Neurospora MUS-30 is an LSH/DDM1 homolog required for normal genome maintenance

LSH/DDM1 enzymes are required for DNA methylation in higher eukaryotes and have poorly defined roles in genome maintenance in yeast, plants, and animals. The filamentous fungus Neurospora crassa is a tractable system that encodes a single LSH/DDM1 homolog (NCU06306). We report that the Neurospora LSH/DDM1 enzyme is encoded by mutagen sensitive-30 (mus-30), a locus identified in a genetic screen over 25 years ago. We show that MUS-30-deficient cells have normal DNA methylation, but are hypersensitive to the DNA damaging agent MMS (methyl methanesulfonate). MUS-30 is a nuclear protein, consistent with its predicted role as a chromatin remodeling enzyme, and levels of MUS-30 are increased following DNA damage. MUS-30 co-purifies with Neurospora WDR76, a homolog of yeast Changed Mutation Rate-1 and mammalian WD40 repeat domain 76. Deletion of wdr76 rescued MMS-hypersensitivity of Dmus-30 strains, demonstrating that the MUS-30-WDR76 interaction is functionally important. DNA damage-sensitivity of Dmus-30 is also partially suppressed by deletion of methyl adenine glycosylase-1, a component of the base excision repair machinery (BER); however, the rate of BER is not affected in Dmus-30 strains. It was reported that mammalian LSH is required for efficient double strand break (DSB) repair. We found that MUS-30-deficient cells were not defective for DSB repair, and we observed a negative genetic interaction between Dmus-30 and Dmei-3, the Neurospora RAD51 homolog required for homologous recombination. These data are consistent with a role for MUS-30 that is independent of DSB repair. Our findings demonstrate that LSH/DDM1 enzymes are key regulators of genome stability in eukaryotes. crf5-1 isolates (two replicates each from the F1 and F2 generation) were grown in Vogel's minimal medium for 48 hours. As a control, two replicates of the wildtype strain were grown under identical conditions.

LSH/DDM1酶（LSH/DDM1 enzymes）是高等真核生物DNA甲基化所必需的，但其在酵母、植物与动物的基因组维持中发挥的功能尚不明确。丝状真菌粗糙脉孢霉（Neurospora crassa）是一种易于操作的研究系统，其基因组仅编码一个LSH/DDM1同源蛋白（NCU06306）。本研究发现，粗糙脉孢霉的LSH/DDM1酶由诱变敏感基因座30（mutagen sensitive-30，mus-30）编码，该位点是25年前通过遗传筛选鉴定得到的。研究表明，缺失MUS-30的细胞其DNA甲基化水平正常，但对DNA损伤剂甲基甲烷磺酸盐（methyl methanesulfonate，MMS）表现出超敏性。MUS-30为核蛋白，这与其作为染色质重塑酶（chromatin remodeling enzyme）的预测功能相符；且其蛋白水平在DNA损伤后会升高。MUS-30可与粗糙脉孢霉WDR76共纯化，WDR76是酵母突变改变率1（Changed Mutation Rate-1）与哺乳动物WD40重复结构域76（WD40 repeat domain 76）的同源蛋白。敲除wdr76可恢复Δmus-30菌株对MMS的超敏表型，这表明MUS-30与WDR76的相互作用具有重要的功能意义。Δmus-30菌株的DNA损伤敏感性还可通过敲除甲基腺嘌呤糖苷酶1（methyl adenine glycosylase-1）得到部分恢复，甲基腺嘌呤糖苷酶1是碱基切除修复（base excision repair，BER）系统的组分；但Δmus-30菌株的BER速率并未受到影响。已有研究表明，哺乳动物LSH对于高效的双链断裂（double strand break，DSB）修复是必需的。本研究发现，缺失MUS-30的细胞并未出现DSB修复缺陷；同时我们观察到Δmus-30与Δmei-3之间存在负向遗传互作，Δmei-3是粗糙脉孢霉中参与同源重组（homologous recombination）的RAD51同源蛋白。上述数据表明，MUS-30的功能不依赖于DSB修复。本研究结果证实，LSH/DDM1酶是真核生物基因组稳定性的关键调控因子。crf5-1分离株（分别取自F1与F2代，各设两个生物学重复）在沃格尔基本培养基（Vogel's minimal medium）中培养48小时。作为对照，野生型菌株（wildtype strain）设置两个重复并在完全相同的条件下培养。