Unlike sgs1, rrm3 is not lethal with mus81 or slx1. Tetrad dissection of diploids heterozygous for deletion of RRM3, SGS1, and MUS81 or SLX1. In panels A and B, circles indicate deletion of the gene of interest (gene X), boxes indicate rrm3 gene X double mutants, and, for comparison, diamonds indicate sgs1 gene X double mutants. In cases in which only two spores grew, inferred mutants are marked if their genotype could be determined.

Interactions of rrm3 with sgs1 and with genes that are synthetically lethal with sgs1. Tetrad dissection of indicated heterozygous diploids. In panels A through D, circles indicate single deletion of the gene of interest (gene X), boxes indicate rrm3 gene X double mutants, and, for comparison, diamonds indicate sgs1 gene X double mutants. In panels A, B, and D, unlabeled microcolonies are rrm3 sgs1 double mutants. In cases in which only two spores grew, inferred mutants are marked if their genotype could be determined.

Intra-S-phase and DNA damage checkpoints. Organization of genes involved in the intra-S-phase and DNA damage checkpoints. The boxed genes were deleted and tested for their ability to confer lethality or slow growth in an rrm3 background. Genes whose deletion had no effect on growth of rrm3 cells are in a dashed box. Genes whose deletion resulted in a modest growth defect in an rrm3 strain are in thin-lined boxes. Genes whose deletion resulted in no or extremely slow growth of rrm3 cells are in boldface and thick-lined boxes. Finally, genes whose deletion resulted in no or extremely slow growth of rrm3 cells at 23°C are marked with an asterisk.

Deletion of RAD51 has no effect on rrm3-dependent replication defects or activation of Rad53p. (A) 2D replication gel analysis of StuI-digested rDNA as previously described (37) from rad51, rrm3 rad51, sgs1 rad51, and rrm3 sgs1 rad51 cells. See the legend to Fig. 6 for details. (B) Western blot analysis of Rad53p hyperphosphorylation. Protein samples from wild-type (Wt), rrm3, sgs1, rad51, rrm3 rad51, sgs1 rad51, and rrm3 sgs1 rad51 cells were analyzed by Western blotting. Detection of Rad53p with anti-Rad53p antibodies is as previously described (35, 58). An arrow points to Rad53p, and P indicates hyperphosphorylated forms of Rad53p.

Model for how cells detect and repair rrm3-dependent DNA damage. Rrm3p functions during DNA replication to allow forks to move past nonnucleosomal protein-DNA complexes. Absence of Rrm3p leads to fork stalling and fork breakage. Stalled forks activate the intra-S-phase checkpoint. Stalled forks are also prone to breakage, which can activate the DNA damage checkpoint. Collapsed and broken forks are restarted or repaired by a Rad51p-dependent and Srs2p-inhibited recombination pathway. The Sgs1p/Top3p complex is needed to resolve the Rad51p-generated intermediates. However, the Mus81p/Mms4p endonuclease does not act on rrm3-induced recombination intermediates. BIR is also capable of restarting broken replication forks.

BIR contributes to rrm3 sgs1 rad51 and rrm3 srs2 rad51 cell growth. Shown are tetrad dissections of diploids homozygous for deletion of RRM3 and heterozygous for deletion of indicated genes. In panels A, B, D, and E, circles indicate rrm3 sgs1 rad51 and rrm3 srs2 rad51 triple mutants and boxes indicate rrm3 sgs1 rad51 rad59 and rrm3 srs2 rad51 rad59 or rrm3 sgs1 rad51 rad1 and rrm3 srs2 rad51 rad1 quadruple mutants. In panel B, diamonds indicate rrm3 rad59 double mutants. In panel E, diamonds indicate rrm3 rad1 double mutants. (C) rrm3 rad59 cells have a modest growth defect. Complete medium was streaked with wild-type (WT), rrm3, rad59, and rrm3 rad59 cells, and the cells were allowed to grow for 2 days at 30°C. The right panel shows a close-up of the colonies.

The intra-S-phase and DNA damage checkpoints are required for normal growth of rrm3 cells. Tetrad dissection of indicated heterozygous diploids. In panels A, C, E, F, and G, hexagons indicate rrm3 cells; circles indicate rad53 sml1, chk1, rad24, rad9, and mrc1 cells; and boxes indicate rrm3 rad53 sml1, rrm3 chk1, rrm3 rad24, rrm3 rad9, and inferred rrm3 mrc1 mutants. In panel A, SML1 was deleted along with RAD53, as deletion of SML1 is required for viability of rad53 cells. In panel E, rrm3 sgs1 spores are in diamonds. For panels F and G, tetrads that could not be genotyped are unmarked. In panels B and D, complete medium was streaked with wild-type (WT), rrm3, rad53 sml1, rrm3 rad53 sml1, chk1, and rrm3 chk1 cells and the cells were allowed to grow for 2 days at 30°C.

rrm3 has little or no sensitivity to DNA-damaging agents. (A) Summary of data for rrm3, sgs1, and srs2 sensitivities to HU, MMS, UV light, and CPT. A superscript 1 indicates that our results are in agreement with previously published results for sgs1 (summarized in reference 7). A superscript 2 indicates previously published results (summarized in reference 7 for srs2). Wt, wild type. (B) Wild-type, rrm3, and sgs1 cells were grown to an optical density at 660 nm of 0.8, serially diluted, and plated onto media containing either 75 mM HU, 0.01% MMS, or 10 μg of CPT per ml. Alternatively, cells were plated on complete medium and treated with 30 J of UV light per m². YC cultures represent the same dilutions plated on complete medium without DNA-damaging agents. Cells were grown at 30°C for 3 days. (C) Tetrad dissection of diploids heterozygous for rrm3, sgs1, and rad27. Circles indicate rad27 single mutants, and boxes indicate rrm3 rad27 double mutants. Unlabeled microcolonies indicate rrm3 sgs1 double mutants.

RRM3 genetic interactions. (A) List of RRM3 genetic interactions. The result “Lethal” indicates that upon dissection, the rrm3 gene X double mutant did not form visible colonies after 3 days at 30°C or formed microcolonies that grew extremely slowly. ++, normal growth rate; +−, moderately reduced growth rate; CS, cold sensitive at 23°C; 1, mutant alone is slow growing, but slow growth is not exacerbated by deletion of RRM3. (B) Assessment of synthetic lethal interactions. In plate sections i to viii, respectively, FOA was streaked with rrm3, rrm3 mrc1, rrm3 mre11, rrm3 rad50, rrm3 xrs2, rrm3 sgs1, rrm3 srs2, and rrm3 top3 spore products carrying pIA20 (RRM3 URA3 ADE3) and the plate was assessed for growth after 3 days at 30°C. Lack of growth on FOA indicates that the strain is inviable in the absence of pIA20.

Deletion of RAD51 (but not RAD52) suppresses some but not all rrm3 synthetic phenotypes. (A) Summary of synthetic lethal phenotypes and their ability to be suppressed by deletion of recombination and checkpoint genes. (B to E) Deletion of RAD51 but not RAD52 rescues the rrm3 sgs1 and rrm3 srs2 lethal phenotypes. Tetrad dissections of indicated heterozygous diploids. Diamonds indicate rrm3 rad51 or rrm3 rad52 double mutants; circles indicate rrm3 sgs1 or rrm3 srs2 double mutants; and boxes indicate rrm3 sgs1 rad51, rrm3 srs2 rad51, rrm3 sgs1 rad52, and rrm3 srs2 rad52 triple mutants. (F) rrm3 rad52 cells have a modest growth defect. Complete medium was streaked with wild-type (WT), rrm3, rad52, and rrm3 rad52 cells, and the cells were allowed to grow for 2 days at 30°C. The right panel shows a close-up of the colonies. (G and H) Deletion of RAD51 does not rescue rrm3 mrc1 lethality and rrm3 rad53 sml1 slow growth. Diploid cells harboring the CEN4 plasmid pIA20, homozygous for deletion of RRM3 and heterozygous for deletion of indicated genes, were dissected. Due to the wild-type copy of RRM3 in pIA20, triple and quadruple mutants were rare. (G) rrm3 mrc1 spores are circled, and rrm3 mrc1 rad51 spores are boxed. Only 1 of 12 rrm3 mrc1 rad51 spores formed a microcolony. (H) rrm3 rad53 sml1 spores are circled, and rrm3 rad53 sml1 rad51 spores are boxed.

Methods for determining synthetic lethality: rrm3 sgs1 cells are dead. (A) Schematic of pIA20 plasmid and sectoring assay. The CEN4 plasmid pIA20 contains the RRM3, URA3, and ADE3 genes. While ade2 ade3 cells generate white colonies, ade2 ade3 cells carrying pIA20 generate red colonies. Loss of pIA20 from ade2 ade3 cells is detected by the presence of white sectors in red colonies. (B) Sectoring assay. Low-adenine plates were streaked with freshly sporulated wild-type, rrm3, sgs1, and rrm3 sgs1 cells carrying pIA20, and the cells were allowed to grow at 30°C for 3 days and at 4°C for 3 days to allow red color development. (C) Tetrad dissection of diploids heterozygous for rrm3 and sgs1. In this and subsequent figures, the four spores from a given tetrad are in a vertical line and dissected spores were allowed to grow at 30°C for 3 days and genotyped by replica plating to test media. Assuming 2:2 segregation of the marker inserted into the deleted genes allows one to identify rrm3 sgs1 double mutants (boxed). (D) Confocal microscope images of YOYO-1-stained rrm3 sgs1 cells from microcolonies shown in panel C.

sgs1 and srs2 cells have a wild-type rDNA replication pattern. (A) Schematic of a small portion of the ∼150-repeat rDNA array. Positions of StuI sites are indicated. The arrowhead and arrow indicate, respectively, the 5S rRNA and the 35S rRNA coding regions. The position of the ARS or replication origin is also indicated. The ARS is active as an origin in only 10 to 20% of rDNA repeats. (B) Schematic of rDNA replication. The left panel shows the progression of rightward-moving forks, with rrm3-dependent pauses indicated by broken lines and lowercase letters c, d, and e in repeats that do not have an active origin. The panel on the right shows bidirectional replication through the StuI fragment in the subset of repeats that have an active origin. StuI fragments with active origins are labeled “BU” in panel C. Leftward-moving forks arrested at the RFB and rightward-moving forks converging at the RFB generate an “X”-like structure (X in panel C). (C) Replication pattern of StuI-digested rDNA from wild-type and rrm3 cells. Lowercase letters c, d, and e correspond to pauses in the 5S rDNA, ARS, and 35S rRNA initiator sequence, respectively (B). BR, breakage products; BU, bubble arc (visible in darker exposures); HJ, putative Holliday junctions; X, converged forks. (D) 2D gel analysis of rDNA from wild-type (panel 1), rrm3 (panel 2), sgs1 (panel 3), and srs2 (panel 4) cells was digested with StuI. Gel conditions, Southern blotting, and probing were as previously described (37).