Genome integrity is maintained by many cellular mechanisms in eukaryotes. One such mechanism functions during the cell cycle and is known as the DNA damage checkpoint. In the filamentous fungus Neurospora crassa, mus-9 and mus-21 are homologes of two key factors of the mammalian DNA damage checkpoint, ATR and ATM, respectively. We previously showed that mus-9 and mus-21 mutants are sensitive to DNA damage and that each mutant shows a characteristic growth defect: conidia from the mus-9 mutant have reduced viability and the mus-21 mutant exhibits slow hyphal growth. However, the relationship between these two genes has not been determined because strains carrying both mus-9 and mus-21 mutations could not be obtained. To facilitate analysis of a strain deficient in both mus-9 and mus-21, we introduced a specific mutation to the kinase domain of MUS-9 to generate a temperature-sensitive mus-9 allele (mus-9(ts)) which shows increased mutagen sensitivity at 37 degrees C. Then we crossed this strain with a mus-21 mutant to obtain a mus-9(ts) mus-21 double mutant. Growth of the mus-9(ts) mus-21 double mutant did not progress at the restrictive temperature (37 degrees C). Even at the permissive temperature (25 degrees C), this strain exhibited a higher mutagen sensitivity than that of the mus-9 and mus-21 single mutants, as well as slow hyphal growth and low viability of conidia. These results indicate that the mus-9(ts) mutation causes hypomorphic phenotypes in the mus-21 mutant and that these two genes regulate different pathways. Interestingly, we observed accumulation of micronuclei in the conidia of this double mutant, and such micronuclei were likely to correlate with spontaneous DSBs. Our results suggest that both mus-9 and mus-21 pathways are involved in DNA damage response, normal growth and maintenance of chromosome integrity, and that at least one of the pathways must be functional for survival.
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