The Drosophila centrosome-associated protein CP190 is essential for viability but not for cell division.

The Drosophila CP190 and CP60 proteins interact with each other and shuttle between the nucleus in interphase and the centrosome in mitosis. Both proteins can bind directly to microtubules in vitro, and have been shown to associate with a specific pattern of loci on salivary gland polytene chromosomes, but their functions are unknown. Here we show that reducing the level of CP190 or CP60 by >90% in tissue culture cells does not significantly interfere with centrosome or microtubule organisation, with cell division, or with cell viability. However, CP190 is an essential protein, as flies homozygous for mutations in the Cp190 gene die at late pupal stages of development. In larval brains of Cp190 mutants, mitosis is not radically perturbed, and a mutated form of CP190 (CP190DeltaM), that cannot bind to microtubules or associate with centrosomes, can rescue the lethality associated with mutations in the Cp190 gene. Thus, CP190 plays an essential role in flies that is independent of its association with centrosomes or microtubules.

Mutation of a Drosophila gamma tubulin ring complex subunit encoded by discs degenerate-4 differentially disrupts centrosomal protein localization.

We have cloned the Drosophila gene discs degenerate-4 (dd4) and find that it encodes a component of the gamma-tubulin ring complex (gammaTuRC) homologous to Spc98 of budding yeast. This provides the first opportunity to study decreased function of a member of the gamma-tubulin ring complex, other than gamma-tubulin itself, in a metazoan cell. gamma-tubulin is no longer at the centrosomes but is dispersed throughout dd4 cells and yet bipolar metaphase spindles do form, although these have a dramatically decreased density of microtubules. Centrosomin (CNN) remains in broad discrete bodies but only at the focused poles of such spindles, whereas Asp (abnormal spindle protein) is always present at the presumptive minus ends of microtubules, whether or not they are focused. This is consistent with the proposed role of Asp in coordinating the nucleation of mitotic microtubule organizing centers. The centrosome associated protein CP190 is partially lost from the spindle poles in dd4 cells supporting a weak interaction with gamma-tubulin, and the displaced protein accumulates in the vicinity of chromosomes. Electron microscopy indicates not only that the poles of dd4 cells have irregular amounts of pericentriolar material, but also that they can have abnormal centrioles. In six dd4 cells subjected to serial sectioning centrioles were missing from one of the two poles. This suggests that in addition to its role in nucleating cytoplasmic and spindle microtubules, the gammaTuRC is also essential to the structure of centrioles and the separation of centrosomes.

The Drosophila mus101 gene, which links DNA repair, replication and condensation of heterochromatin in mitosis, encodes a protein with seven BRCA1 C-terminus domains.

The mutagen-sensitive-101 (mus101) gene of Drosophila melanogaster was first identified 25 years ago through mutations conferring larval hypersensitivity to DNA-damaging agents. Other alleles of mus101 causing different phenotypes were later isolated: a female sterile allele results in a defect in a tissue-specific form of DNA synthesis (chorion gene amplification) and lethal alleles cause mitotic chromosome instability that can be observed genetically and cytologically. The latter phenotype presents as a striking failure of mitotic chromosomes of larval neuroblasts to undergo condensation of pericentric heterochromatic regions, as we show for a newly described mutant carrying lethal allele mus101(lcd). To gain further insight into the function of the Mus101 protein we have molecularly cloned the gene using a positional cloning strategy. We report here that mus101 encodes a member of the BRCT (BRCA1 C terminus) domain superfamily of proteins implicated in DNA repair and cell cycle checkpoint control. Mus101, which contains seven BRCT domains distributed throughout its length, is most similar to human TopBP1, a protein identified through its in vitro association with DNA topoisomerase IIbeta. Mus101 also shares sequence similarity with the fission yeast Rad4/Cut5 protein required for repair, replication, and checkpoint control, suggesting that the two proteins may be functional homologs.

Mutual correction of faulty PCNA subunits in temperature-sensitive lethal mus209 mutants of Drosophila melanogaster.

Proliferating cell nuclear antigen (PCNA) functions in DNA replication as a processivity factor for polymerases delta and epsilon, and in multiple DNA repair processes. We describe two temperature-sensitive lethal alleles (mus209(B1) and mus209(2735)) of the Drosophila PCNA gene that, at temperatures permissive for growth, result in hypersensitivity to DNA-damaging agents, suppression of position-effect variegation, and female sterility in which ovaries are underdeveloped and do not produce eggs. We show by mosaic analysis that the sterility of mus209(B1) is partly due to a failure of germ-line cells to proliferate. Strikingly, mus209(B1) and mus209(2735) interact to restore partial fertility to heteroallelic females, revealing additional roles for PCNA in ovarian development, meiotic recombination, and embryogenesis. We further show that, although mus209(B1) and mus209(2735) homozygotes are each defective in repair of transposase-induced DNA double-strand breaks in somatic cells, this defect is substantially reversed in the heteroallelic mutant genotype. These novel mutations map to adjacent sites on the three-dimensional structure of PCNA, which was unexpected in the context of this observed interallelic complementation. These mutations, as well as four others we describe, reveal new relationships between the structure and function of PCNA.

DNA repair defects and other (mus)takes in Drosophila melanogaster.

Preservation of the structural integrity of DNA in any organism is crucial to its health and survival. Such preservation is achieved by an extraordinary cellular arsenal of damage surveillance and repair functions, many of which are now being defined at the gene and protein levels. Mutants hypersensitive to the killing effects of DNA-damaging agents have been instrumental in helping to identify DNA repair-related genes and to elucidate repair mechanisms. In Drosophila melanogaster, such strains are generally referred to as mutagen-sensitive (mus) mutants and currently define more than 30 genetic loci. Whereas most mus mutants have been recovered on the basis of hypersensitivity to the monofunctional alkylating agent methyl methanesulfonate, they nevertheless constitute a phenotypically diverse group, with many mutants having effects beyond mutagen sensitivity. These phenotypes include meiotic dysfunctions, somatic chromosome instabilities, chromatin abnormalities, and cell proliferation defects. Within the last few years numerous mus and other DNA repair-related genes of Drosophila have been molecularly cloned, providing new insights into the functions of these genes. This article outlines strategies for isolating mus mutations and reviews recent advances in the Drosophila DNA repair field, emphasizing mutant analysis and gene cloning.

Chromosome fragmentation resulting from an inability to repair transposase-induced DNA double-strand breaks in PCNA mutants of Drosophila.

Proliferating cell nuclear antigen (PCNA) has several roles in progression through S phase: it is required for the function of DNA polymerases delta and epsilon and physically associates with the structure-specific nuclease FEN-1 that is essential for Okazaki fragment processing. The cyclindependent kinase inhibitor p21 appears to displace FEN-1 from PCNA to inhibit DNA replication and possibly permit participation of PCNA in nucleotide excision repair. Here we show that PCNA is also indispensable for repair of DNA double-strand breaks (DSBs), lesions which are not corrected by excision repair processes. When PCNA-deficient Drosophila mutants are incorporated into a genetic system that induces chromosomal site-specific DSBs upon mobilization of transposable P elements they fail to undertake DSB repair. This has dominant lethal effects: DSBs are converted into chromosome breaks that can be seen at mitosis.

Mutagen sensitivity and suppression of position-effect variegation result from mutations in mus209, the Drosophila gene encoding PCNA.

The mus209B1 mutant of Drosophila melanogaster exhibits a complex pleiotropy of temperature-sensitive (ts) lethality, hypersensitivity to DNA-damaging agents such as ionizing radiation and methyl methanesulfonate, suppression of position-effect variegation (PEV), and female sterility. Our discovery that mus209 encodes proliferating cell nuclear antigen (PCNA), which is an indispensable component of the DNA replication apparatus, suggests that alterations to chromosome replication may underlie that pleiotropy. Nine lethal mutations, three of them ts, genetically define the Pcna locus. Temperature shift studies reveal that the vital function of PCNA is required throughout virtually all stages of fly development, and that maternally encoded PCNA is essential for embryogenesis. All three ts mutants strongly suppress PEV, which suggests a role for PCNA in chromatin assembly or modification.

The inhibitor of DNA replication encoded by the Drosophila gene plutonium is a small, ankyrin repeat protein.

The plutonium (plu) gene product controls DNA replication early in Drosophila development. plu mutant females lay unfertilized eggs that have undergone extensive DNA synthesis. In fertilized embryos from plu mutant mothers, S-phase is uncoupled from mitosis. The gene is expressed only in ovaries and embryos, null alleles are strict maternal effect mutations, and the phenotype of inappropriate DNA replication is the consequence of loss-of-gene function. plu therefore negatively regulates S-phase at a time in early development when commitment to S-phase does not depend on cyclic transcription. plu encodes a protein with two ankyrin-like repeats, a domain for protein-protein interaction. plu is immediately adjacent to, but distinct from, the PCNA gene.

Characterization of the mus308 gene in Drosophila melanogaster.

Among the available mutagen-sensitive mutations in Drosophila, those at the mus308 locus are unique in conferring hypersensitivity to DNA cross-linking agents but not to monofunctional agents. Those mutations are also associated with an elevated frequency of chromosomal aberrations, altered DNA metabolism and the modification of a deoxyribonuclease. This spectrum of phenotypes is shared with selected mammalian mutations including Fanconi anemia in humans. In anticipation of the molecular characterization of the mus308 gene, it has been localized cytogenetically to 87C9-87D1,2 on the right arm of chromosome three. Nine new mutant alleles of the gene have been generated by X-ray mutagenesis and one was recovered following hybrid dysgenesis. Characterization of these new alleles has uncovered additional phenotypes of mutations at this locus. Homozygous mus308 flies that have survived moderate mutagen treatment exhibit an altered wing position that is correlated with reduced flight ability and an altered mitochondrial morphology. In addition, observations of elevated embryo mortality are potentially explained by an aberrant distribution of nuclear material in early embryos which is similar to that seen in the mutant giant nuclei.

A rapid somatic genotoxicity assay in Drosophila melanogaster using multiple mutant mutagen-sensitive (mus) strains.

Mutagen-sensitive (mus) mutations in Drosophila melanogaster render developing flies hypersensitive to the lethal effects of DNA-damaging agents. In principle, multiply mutant mus strains might then serve as sensitive in vivo indicators of a wide range of mutagens and genotoxic carcinogens. As a first step to evaluate that potential we characterized interactions between mus mutations in eight double mutants containing combinations of the second chromosomal mutations mus201D1, mus205B1, mus208B1, mus210B1 and mus211B1. We found that (i) all double mutants are fully viable in the absence of mutagen exposure, (ii) mus205B1 is epistatic to any other mus mutation with respect to methyl methanesulfonate (MMS) sensitivity, and (iii) in double mutants carrying any combination of mus201D1, mus210B1 or mus211B1, MMS sensitivity is increased in a synergistic manner. Based on those results, and on mutagen cross-sensitivity data of single mutants generated in previous studies, we constructed two triple mutant mus strains for use as testers in a simple genotoxicity assay. That assay measures the survival of DNA repair-deficient mus homozygotes relative to their repair-proficient heterozygous siblings. Those two classes of fly are easily distinguished from one another by their phenotypic markers. In addition, the heterozygotes serve as a relatively mutagen-insensitive internal control in all test vials. One tester strain (mus208B1 mus210B1 mus211B2) identified 11 of 12 chemical carcinogens as genotoxic (benzo[a]pyrene, cyclophosphamide, 1,2,3,4-diepoxybutane, diethylnitrosamine, dimethylnitrosamine, ethyl methanesulfonate, formaldehyde, hexamethylphosphoramide, methyl methanesulfonate, methylnitrosourea and N-methyl-N'-nitro-N-nitrosoguanidine). Safrole and two noncarcinogens (benzo[e]pyrene and caprolactam) tested as nongenotoxic.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and characterization of second chromosome mutagen-sensitive mutations in Drosophila melanogaster.

We have undertaken the study of a collection of 32 Drosophila melanogaster mus strains selected on the basis of developmental sensitivity to the DNA-damaging agents, methyl methanesulfonate (MMS), N-acetyl-2-aminofluorene (AAF), nitrogen mustard (HN2), and gamma-radiation. In total, 18 of these strains are sensitive to MMS. In turn, 14 of these exhibit unconditional MMS sensitivity (one of the latter mutants is lethal at 29 degrees C), whereas the other 4 are sensitive to MMS only at higher temperatures. Detailed analysis of the 7 strongest MMS-sensitive strains reveals that they identify 4 new second chromosome mus loci. Two mus loci are each represented by two alleles. One mutant (mus205B1) is allelic to a previously characterized mus locus. Different MMS-sensitive mutants display patterns of mutagen cross-sensitivity (to AAF, HN2, benzo[a]pyrene (BP), and gamma-rays) that parallel the range of responses seen in previously recovered X-linked and autosomal mus loci. In general, mutations that are strongly sensitive to MMS are also sensitive to one or both of the procarcinogens, AAF and BP, as opposed to HN2 and gamma-radiation. In contrast, the moderately MMS-sensitive mutations are sensitive to HN2 and gamma-rays, but not to AAF or BP. Of the 14 mus strains that are not sensitive to MMS, 5 are sensitive to AAF, another 5 are sensitive to HN2, and the remaining 4 are sensitive to gamma-rays.