A novel phenotype of an excision-repair mutant in Neurospora crassa: Mutagen sensitivity of the mus-18 mutant is specific to UV

Abstract

A UV-sensitive mutant has been isolated from UV-mutagenized conidia of Neurospora crassa. The mutation responsible for the lesion was mapped in linkage group VL, proximal to the nucleolus organizer region. We designated the mutant mus-18. The sensitivity of the mus-18 mutant to UV-irradiation was not particularly high, being less than twice that of the wild-type strain. However, the frequency of mutations at the ad-3 loci induced by UV was extremely high even at low doses, under conditions where survival rates of mus-18 cells were almost identical to those of wild-type cells. Photo-reactivation of UV damage was normal in the mus-18 mutant. Sensitivity to other mutagens, such as gamma rays, 4-nitroquinoline-1-oxide, N-methyl-N'-nitro-N-nitrosoguanidine, mitomycin C and methyl methanesulfonate, was similar to that of the wild type. Fertility of the mus-18 mutant was normal in homozygous crosses. These results suggest that mus-18 is an excision-repair mutant. Measurement of endonuclease-sensitive sites (ESS) after liquid-holding recovery from UV damage revealed that ESS remained unrepaired for longer than 18 h in the mus-18 mutant, while most were eliminated within 6 h in wild-type cells and in other UV-sensitive mutants. This result suggests that mus-18 is defective in the incision step of dimer excision. The mus-18 mutant provides the first example of an excision-defective mutation in eukaryotes, which is specific to UV damage.

Genetic analysis of the Neurospora crassa RAD14 homolog mus-43 and the RAD10 homolog mus-44 reveals that they belong to the mus-38 pathway of two nucleotide excision repair systems

“…In addition to repair of UV damage, the NER pathway appears to be necessary for the repair of the chemical adducts since the mus-43 and mus-44 mutants are both sensitive to 4NQO (Fig. 1A), whereas the mus-18 mutant does not exhibit sensitivity (Ishii et al, 1991).…”

“…This gene, mus-38, was characterized as a homolog of S. cerevisiae RAD1 (Hatakeyama et al, 1998;Inoue, 1999). Interestingly, N. crassa has another excision repair mechanism for the removal of UVinduced damage that is mediated by the MUS-18 protein (Ishii et al, 1991;Yajima et al, 1995). This protein is a homolog of Schizosaccharomyces pombe UVDE (Yonemasu et al, 1997).…”

Alternative repair pathways for UV-induced DNA damage

“…15 Recently, however, a mutant, designated mus-38, has been shown to be deficient in a gene homologue of the human NER gene, XPF (Ishii and Inoue, unpublished observations). Like the uvde -rad13 -double mutant in S. pombe, a double mutant between mus-18 and mus-38 exhibits a hypersensitivity to UV and fails to remove any UV damage.…”

“…15 The existence of this unique UV-sensitive mutant suggested that a novel UV damage-specific enzyme may be present in N. crassa. Therefore, a cDNA library made from wild-type N. crassa was introduced into Escherichia coli strain SY2 (uvrA recA phr), which is completely deficient in repair of UV damage.…”

“…So far, however, in vivo experiments using N. crassa have not identified any chemical or physical agents other than UV as potential substrate for UVDE. 15 Therefore, judging by its efficient specific incision activity against UV damage, UVDE may have evolved for protection against solar UV radiation in an early period of evolution.…”