TRF4 is involved in polyadenylation of snRNAs in Drosophila melanogaster.

The Saccharomyces cerevisiae poly(A) polymerases Trf4 and Trf5 are involved in an RNA quality control mechanism, where polyadenylated RNAs are degraded by the nuclear exosome. Although Trf4/5 homologue genes are distributed throughout multicellular organisms, their biological roles remain to be elucidated. We isolated here the two homologues of Trf4/5 in Drosophila melanogaster, named DmTRF4-1 and DmTRF4-2, and investigated their biological function. DmTRF4-1 displayed poly(A) polymerase activity in vitro, whereas DmTRF4-2 did not. Gene knockdown of DmTRF4-1 by RNA interference is lethal in flies, as is the case for the trf4 trf5 double mutants. In contrast, disruption of DmTRF4-2 results in viable flies. Cellular localization analysis suggested that DmTRF4-1 localizes in the nucleolus. Abnormal polyadenylation of snRNAs was observed in transgenic flies overexpressing DmTRF4-1 and was slightly increased by the suppression of DmRrp6, the 3'-5' exonuclease of the nuclear exosome. These results suggest that DmTRF4-1 and DmRrp6 are involved in the polyadenylation-mediated degradation of snRNAs in vivo.

Characterization of a second proliferating cell nuclear antigen (PCNA2) from Drosophila melanogaster.

The eukaryotic DNA polymerase processivity factor, proliferating cell nuclear antigen, is an essential component in the DNA replication and repair machinery. In Drosophila melanogaster, we cloned a second PCNA cDNA that differs from that encoded by the gene mus209 (for convenience called DmPCNA1 in this article). The second PCNA cDNA (DmPCNA2) encoded a 255 amino acid protein with 51.7% identity to DmPCNA1, and was ubiquitously expressed during Drosophila development. DmPCNA2 was localized in nuclei as a homotrimeric complex and associated with Drosophila DNA polymerase delta and epsilonin vivo. Treatment of cells with methyl methanesulfonate or hydrogen peroxide increased the amount of both DmPCNA2 and DmPCNA1 associating with chromatin, whereas exposure to UV light increased the level of association of only DmPCNA1. Our observations suggest that DmPCNA2 may function as an independent sliding clamp of DmPCNA1 when DNA repair occurs.

Drosophila DNA polymerase zeta interacts with recombination repair protein 1, the Drosophila homologue of human abasic endonuclease 1.

Abasic (AP) sites are a threat to cellular viability and genomic integrity, since they impede transcription and DNA replication. In mammalian cells, DNA polymerase (pol) beta plays an important role in the repair of AP sites. However, it is known that many organisms, including Drosophila melanogaster, do not have a pol beta homologue, and it is unclear how they repair AP sites. Here, we screened for DNA polymerases that interact with the Drosophila AP endonuclease 1 homologue, Rrp1 (recombination repair protein 1), and found that Drosophila pol zeta (Dmpol zeta), DmREV3 and DmREV7 bound to Rrp1 in a protein affinity column. Rrp1 directly interacted with DmREV7 in vitro and in vivo but not with DmREV3. These findings suggest that the DNA polymerase partner for Rrp1 is Dmpol zeta and that this interaction occurs through DmREV7. Interestingly, DmREV7 bound to the N-terminal region of Rrp1, which has no known protein homologue, suggesting that this binding is a species-specific event. Moreover, DmREV7 could stimulate the AP endonuclease activity of Rrp1, but not the 3'-exonuclease activity, and form a homomultimer. DmREV3 could not incorporate nucleotides at the 5'-incised tetrahydrofran sites but did show strand displacement activity for one-nucleotide-gapped DNA, which was not influenced by either DmREV7 or Rrp1. Methyl methanesulfonate and hydrogen peroxide treatments increased mRNA levels of DmREV3 and DmREV7. On the basis of the direct interaction between DmREV7 and Rrp1, we suggest that Dmpol zeta may be involved in the repair pathway of AP sites in DNA.

Drosophila damaged DNA binding protein 1 contributes to genome stability in somatic cells.

The damaged DNA-binding protein (DDB) complex consists of a heterodimer of p127 (DDB1) and p48 (DDB2) subunits and is believed to have a role in nucleotide excision repair (NER). We used the GAL4-UAS targeted expression system to knock down DDB1 in wing imaginal discs of Drosophila. The knock-down was achieved in transgenic flies using over-expression of inverted repeat RNA of the D-DDB1 gene [UAS-D-DDB1(650)-dsRNA]. As a consequence of RNA interference (RNAi), the fly had a shrunken wing phenotype. The wing spot test showed induced genome instability in transgenic flies with RNAi knock-down of D-DDB1 in wing imaginal discs. When Drosophila larvae with RNAi knock-down of D-DDB1 in wing imaginal discs were treated with the chemical mutagen methyl methanesulfonate (MMS), the frequency of flies with a severely shrunken wing phenotype increased compared to non-treated transgenic flies. These results suggested that DDB1 plays a role in the response to DNA damaged with MMS and in genome stability in Drosophila somatic cells.

DmGEN, a novel RAD2 family endo-exonuclease from Drosophila melanogaster.

A novel endo-exonuclease, DmGEN (Drosophila Melanogaster XPG-like endonuclease), was identified in D.melanogaster. DmGEN is composed of five exons and four introns, and the open reading frame encodes a predicted product of 726 amino acid residues with a molecular weight of 82.5 kDa and a pI of 5.36. The gene locus on Drosophila polytene chromosomes was detected at 64C9 on the left arm of chromosome 3 as a single site. The encoded protein showed a relatively high degree of sequence homology with the RAD2 nucleases, especially XPG. Although the XPG-N- and XPG-I-domains are highly conserved in sequence, locations of the domains are similar to those of FEN-1 and EXO-1, and the molecular weight of the protein is close to that of EXO-1. In vitro, DmGEN showed endonuclease and 3'-5' exonuclease activities with both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), but the endonuclease action with dsDNA was quite specific: 5'-3' exonuclease activity was found to occur with nicked DNA, while dsDNA was endonucleolytically cut at 3-4 bp from the 5' end. Homologs are widely found in mammals and higher plants. The data suggest that DmGEN belongs to a new class of RAD2 nuclease.

Damaged DNA binding protein 1 in Drosophila defense reactions.

We have focused attention on functions of Drosophila damaged DNA binding protein 1 (D-DDB1) in Drosophila hematopoiesis and previously reported that its whole body dsRNA over-expression using a GAL4-UAS targeted expression system results in melanotic tumors and complete lethality. Since the lesions appear to arise as a normal and heritable response to abnormal development, forming groups of cells that are recognized by the immune system and encapsulated in melanized cuticle, D-DDB1 appears to be an essential development-associated factor in Drosophila. To probe the possibility that it contributes to hemocyte development, we used a collagen promoter-GAL4 strain to over-express dsRNA of D-DDB1 in Drosophila hemocytes. The D-DDB1 gene silencing caused melanotic tumors and mortality at the end of larval development. Similarly, it interfered with melanization and synthesis of antimicrobial peptides. Transgenic flies with D-DDB1 gene silencing were found to accumulate abnormal large blood cells, reminiscent of human leukemia, suggesting that D-DDB1 has functions in hemocyte development.

Purification of Drosophila DNA polymerase zeta by REV1 protein-affinity chromatography.

Studies on the biochemical properties of very-large-size eukaryotic DNA polymerases have been limited by the difficulty in obtaining sufficient purified forms of each enzyme. Our aim was to determine and elucidate the biochemical properties of one such polymerase, pol zeta (DNA polymerase zeta) from Drosophila melanogaster (Dmpol zeta). Using an REV1 (UV-revertible gene 1) protein-affinity column, we have isolated the enzyme directly from Drosophila embryos. Completely purified Dmpol zeta was found to have a molecular mass of approx. 240 kDa, and to be sensitive to aphidicolin and resistant to ddTTP (2',3'-dideoxythymidine-5-triphosphate) and N-ethylmaleimide. The enzyme has a preference for poly(dA)/oligo(dT)(10:1) as a template primer and has high processivity for DNA synthesis. Moreover, Dmpol zeta showed significantly higher fidelity compared with Rattus norvegicus DNA polymerase, an error-prone DNA polymerase, in an M13 forward mutation assay. The activities of bypassing pyrimidine dimers and (6-4) photoproducts and extending from mismatched primer-template termini in (6-4) photoproduct by Dmpol zeta were not detected. Drosophila REV7 interacted with Dmpol zeta in vitro, but did not influence the DNA synthesis activity of Dmpol zeta. The present study is the first report about characterization of purified pol zeta from multicellular organisms, and the second concerning the characterization of yeast pol zeta.

Spatio-temporal expression of Drosophila mitochondrial transcription factor A during development.

We previously reported cloning of the Drosophila mitochondrial transcription factor A (D-mtTFA) gene, and characterization of a recombinant protein. In this report, the expression and distribution patterns of D-mtTFA during development are described. D-mtTFA mRNA and its protein product were found to be abundant in cells of tissues undergoing both proliferation and polytenization. Furthermore, a DRE-like sequence present in the D-mtTFA gene showed promotor activity in Drosophila Kc cells. In addition, D-mtTFA was detected in brain throughout the developmental process, as well as in non-proliferating tissues, such as flight muscle and cardia, and was also found in spermatids of imagos. D-mtTFA possesses a nuclear-targeting sequence, and is present in the nucleus at the syncytial stage, where bundles of 64 spermatids are present during spermatogenesis. The results suggest that D-mtTFA not only contributes to the mitochondrial DNA transcription and replication system, but also has a role in cell proliferation and development, including spermatogenesis.