Srs2 and RecQ homologs cooperate in mei-3-mediated homologous recombination repair of Neurospora crassa

Abstract

Homologous recombination and post-replication repair facilitate restart of stalled or collapsed replication forks. The SRS2 gene of Saccharomyces cerevisiae encodes a 3'-5' DNA helicase that functions both in homologous recombination repair and in post-replication repair. This study identifies and characterizes the SRS2 homolog in Neurospora crassa, which we call mus-50. A knockout mutant of N.crassa, mus-50, is sensitive to several DNA-damaging agents and genetic analyses indicate that it is epistatic with mei-3 (RAD51 homolog), mus-11 (RAD52 homolog), mus-48 (RAD55 homolog) and mus-49 (RAD57 homolog), suggesting a role for mus-50 in homologous recombination repair. However, epistasis evidence has presented that MUS50 does not participate in post-replication repair in N.crassa. Also, the N.crassa mus-25 (RAD54 homolog) mus-50 double mutant is viable, which is in contrast to the lethal phenotype of the equivalent rad54 srs2 mutant in S.cerevisiae. Tetrad analysis revealed that mus-50 in combination with mutations in two RecQ homologs, qde-3 and recQ2, is lethal, and this lethality is suppressed by mutation in mei-3, mus-11 or mus-25. Evidence is also presented for the two independent pathways for recovery from camptothecin-induced replication fork arrest: one pathway is dependent on QDE3 and MUS50 and the other pathway is dependent on MUS25 and RECQ2.

Figure 1

Schematic alignment of Srs2 and RecQ helicases. The names of species and gene products are shown on the left-hand side, while protein size (number of amino acid residues) is shown on the right-hand side. (A) Schematic alignment of Srs2 helicases. Their conserved helicase domains are shown in black. (B) Schematic alignment of RecQ helicases. Their conserved helicase, RQC and HRDC domains are shown in black, dark gray and light gray, respectively.

Figure 2

Characterization of mus-50. (A) Northern-blot analyses of mus-50 transcripts. Upper panel: germinating mycelia were irradiated with UV at a dose of 100 J/m². At indicated time intervals after irradiation, a sample was collected and the level of mus-50 transcripts was analyzed by northern-blot hybridization. Lower panel: germinating mycelia were treated with 0.05% MMS. At indicated intervals after MMS treatment, a sample was collected and the level of mus-50 transcripts was analyzed by northern-blot hybridization. The 28S rRNA was estimated by ethidium bromide staining to confirm equal loading of all samples (data not shown). (B) Complementation of the UV- and MMS-sensitivities of the S.cerevisiae srs2Δ mutant by the N.crassa mus-50 gene. Stationary cells were UV-irradiated at the indicated dose. UV-irradiated cells were diluted and plated at 300 colonies per plate. Plates were incubated at 30°C for 2 days. To assay MMS-sensitivity, 30 μl of MMS was added to 20 ml of yeast suspension. Yeast suspensions were shaken continuously at 30°C for the indicated time period. Dilution, plating and incubation were as described above. Closed circles, wild type; open circles, srs2Δ; closed squares, srs2Δ+pAS2-1; and open squares, srs2Δ+pAS2-NCSRS2. The error bar of each point shows the standard deviation calculated from the data of those independent experiments. (C) Sensitivity to UV, HU, MMS, CPT and BLM of the wild-type and mus-50 strains. A conidial suspension was irradiated with UV at indicated dose or mixed with medium containing HU, MMS, CPT or BLM at indicated concentration. Colonies were counted after incubation at 30°C for 3 days. Closed circles, wild type and open circles, mus-50. The error bar of each point shows the standard deviation calculated from the data of those independent experiments. (D) Comparison of the UV- and MMS-induced pan2 reversion frequency. The B36 and OGW1 alleles of pan-2 have base substitution and frameshift mutations in the pan-2 locus, respectively. The y-axis indicates the number of revertants per 1 × 10⁷ survivors. The x-axis indicates UV dose or MMS concentration. Closed circles, pan-2 (B36); closed squares, pan-2 (OGW1); open squares, pan-2 (B36) mus-50; and open squares, pan-2 (OGW1) mus-50. The error bar of each point shows the standard deviation calculated from the data of those independent experiments.

Figure 3

Epistasis analysis of mus-50 and recombination repair defective. Cells were scored for MMS-sensitivity as described in the legend to Figure 2C. The error bar of each point shows the standard deviation calculated from the data of those independent experiments. (A) Closed circles, wild type; open circles, mus-50; closed squares, mei-3; and open squares, mei-3 mus-50. (B) Closed circles, wild type; open circles, mus-50; closed squares, mus-11; and open squares, mus-11 mus-50. (C) Closed circles, wild type; open circles, mus-50; closed squares, mus-25; and open squares, mus-25 mus-50. (D) Closed circles, wild type; open circles, mus-50; closed squares, uvs-6; and open squares, uvs-6 mus-50.

Figure 4

Epistasis analysis of mus-50 and PRR defective. Cells were scored for MMS-sensitivity as described in the legend to Figure 2C. The error bar of each point shows the standard deviation calculated from the data of those independent experiments. (A) Closed circles, wild type; open circles, mus-50; closed squares, mus-8; and open squares, mus-8 mus-50. (B) Closed circles, wild type; open circles, mus-50; closed squares, uvs-2; and open squares, uvs-2 mus-50. (C) Closed circles, wild type; open circles, mus-50; closed squares, mus-41; and open squares, mus-41 mus-50.

Figure 5

Genetic interactions between mus-50 and RecQ helicase mutants. (A) Epistasis analysis of mus-50 and qde-3 mutant. Cells were scored for MMS-sensitivity as described in the legend to Figure 2C. Closed circles, wild type; open circles, mus-50; closed squares, qde-3; and open squares, qde-3 mus-50. The error bar of each point shows the standard deviation calculated from the data of those independent experiments. (B) Epistasis analysis of mus-50 mutant and recQ2 mutant. Cells were scored for MMS-sensitivity as described in the legend to Figure 2C. Closed circles, wild type; open circles, mus-50; closed squares, recQ2; and open squares, recQ2 mus-50. The error bar of each point shows the standard deviation calculated from the data of those independent experiments. (C) Tetrad analysis of spores from a cross between qde-3 mus-50 and recQ2 mus-50 double mutant strains. In the N.crassa eight ascospores are produced in an ascus. After dissected spores were incubated for 1 week at 25°C, spot test was carried out to identify the genotypes of the progeny. mus-50 single mutant (s), qde-3 mus-50 double mutant (d1), recQ2 mus-50 double mutant (d2) and qde-3 recQ2 mus-50 triple mutant (t).

Figure 6

The synthetic lethality of the qde-3 recQ2 mus-50 triple mutant strain is rescued by the deletion of mei-3, mus-11 or mus-25. (A) Tetrad analysis of spores from a cross between qde-3 mus-50 and mei-3 recQ2 mus-50 mutant strains. mus-50 single mutant (s), qde-3 mus-50 double mutant (d1), recQ2 mus-50 double mutant (d2), mei-3 mus-50 double mutant (d3), qde-3 recQ2 mus-50 triple mutant (t), mei-3 qde-3 mus-50 triple mutant (t1), mei-3 recQ2 mus-50 triple mutant (t2) and mei-3 qde-3 recQ2 mus-50 quadruple mutant (q). (B) Tetrad analysis of spores from a cross between qde-3 mus-50 and mus-11 recQ2 mus-50 mutant strains. mus-50 single mutant (s), qde-3 mus-50 double mutant (d1), recQ2 mus-50 double mutant (d2), mus-11 mus-50 double mutant (d3), qde-3 recQ2 mus-50 triple mutant (t), mus-11 qde-3 mus-50 triple mutant (t1), mus-11 recQ2 mus-50 triple mutant (t2) and mus-11 qde-3 recQ2 mus-50 quadruple mutant (q). (C) Tetrad analysis of spores from a cross between qde-3 mus-50 and mus-25 recQ2 mus-50 mutant strains. mus-50 single mutant (s), recQ2 mus-50 double mutant (d2), mus-25 mus-50 double mutant (d3), qde-3 recQ2 mus-50 triple mutant (t), mus-25 qde-3 mus-50 triple mutant (t1) and mus-25 qde-3 recQ2 mus-50 quadruple mutant (q).

Figure 7

Epistasis analysis of mei-3, mus-25, mus-50, qde-3 and recQ2 for MMS-sensitivity. Cells were scored for MMS-sensitivity as described in the legend to Figure 2C. The error bar of each point shows the standard deviation calculated from the data of those independent experiments. (A) Epistasis analysis of mei-3 and mus-50, qde-3 and recQ2. Closed circles, mei-3; open circles, mei-3 mus-50; closed squares, mei-3 qde-3; and open squares, mei-3 recQ2. (B) Epistasis analysis of mus-25 and mus-50, qde-3 and recQ2. Closed circles, mus-25; open circles, mus-25 mus-50; closed squares, mus-25 qde-3; and open squares, mus-25 recQ2. (C) Epistasis analysis of mei-3, mus-50 and mus-25. Closed circles, mei-3; open circles, mei-3 mus-50; closed squares, mus-25; open squares, mei-3 mus-25; closed triangles, mus-25 mus-50; and open triangles, mei-3 mus-25 mus-50.

Figure 8

Epistasis analysis of mei-3, mus-25, mus-50, qde-3 and recQ2 for CPT-sensitivity. Cells were scored for CPT-sensitivity as described in the legend to Figure 2C. The error bar of each point shows the standard deviation calculated from the data of those independent experiments. (A) Epistasis analysis of mus-50, qde-3 and recQ2. Closed circles, wild type; open circles, mus-50; closed squares, qde-3; open squares, qde-3 mus-50; closed triangles, recQ2; and open triangles, recQ2 mus-50. (B) Epistasis analysis between mei-3 and mus-50, qde-3, recQ2, mus-25. Closed circles, mei-3; open circles, mei-3 mus-50; closed squares, mei-3 qde-3; open squares, mei-3 recQ2; and open triangles, mei-3 mus-25. (C) Epistasis analysis between mus-25 and mus-50, qde-3, recQ2 or between mei-3 and mus-25 mus-50. Closed circles, mus-25; open circles, mus-25 mus-50; closed squares, mus-25 qde-3; open squares, mus-25 recQ2; closed triangles, mei-3; and open triangles, mei-3 mus-25 mus-50.