Targeted mutagenesis by homologous recombination in D. melanogaster

Journal ArticleWe used a recently developed method to produce mutant alleles of five endogenous Drosophila genes, including the homolog of the p53 tumor suppressor. Transgenic expression of the FLP site-specific recombinase and the I-SceI endonuclease generates extrachromosomal linear DNA molecules in vivo

The processivity factor Pol32 mediates nuclear localization of DNA polymerase delta and prevents chromosomal fragile site formation in Drosophila development

The Pol32 protein is one of the universal subunits of DNA polymerase (Pol ), which is responsible for genome replication in eukaryotic cells. Although the role of Pol32 in DNA repair has been well-characterized, its exact function in genome replication remains obscure as studies in single cell systems have not established an essential role for Pol32 in the process. Here we characterize Pol32 in the context of Drosophila melanogaster development. In the rapidly dividing embryonic cells, loss of Pol32 halts genome replication as it specifically disrupts Pol localization to the nucleus. This function of Pol32 in facilitating the nuclear import of Pol would be similar to that of accessory subunits of DNA polymerases from mammalian Herpes viruses. In post-embryonic cells, loss of Pol32 reveals mitotic fragile sites in the Drosophila genome, a defect more consistent with Pol32's role as a polymerase processivity factor. Interestingly, these fragile sites do not favor repetitive sequences in heterochromatin, with the rDNA locus being a striking exception. Our study uncovers a possibly universal function for DNA polymerase ancillary factors and establishes a powerful system for the study of chromosomal fragile sites in a non-mammalian organism. Author summary Cancer etiological studies suggest that the majority of pathological mutations occurred under near normal DNA replication conditions, emphasizing the importance of understanding replication regulation under non-lethal conditions. To gain such a better understanding, we investigated the function of Pol32, a conserved ancillary subunit of the essential DNA polymerase Delta complex, through the development of the fruit fly Drosophila. We uncovered a previously unappreciated function of Pol32 in regulating the nuclear import of the polymerase complex, and this function is developmentally regulated. By utilizing mutations in pol32 and other replication factors, we have started to define basic features of Chromosome Fragile Sites (CFS) in Drosophila somatic cells. CFS is a major source of genome instability associated with replication stresses, and has been an important topic of cancer biology. We discovered that CFS formation does not favor genomic regions with repetitive sequences except the highly transcribed locus encoding ribosomal RNA. Our work lays the groundwork for future studies using Drosophila as an alternative system to uncover the most fundamental features of CFS.National Key R&D Program of China [2018YFA0107000]; National Natural Science Foundation of China [31830046, 31601162]; National Cancer Institute of America; Science and Technology Program of Guangzhou, China [201707010022]; National Institutes of Health of America [R01GM123640]Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

The Drosophila Citrate Lyase Is Required for Cell Division during Spermatogenesis

The Drosophila melanogaster DmATPCL gene encodes for the human ATP Citrate Lyase (ACL) ortholog, a metabolic enzyme that from citrate generates glucose-derived Acetyl-CoA, which fuels central biochemical reactions such as the synthesis of fatty acids, cholesterol and acetylcholine, and the acetylation of proteins and histones. We had previously reported that, although loss of Drosophila ATPCL reduced levels of Acetyl-CoA, unlike its human counterpart, it does not affect global histone acetylation and gene expression, suggesting that its role in histone acetylation is either partially redundant in Drosophila or compensated by alternative pathways. Here, we describe that depletion of DmATPCL affects spindle organization, cytokinesis, and fusome assembly during male meiosis, revealing an unanticipated role for DmATPCL during spermatogenesis. We also show that DmATPCL mutant meiotic phenotype is in part caused by a reduction of fatty acids, but not of triglycerides or cholesterol, indicating that DmATPCL-derived Acetyl-CoA is predominantly devoted to the biosynthesis of fatty acids during spermatogenesis. Collectively, our results unveil for the first time an involvement for DmATPCL in the regulation of meiotic cell division, which is likely conserved in human cells

Drosophila melanogaster MNK/Chk2 and p53 regulate multiple DNA repair and apoptotic pathways following DNA damage

We have used genetic and microarray analysis to determine how ionizing radiation (IR) induces p53-dependent transcription and apoptosis in Drosophila melanogaster. IR induces MNK/Chk2-dependent phosphorylation of p53 without changing p53 protein levels, indicating that p53 activity can be regulated without an Mdm2-like activity. In a genome-wide analysis of IR-induced transcription in wild-type and mutant embryos, all IR-induced increases in transcript levels required both p53 and the Drosophila Chk2 homolog MNK. Proapoptotic targets of p53 include hid, reaper, sickle, and the tumor necrosis factor family member EIGER: Overexpression of Eiger is sufficient to induce apoptosis, but mutations in Eiger do not block IR-induced apoptosis. Animals heterozygous for deletions that span the reaper, sickle, and hid genes exhibited reduced IR-dependent apoptosis, indicating that this gene complex is haploinsufficient for induction of apoptosis. Among the genes in this region, hid plays a central, dosage-sensitive role in IR-induced apoptosis. p53 and MNK/Chk2 also regulate DNA repair genes, including two components of the nonhomologous end-joining repair pathway, Ku70 and Ku80. Our results indicate that MNK/Chk2-dependent modification of Drosophila p53 activates a global transcriptional response to DNA damage that induces error-prone DNA repair as well as intrinsic and extrinsic apoptosis pathways

A Single Residue Mutation in the Gαq Subunit of the G Protein Complex Causes Blindness in Drosophila.

Heterotrimeric G proteins play central roles in many signaling pathways, including the phototransduction cascade in animals. However, the degree of involvement of the G protein subunit Gαq is not clear since animals with previously reported strong loss-of-function mutations remain responsive to light stimuli. We recovered a new allele of Gαq in Drosophila that abolishes light response in a conventional electroretinogram assay, and reduces sensitivity in whole-cell recordings of dissociated cells by at least five orders of magnitude. In addition, mutant eyes demonstrate a rapid rate of degeneration in the presence of light. Our new allele is likely the strongest hypomorph described to date. Interestingly, the mutant protein is produced in the eyes but carries a single amino acid change of a conserved hydrophobic residue that has been assigned to the interface of interaction between Gαq and its downstream effector, PLC. Our study has thus uncovered possibly the first point mutation that specifically affects this interaction in vivo

Competition between Replicative and Translesion Polymerases during Homologous Recombination Repair in Drosophila

In metazoans, the mechanism by which DNA is synthesized during homologous recombination repair of double-strand breaks is poorly understood. Specifically, the identities of the polymerase(s) that carry out repair synthesis and how they are recruited to repair sites are unclear. Here, we have investigated the roles of several different polymerases during homologous recombination repair in Drosophila melanogaster. Using a gap repair assay, we found that homologous recombination is impaired in Drosophila lacking DNA polymerase zeta and, to a lesser extent, polymerase eta. In addition, the Pol32 protein, part of the polymerase delta complex, is needed for repair requiring extensive synthesis. Loss of Rev1, which interacts with multiple translesion polymerases, results in increased synthesis during gap repair. Together, our findings support a model in which translesion polymerases and the polymerase delta complex compete during homologous recombination repair. In addition, they establish Rev1 as a crucial factor that regulates the extent of repair synthesis

Loss of the Histone Variant H2A.Z Restores Capping to Checkpoint-Defective Telomeres in Drosophila

The conserved histone variant H2A.Z fulfills many functions by being an integral part of the nucleosomes placed at specific regions of the genome. Telomeres cap natural ends of chromosomes to prevent their recognition as double-strand breaks. At yeast telomeres, H2A.Z prevents the spreading of silent chromatin into proximal euchromatin. A role for H2A.Z in capping, however, has not been reported in any organism. Here, I uncover such a role for Drosophila H2A.Z. Loss of H2A.Z, through mutations in either its gene or the domino gene for the Swr1 chromatin-remodeling protein, suppressed the fusion of telomeres that lacked the protection of checkpoint proteins: ATM, ATR, and the Mre11–Rad50–NBS complex. Loss of H2A.Z partially restores the loading of the HOAP capping protein, possibly accounting for the partial restoration in capping. I propose that, in the absence of H2A.Z, checkpoint-defective telomeres adopt alternative structures, which are permissive for the loading of the capping machinery at Drosophila telomeres

A Genetic Screen For DNA Double-Strand Break Repair Mutations in Drosophila

The study of DNA double-strand break (DSB) repair has been greatly facilitated by the use of rare-cutting endonucleases, which induce a break precisely at their cut sites that can be strategically placed in the genome. We previously established such a system in Drosophila and showed that the yeast I-SceI enzyme cuts efficiently in Drosophila cells and those breaks are effectively repaired by conserved mechanisms. In this study, we determined the genetic requirements for the repair of this I-SceI-induced DSB in the germline. We show that Drosophila Rad51 and Rad54 are both required for homologous repair by gene conversion, but are dispensable for single-strand annealing repair. We provided evidence suggesting that Rad51 is more stringently required than Rad54 for intersister gene conversion. We uncovered a significant role of DNA ligase IV in nonhomologous end joining. We conducted a screen for candidate mutations affecting DSB repair and discovered novel mutations in genes that include mutagen sensitive 206, single-strand annealing reducer, and others. In addition, we demonstrated an intricate balance among different repair pathways in which the cell differentially utilizes repair mechanisms in response to both changes in the genomic environment surrounding the break and deficiencies in one or the other repair pathways

Targeted mutagenesis by homologous recombination in D. melanogaster

Journal ArticleWe used a recently developed method to produce mutant alleles of five endogenous Drosophila genes, including the homolog of the p53 tumor suppressor. Transgenic expression of the FLP site-specific recombinase and the I-SceI endonuclease generates extrachromosomal linear DNA molecules in vivo. These molecules undergo homologous recombination with the corresponding chromosomal locus to generate targeted alterations of the host genome. The results address several questions about the general utility of this technique. We show that genes not near telomeres can be efficiently targeted; that no knowledge of the mutant phenotype is needed for targeting; and that insertional mutations and allelic substitutions can be easily produced