ATM and ATR homologes of Neurospora crassa are essential for normal cell growth and maintenance of chromosome integrity.

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.

High efficient gene targeting on the AGAMOUS gene in an ArabidopsisAtLIG4 mutant.

Gene targeting induced by homologous integration of a foreign DNA segment into a chromosomal target sequence enables precise disruption or replacement of genes of interest and provides an effective means to analyze gene function, and also becomes an useful technique for breeding. But, integration of introduced DNA fragments is predominantly non-homologous in most species. However, we presented high-efficient homologous integration in disruptants of non-homologous end joining (NHEJ), that is, the Ku70-, Ku80- or Lig4-homologs deficient strain, in a model fungus Neurospora crassa. When the effect of NHEJ-defective plants for gene targeting was therefore examined in a model plant Arabidopsis (Arabidopsis thaliana), the efficiencies of gene targeting in the Atlig4/Atlig4 plant were 2/7 (28.6%) against calli obtained a selection-marker gene, 2/16 (12.5%) against selected calli, and about 2/540 (0.004%) against total cell particles at the starting point for transformation. The results of this paper show that the NHEJ-deficient system might cause a decrease in the efficiency of transformation but gives true targeted transformants with high efficiency in plant cell.

Genetic analysis of CHK1 and CHK2 homologues revealed a unique cross talk between ATM and ATR pathways in Neurospora crassa.

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.

The Neurospora crassa UVS-3 epistasis group encodes homologues of the ATR/ATRIP checkpoint control system.

The mutagen sensitive uvs-3 and mus-9 mutants of Neurospora show mutagen and hydroxyurea sensitivity, mutator effects and duplication instability typical of recombination repair and DNA damage checkpoint defective mutants. To determine the nature of these genes we used cosmids from a genomic library to clone the uvs-3 gene by complementation for MMS sensitivity. Mutation induction by transposon insertion and RIP defined the coding sequence. RFLP analysis confirmed that this sequence maps in the area of uvs-3 at the left telomere of LG IV. Analysis of the cDNA showed that the UVS-3 protein contains an ORF of 969 amino acids with one intron. It is homologous to UvsD of Aspergillus nidulans, a member of the ATRIP family of checkpoint proteins. It retains the N' terminal coiled-coil motif followed by four basic amino acids typical of these proteins and shows the highest homology in this region. The uvsD cDNA partially complements the defects of the uvs-3 mutation. The uvs-3 mutant shows a higher level of micronuclei in conidia and failure to halt germination and nuclear division in the presence of hydroxyurea than wild type, suggesting checkpoint defects. ATRIP proteins bind tightly to ATR PI-3 kinase (phosphatidylinositol 3-kinase) proteins. Therefore, we searched the Neurospora genome sequence for homologues of the Aspergillus nidulans ATR, UvsB. A uvsB homologous sequence was present in the right arm of chromosome I where the mus-9 gene maps. A cosmid containing this genomic DNA complemented the mus-9 mutation. The putative MUS-9 protein is 2484 amino acids long with eight introns. Homology is especially high in the C-terminal 350 amino acids that correspond to the PI-3 kinase domain. In wild type a low level of constitutive mRNA is present for both genes. It is transiently induced upon UV exposure.

The novel gene mus7(+) is involved in the repair of replication-associated DNA damage in fission yeast.

The progression of replication forks is often impeded by obstacles that cause them to stall or collapse, and appropriate responses to replication-associated DNA damage are important for genome integrity. Here we identified a new gene, mus7(+), that is involved in the repair of replication-associated DNA damage in the fission yeast Schizosaccharomyces pombe. The Deltamus7 mutant shows enhanced sensitivity to methyl methanesulfonate (MMS), camptothecin, and hydroxyurea, agents that cause replication fork stalling or collapse, but not to ultraviolet light or X-rays. Epistasis analysis of MMS sensitivity indicates that Mus7 functions in the same pathway as Mus81, a subunit of the Mus81-Eme1 structure-specific endonuclease, which has been implicated in the repair of the replication-associated DNA damage. In Deltamus7 and Deltamus81 cells, the repair of MMS-induced DNA double-strand breaks (DSBs) is severely impaired. Moreover, some cells with either mutation are hyper-elongated or enlarged, and most of these cells accumulate in late G2 phase. Spontaneous Rad22 (recombination mediator protein RAD52 homolog) foci increase in S phase to late G2 phase in Deltamus7 and Deltamus81 cells. These results suggest that replication-associated DSBs accumulate in these cells and that Rad22 foci form in the absence of Mus7 or Mus81. We also found that the rate of spontaneous conversion-type recombination is reduced in mitotic Deltamus7 cells, suggesting that Rhp51- (RAD51 homolog) dependent homologous recombination is disturbed in this mutant. From these data, we propose that Mus7 functions in the repair of replication-associated DSBs by promoting RAD51-dependent conversion-type recombination downstream of Rad22 and Mus81.

Genetic and molecular analysis of the temperature-sensitive mutant un-17 carrying a mutation in the gene encoding poly(A)-polymerase in Neurospora crassa.

The un-17 mutant was originally isolated as an irreparable temperature-sensitive (ts) mutant in Neurospora crassa. Early experiments showed that cells of this mutant immediately stopped growing and died when the temperature of the culture was shifted from a permissive temperature (25 degrees C) to non-permissive temperature (35 degrees C). This ts phenotype is suppressed by addition of cycloheximide or in some conditions of growth repression. Even at the permissive temperature, it shows a female sterile phenotype and is deficient in production of exocellular superoxide dismutase SOD4 (EC 1.15.1.1). By searching for a DNA fragment that complements the ts phenotype of the un-17 mutant from a N. crassa genome library, we found the un-17 gene. The cloned un-17 gene encodes a homolog of the Saccharomyces cerevisiae poly(A) polymerase (PAP). The un-17 mutant had a one-base substitution mutation in the gene. The cloned un-17 genes from the wild-type strain and the un-17 mutant were introduced into both the un-17 mutant and wild-type strain. The un-17 mutant introduced by un-17 DNA from the wild-type strain showed recovery of both the ts and female sterile phenotypes. Moreover, the purified product derived from the wild-type strain showed PAP activity in vitro. These findings indicate that the un-17 mutant carries a ts mutation in the gene encoding PAP.

Highly efficient gene replacements in Neurospora strains deficient for nonhomologous end-joining.

Gene disruption and overexpression play central roles in the analysis of gene function. Homologous recombination is, in principle, the most efficient method of disrupting, modifying, or replacing a target gene. Although homologous integration of exogenous DNA into the genome occurs readily in Saccharomyces cerevisiae, it is rare in many other organisms. We identified and disrupted Neurospora crassa genes homologous to human KU70 and KU80, which encode proteins that function in nonhomologous end-joining of double-stranded DNA breaks. The resulting mutants, named mus-51 and mus-52, showed higher sensitivity to methyl methanesulfonate, ethyl methanesulfonate, and bleomycin than wild type, but not to UV, 4-nitroquinoline 1-oxide, camptothecin, or hydroxyurea. Vegetative growth, conidiation, and ascospore production in homozygous crosses were normal. The frequency of integration of exogenous DNA into homologous sequences of the genome in the KU disruption strains of N. crassa was compared with that in wild type, mei-3, and mus-11. In mei-3 and mus-11, which are defective in homologous recombination, none or few homologous integration events were observed under any conditions. When mtr target DNA with approximately 2-kb 5' and 3' flanking regions was used for transformation of the KU disruption strains, 100% of transformants exhibited integration at the homologous site, compared to 10 to 30% for a wild-type recipient. Similar results were obtained when the ad-3A gene was targeted for disruption. These results indicate that KU disruption strains are efficient recipients for gene targeting.

Lessons from the genome sequence of Neurospora crassa: tracing the path from genomic blueprint to multicellular organism.

We present an analysis of over 1,100 of the approximately 10,000 predicted proteins encoded by the genome sequence of the filamentous fungus Neurospora crassa. Seven major areas of Neurospora genomics and biology are covered. First, the basic features of the genome, including the automated assembly, gene calls, and global gene analyses are summarized. The second section covers components of the centromere and kinetochore complexes, chromatin assembly and modification, and transcription and translation initiation factors. The third area discusses genome defense mechanisms, including repeat induced point mutation, quelling and meiotic silencing, and DNA repair and recombination. In the fourth section, topics relevant to metabolism and transport include extracellular digestion; membrane transporters; aspects of carbon, sulfur, nitrogen, and lipid metabolism; the mitochondrion and energy metabolism; the proteasome; and protein glycosylation, secretion, and endocytosis. Environmental sensing is the focus of the fifth section with a treatment of two-component systems; GTP-binding proteins; mitogen-activated protein, p21-activated, and germinal center kinases; calcium signaling; protein phosphatases; photobiology; circadian rhythms; and heat shock and stress responses. The sixth area of analysis is growth and development; it encompasses cell wall synthesis, proteins important for hyphal polarity, cytoskeletal components, the cyclin/cyclin-dependent kinase machinery, macroconidiation, meiosis, and the sexual cycle. The seventh section covers topics relevant to animal and plant pathogenesis and human disease. The results demonstrate that a large proportion of Neurospora genes do not have homologues in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. The group of unshared genes includes potential new targets for antifungals as well as loci implicated in human and plant physiology and disease.

Isolation and genetic characterization of the Neurospora crassa REV1 and REV7 homologs: evidence for involvement in damage-induced mutagenesis.

In a previous paper, we reported that the Neurospora crassa upr-1 gene is a homolog of the yeast gene REV3, which encodes the catalytic subunit of DNA polymerase zeta (polzeta). Characterization of the upr-1 mutant indicated that the UPR1 protein plays a role in DNA repair and mutagenesis. To help understand the mechanisms of mutagenic DNA repair in the N. crassa more extensively, we identified N. crassa homologs of yeast REV1 and REV7 and obtained mutants ncrev1 or ncrev7, which had similar phenotypes to the upr-1 mutant. Mutant carrying ncrev7 was more sensitive to UV and 4NQO, and slightly sensitive to MMS than the wild-type. The sensitivity to UV and MMS of the ncrev1 mutant was moderately higher than that of the wild-type, but the sensitivity to 4NQO of the mutant was similar to that of the wild-type. In reversion assay using testers with base substitution or frameshift mutation at the ad-3A locus, each of ncrev1 and ncrev7 mutants showed lower induced-mutability than the wild-type. Expression of ncrev1 and ncrev7 was found to be UV-inducible like the case of upr-1. Genetic analyses showed that the ncrev7 was identical to mus-26, which belongs to the upr-1 epistasis group, and that the ncrev1 was a newly identified DNA repair gene and designated as mus-42. Interestingly, all three mutants have a normal CPD photolyase gene, however, they showed a partial photoreactivation defect (PPD) phenotype, not completely defective but inefficient in photoreactivation. These results suggest that N. crassa REV homolog genes function in DNA repair and UV mutagenesis through the bypass of (6-4) photoproducts.