (A and B) dna2–22 cells lose viability at 37°C but not 30°C. (A) Strains YDF100 (DNA2⁺) and YDF102 (dna2–22) were synchronized with α factor and released at 30°C. In panel B, YDF102 (dna2–22) cells were synchronized as in panel A but then preincubated at 37°C for various times before α factor release. Aliquots were taken at the times shown and assayed for colony-forming ability at 30°C. All numbers are normalized to values at t = 0 after α factor release. The black bar in panel B represents the time of DNA synthesis (see panel C) in the 1-h preincubation samples. (C) Kinetics of DNA synthesis in the dna2–22 mutant. Aliquots of cells from the 1-h preincubation protocol in panel B were taken every 10 min after α factor release and analyzed for DNA content by FACS analysis.

Figure 10

The Dna2p execution step depends upon S phase but not mitosis. (A) DNA2 executes its function after the HU-dependent step. Strains YDF100 (DNA2⁺) and YDF102 (dna2–22) were synchronized with α factor, shifted to 37°C for 3 h, and released from α factor at 37°C into YPD (control) or YPD+0.2 mM HU (+HU). At t = 0 and 60 min after release, cells were diluted into YPD and plated at 30°C. Colonies were counted after 2 d. (B) DNA2 executes its function independent of the nocodazole-dependent step. Cells were treated as in panel A except that, after α factor release, the strains were incubated in either YPD (control) or YPD+15 μg/ml nocodazole +nocodazole). Cells were then diluted and plated as in panel A. Results are expressed as the number of colonies formed at t = 60 min after α factor release divided by the number of colonies formed at t = 0 min after release.

Figure 11

dna2 mutants replicate the bulk of their DNA. Strains YDF100 (DNA2⁺), YDF102 (dna2–22), and tsDNA2 (Budd and Campbell, 1995) were grown to OD 0.25 in YPD and arrested at 30°C with 10 μg/ml α factor. After 3.5 h, cultures were spun down and resuspended in prewarmed (37°C) YPD+0.2 mM HU and incubated at 37°C for 3 h. Cells were then washed and resuspended in prewarmed (37°C) YPD, and 5-ml aliquots were taken and analyzed for DNA content.