Growth defect and mutator phenotypes of RecQ-deficient Neurospora crassa mutants separately result from homologous recombination and nonhomologous end joining during repair of DNA double-strand breaks

Abstract

RecQ helicases function in the maintenance of genome stability in many organisms. The filamentous fungus Neurospora crassa has two RecQ homologs, QDE3 and RECQ2. We found that the qde-3 recQ2 double mutant showed a severe growth defect. The growth defect was alleviated by mutation in mei-3, the homolog of yeast RAD51, which is required for homologous recombination (HR), suggesting that HR is responsible for this phenotype. We also found that the qde-3 recQ2 double mutant showed a mutator phenotype, yielding mostly deletions. This phenotype was completely suppressed by mutation of mus-52, a homolog of the human KU80 gene that is required for nonhomologous end joining (NHEJ), but was unaffected by mutation of mei-3. The high spontaneous mutation frequency in the double mutant is thus likely to be due to NHEJ acting on an elevated frequency of double-strand breaks (DSBs) and we therefore suggest that QDE3 and RECQ2 maintain chromosome stability by suppressing the formation of spontaneous DSBs.

Figure 1.

Sensitivity of the qde-3, recQ2, and qde-3 recQ2 double mutants to mutagens and other chemicals. (A, B, and D) Approximately 10⁵, 10⁴, 10³, or 10² viable conidia were spotted from left to right onto the agar plates containing the indicated chemicals. The final concentration of each chemical was as indicated. In the test of UV sensitivity, conidia were irradiated after spotting. (C) UV sensitivity of the wild type (open circles), the qde-3 mutant (solid squares), the recQ2 mutant (solid triangles), and the qde-3 recQ2 double mutant (solid diamonds) was measured quantitatively. Error bars indicate the standard errors calculated from the data for three independent experiments.

Figure 1.

Sensitivity of the qde-3, recQ2, and qde-3 recQ2 double mutants to mutagens and other chemicals. (A, B, and D) Approximately 10⁵, 10⁴, 10³, or 10² viable conidia were spotted from left to right onto the agar plates containing the indicated chemicals. The final concentration of each chemical was as indicated. In the test of UV sensitivity, conidia were irradiated after spotting. (C) UV sensitivity of the wild type (open circles), the qde-3 mutant (solid squares), the recQ2 mutant (solid triangles), and the qde-3 recQ2 double mutant (solid diamonds) was measured quantitatively. Error bars indicate the standard errors calculated from the data for three independent experiments.

Figure 2.

Apical growth and morphology of the RecQ-homolog mutants. (A) Apical growth of hyphae was measured by marking the growth front once or twice per day. When growth reached the end of a tube, mycelia from the growth front were transferred to a new tube. Crosses, the wild-type strain; open squares, qde-3 mutant; open triangles, recQ2 mutant; solid circles, qde-3 recQ2 double mutant; solid diamonds, qde-3 recQ2 mei-3 triple mutant; solid triangles, qde-3 recQ2 mus-52 triple mutant. (B) Appearance of the wild type (a and b, top) and the qde-3 recQ2 double mutant (a and b, bottom). Photographs were taken from above (a) and beneath (b) the agar. (C) Micrographs of the hyphal tips. Nuclei were stained with SYBR gold and observed by fluorescence microscopy (bottom). Bars, 10 μm. (D) Colony morphology on agar medium after incubation at 30° for 2 days. Bar, 1 cm.

Figure 2.

Apical growth and morphology of the RecQ-homolog mutants. (A) Apical growth of hyphae was measured by marking the growth front once or twice per day. When growth reached the end of a tube, mycelia from the growth front were transferred to a new tube. Crosses, the wild-type strain; open squares, qde-3 mutant; open triangles, recQ2 mutant; solid circles, qde-3 recQ2 double mutant; solid diamonds, qde-3 recQ2 mei-3 triple mutant; solid triangles, qde-3 recQ2 mus-52 triple mutant. (B) Appearance of the wild type (a and b, top) and the qde-3 recQ2 double mutant (a and b, bottom). Photographs were taken from above (a) and beneath (b) the agar. (C) Micrographs of the hyphal tips. Nuclei were stained with SYBR gold and observed by fluorescence microscopy (bottom). Bars, 10 μm. (D) Colony morphology on agar medium after incubation at 30° for 2 days. Bar, 1 cm.

Figure 3.

(A) PCR primers used to amplify the ad-3A ORF and flanking regions. Arrows indicate the positions of PCR primers. The shaded box indicates the ad-3A ORF and the open boxes indicate DNA fragments amplified by PCR using these primers. (B) PCR primers used to amplify the ad-3B ORF. Arrows indicate the positions of PCR primers. The shaded box indicates the ad-3B ORF and the open box indicates the DNA fragment amplified by PCR using these primers.

Figure 4.

Sequence of spontaneous mutations within the ad-3A ORF. ad-3A mutants arising in the qde-3 or the qde-3 recQ2 double mutant were subcultured and their ad-3A genes were sequenced. The numbers above the sequences show the nucleotide position from the first base of the start codon (ATG) of the ad-3A ORF.

Figure 5.

A model to explain how the loss of function of RecQ helicases results in a growth defect and mutator phenotype: In the wild type, Holliday junctions formed by regressed replication forks are mainly resolved by QDE3 and RECQ2, and few or no DSBs result. If QDE3 and RECQ2 are not functional, Holliday junctions at stalled replication forks are resolved by an endonuclease, creating DSBs. Repair of these DSBs uses components of HR or NHEJ. However, HR is unsuccessful without QDE3 and RECQ2 and proliferation-inhibiting DNA structures remain, causing the growth defect in the qde-3 recQ2 double mutant. Without QDE3 and RECQ2, NHEJ repairs the DSBs, causing large deletions, a characteristic of the qde-3 recQ2 double mutant. When QDE3 and RECQ2 are fully functional, the few DSBs that arise from endonuclease activity are repaired normally by HR or NHEJ.