Wakabayashi Michiyoshi, Ishii Chizu, Inoue Hirokazu, Tanaka Shuuitsu
Laboratory of Genetics, Saitama University, Japan.
DNA Repair (Amst). 2008 Dec 1;7(12):1951-61. doi: 10.1016/j.dnarep.2008.08.004. Epub 2008 Sep 27.
DNA damage checkpoint is an important mechanism for organisms to maintain genome integrity. In Neurospora crassa, mus-9 and mus-21 are homologues of ATR and ATM, respectively, which are pivotal factors of DNA damage checkpoint in mammals. A N. crassa clock gene prd-4 has been identified as a CHK2 homologue, but its role in DNA damage response had not been elucidated. In this study, we identified another CHK2 homologue and one CHK1 homologue from the N. crassa genome database. As disruption of these genes affected mutagen tolerance, we named them mus-59 and mus-58, respectively. The mus-58 mutant was sensitive to hydroxyurea (HU), but the mus-59 and prd-4 mutants showed the same HU sensitivity as that of the wild-type strain. This indicates the possibility that MUS-58 is involved in replication checkpoint and stabilization of stalled forks like mammalian CHK1. Phosphorylation of MUS-58 and MUS-59 was observed in the wild-type strain in response to mutagen treatments. Genetic relationships between those three genes and mus-9 or mus-21 indicated that the mus-9 mutation was epistatic to mus-58, and mus-21 was epistatic to prd-4. These relationships correspond to two signal pathways, ATR-CHK1 and ATM-CHK2 that have been established in mammalian cells. However, both the mus-9 mus-59 and mus-21 mus-58 double mutants showed an intermediate level between the two parental strains for CPT sensitivity. Furthermore, these double mutants showed severe growth defects. Our findings suggest that the DNA damage checkpoint of N. crassa is controlled by unique mechanisms.
DNA损伤检查点是生物体维持基因组完整性的重要机制。在粗糙脉孢菌中，mus-9和mus-21分别是ATR和ATM的同源物，而ATR和ATM是哺乳动物DNA损伤检查点的关键因子。粗糙脉孢菌的一个生物钟基因prd-4已被鉴定为CHK2的同源物，但其在DNA损伤应答中的作用尚未阐明。在本研究中，我们从粗糙脉孢菌基因组数据库中鉴定出另一个CHK2同源物和一个CHK1同源物。由于这些基因的破坏影响了对诱变剂的耐受性，我们分别将它们命名为mus-59和mus-58。mus-58突变体对羟基脲（HU）敏感，但mus-59和prd-4突变体对HU的敏感性与野生型菌株相同。这表明MUS-58可能像哺乳动物CHK1一样参与复制检查点和停滞复制叉的稳定。在野生型菌株中，诱变剂处理后可观察到MUS-58和MUS-59的磷酸化。这三个基因与mus-9或mus-21之间的遗传关系表明，mus-9突变对mus-58上位，mus-21对prd-4上位。这些关系对应于哺乳动物细胞中已建立的两条信号通路，即ATR-CHK1和ATM-CHK2。然而，mus-9 mus-59和mus-21 mus-58双突变体对喜树碱（CPT）的敏感性在两个亲本菌株之间呈中间水平。此外，这些双突变体表现出严重的生长缺陷。我们的研究结果表明，粗糙脉孢菌的DNA损伤检查点受独特机制的控制。
