PMC

Strains used in these experiments are listed in , and were isogenic or congenic with W303 RAD5 ⁺. Segregants of a MEC1/mec1Δ TEL1/tel1Δ DNA2/dna2–2 SML1/sml1Δ diploid were placed on a YPD plate incubated at 30 °C (A) or 37 °C (B).

Data were compiled with the Payek program using the dna2 screen results in and the results of the previous screens [,,], as well as data compiled for candidate genes synthetically lethal with rrm3Δ [,]. The data are presented in tabular form in .

Strain W303 dna2–1 was transformed with the plasmids pRS424G and pRS424G-EXO1, an empty plasmid vector and a plasmid expressing ExoIp from the GAL110 promoter, respectively. W303RAD5 strains dna2–1 7A(pRS424G) and dna2–1(pRS424G-EXO1) were grown to mid log phase and serially diluted on yeast-dextrose galactose- and raffinose-containing plates and incubated at 30 °C for 5 d.

mrc1Δ experiments were carried out in the isogenic 4741 strain and are listed in . mrc1AQ experiments were carried out in strains isogenic with W303 RAD5 ⁺.
Panel 1 (top): segregants from a DNA2/dna2–2 MRC1/mrc1Δ diploid. Segregants are isogenic with 4741. d, dna2–2; m, mrc1Δ.
Panel 2: segregants from a MRC1/mrc1AQ RAD9/rad9Δ DNA2/dna2–2 diploid. d, dna2–2; m, mrc1AQ; r, rad9Δ.
Panel 3: segregants from a DNA2/dna2–1 MRC1/mrc1AQ RAD9/rad9Δ strain. d, dna2–1; m, mrc1AQ; r, rad9Δ.
Panel 4 (bottom): segregants from a RRM3/rrm3Δ MRC1/mrc1AQ diploid. m, mrc1AQ; r, rrm3.

(A) WT, pol32Δ, dna2–1 pol32Δ, and dna2–1 strains were grown to log phase, serially diluted, and plated on YPD plates and incubated at 23 °C, 30 °C, and 37 °C for 5 d.
(B) WT, pol32Δ, dna2–2, and pol32Δ dna2–2 strains were grown to log phase, serially diluted, and incubated on MMS-containing YPD plates for 3 d at 30 °C.
(C) WT, pol32Δ, dna2–1 pol32Δ, and dna2–1 strains were grown to log phase, serially diluted, and grown on MMS-containing YPD plates for 5 d at 23 °C.
All strains are isogenic with strain 4741 (). (dna2–1 grows slowly even at 23 °C, and plates at 23 °C were photographed before they were fully grown so that the other strains would not be overgrown.)

Strain W303 dna2–1 carrying a TRP1 CEN3 DNA2 plasmid was transformed with an integrating URA3 pol3–01 plasmid [] cut with BamH1. The transformants were streaked on 5-FOA medium to excise the WT POL3 gene and identify clones with the pol3–01 mutation. Three transformants carrying the pol3–01 mutation (isolates 2, 8, and 11) were restreaked on YPD medium containing 2-amino-5-fluorobenzoic acid (FAA), which selects for strains that have lost the DNA2 TRP1–containing plasmid []. Three dna2–1 pol3–01 colonies showing some residual growth on the FAA plates were restreaked on YPD-containing medium. The same three isolates of dna2–1 pol3–01 but containing the DNA2 TRP plasmid, i.e., that had not been grown in the presence of FAA, are shown as controls, as indicated.

Hierarchical two-dimensional clustering analysis was applied to the DNA2 interactions and those of 43 genes synthetically lethal with DNA2 (see ). The interactions were clustered with respect to the results of the current screen and 43 previous screens using these genes as queries. A total of 322 genes and 876 interactions, each indicated by a red box, were identified. This panel shows a zoom into the region of most significant overlap of shared genetic interactions. In addition to including reduced fitness genes, this analysis includes genes that give increased fitness with dna2, such as pol32Δ.

Top panel: Tetrads from the dissection of a DNA2/dna2–2 CTF4/ctf4Δ FOB1/fob1Δ heterozygote. c, ctf4Δ; d, dna2Δ; f, fob1Δ; WT, DNA2 CTF4 FOB1. The following spores were recovered: 11 WT, 21 dna2–2, 26 fob1Δ, 17 ctf4Δ, 18 dna2–2 fob1Δ, 20 ctf4Δ fob1Δ, zero ctf4Δ dna2–2, and six dna2–2 ctf4Δ fob1Δ. Since the triple mutant grew slowly at 30 °C, another 27 spores were dissected and incubated at 23 °C. The following spores were recovered: 14 WT, nine fob1Δ, 14 ctf4Δ, seven dna2–2, 14 dna2–2 fob1Δ, nine ctf4Δ fob1Δ, zero dna2–2 ctf4Δ, and nine dna2–2 ctf4Δ fob1Δ. The triple mutant did not grow at 37 °C.
Bottom panel: X-ray sensitivity of dna2–2 ctf4Δ fob1Δ. Strains with genotype WT, fob1Δ, dna2–2 fob1Δ, ctf4Δ fob1Δ, and dna2–2 ctf4Δ fob1Δ were grown to log phase, irradiated as described in Materials and Methods, serially diluted, plated on YPD plates, and incubated at 30 °C. Strains are isogenic or congenic with 4741.