Bloom syndrome, characterized by a predisposition to cancer, is caused by mutation of the RecQ DNA helicase gene BLM. The precise function of BLM remains unclear. Previous research suggested that Drosophila BLM functions in the repair of DNA double-strand breaks. Most double-strand breaks in flies are repaired by homologous recombination through the synthesis-dependent strand-annealing pathway. Here, we demonstrate that Drosophila BLM mutants are severely impaired in their ability to carry out repair DNA synthesis during synthesis-dependent strand annealing. Consequently, repair in the mutants is completed by error-prone pathways that create large deletions. These results suggest a model in which BLM maintains genomic stability by promoting efficient repair DNA synthesis and thereby prevents double-strand break repair by less precise pathways.

DNA double-strand breaks (DSBs), a major source of genome instability, are often repaired through homologous recombination pathways. Models for these pathways have been proposed, but the precise mechanisms and the rules governing their use remain unclear. In Drosophila, the synthesis-dependent strand annealing (SDSA) model can explain most DSB repair. To investigate SDSA, we induced DSBs by excision of a P element from the male X chromosome, which produces a 14-kb gap relative to the sister chromatid. In wild-type males, repair synthesis tracts are usually long, resulting in frequent restoration of the P element. However, repair synthesis is often incomplete, resulting in internally deleted P elements. We examined the effects of mutations in spn-A, which encodes the Drosophila Rad51 ortholog. As expected, there is little or no repair synthesis in homozygous spn-A mutants after P excision. However, heterozygosity for spn-A mutations also resulted in dramatic reductions in the lengths of repair synthesis tracts. These findings support a model in which repair DNA synthesis is not highly processive. We discuss a model wherein repair of a double-strand gap requires multiple cycles of strand invasion, synthesis, and dissociation of the nascent strand. After dissociation, the nascent strand may anneal to a complementary single strand, reinvade a template to be extended by additional synthesis, or undergo end joining. This model can explain aborted SDSA repair events and the prevalence of internally deleted transposable elements in genomes.

The importance of this family is highlighted by the observation that mutations in three of the five human RecQ helicases, WRN, BLM, and RECQ4, cause the Werner, Bloom, and RothmundThomson syndromes, respectively (2-4). These three rare disorders are characterized by genomic instability and cancer predisposition, and patients with Werner and RothmundThomson syndromes also display symptoms of premature aging.Bloom syndrome (BS) patients develop a wide spectrum of cancers typical of those found in older individuals of the general population, including sarcomas, lymphomas, and epithelial cancers (reviewed in ref. 5). A hallmark of BS cells is a large increase in sister chromatid exchanges (6). In addition, BS cells show elevated levels of chromosome breaks, rearrangements, and deletions (7). Both in vivo and in vitro experiments with broken plasmids demonstrate that repair of double-strand breaks (DSBs) in the absence of BLM results in products with large deletions (8, 9). Therefore, a clearer understanding of the underlying events that cause chromosomal deletions in BS cells may provide insight into how the BLM protein prevents cancer in normal cells.Accumulating evidence suggests that the BLM protein functions in homologous recombination repair pathways to promote genomic stability. In humans, BLM interacts with RAD51, a protein required for the strand invasion step that initiates DSB repair by homologous recombination (10). Sgs1p, the Saccharomyces cerevisiae homologue of BLM, also interacts with Rad51p (10), and sgs1 mutants have an increased rate of chromosomal rearrangements such as translocations and deletions. Interestingly, Sgs1p suppresses recombination between DNA sequences with imperfect homology (11), consistent with the notion that it functions both to promote accurate recombination and to suppress inappropriate recombination.In vitro experiments provide further evidence that BLM functions during recombination. The human BLM helicase preferentially unwinds Holliday junctions, branched DNA structures, and other homologous recombination intermediates (12)(13)(14). Interestingly, BLM has also been shown to be adept at binding to and unwinding D-loops (15), which are thought to be the initial intermediate in DSB repair by homologous recombination.The Drosophila melanogaster ortholog of BLM, DmBlm, is encoded by the mus309 gene (16). Mutations in mus309 cause increased sensitivity to ionizing radiation and defects in DSB repair. Reminiscent of the human phenotype, deletions flanking a DSB site on a plasmid are frequently observed in mus309 mutants (17, 18). We recently used a chromosomal DSB repair assay to demonstrate that mus309 mutants are defective in the repair of double-strand gaps created by the excision of a P transposable element (19). Double-strand gaps generated by P element excision are repaired predominantly through a homologous recombination pathway termed synthesis-dependent strand annealing (SDSA) (Fig. 1). During SDSA, single-stranded DNA is generated by 5Ј to 3Ј resection of each end. One ...

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P-element somatic inhibitor (PSI) is a KH domain-containing splicing factor highly expressed in Drosophila somatic tissues. Here we have identified a direct association of PSI with the spliceosomal U1 small nuclear ribonucleoprotein (snRNP) particle in somatic nuclear extracts. This interaction is mediated by highly conserved residues within the PSI C-terminal AB motif and the U1 snRNP-specific 70K protein. Through the AB motif, PSI modulates U1 snRNP binding on the P-element third intron (IVS3) 5' splice site and its upstream exonic regulatory element. Ectopic expression experiments in the Drosophila female germline demonstrate that the AB motif also contributes to IVS3 splicing inhibition in vivo. These data show that the processing of specific target transcripts, such as the P-element mRNA, is regulated by a functional PSI-U1 snRNP interaction in Drosophila.

Splicing of the Drosophila P-element third intron (IVS3) is inhibited in somatic cells, restricting transposase expression to the germ line. Somatic inhibition of IVS3 splicing involves the assembly of a multiprotein complex on a regulatory sequence in the IVS3 5' exon. The P-element somatic inhibitor protein (PSI) is a component of this ribonucleoprotein complex and is required for inhibition of IVS3 splicing in vitro. The soma-specific expression pattern of PSI suggests that its low abundance in the germ line allows IVS3 splicing. We demonstrate that ectopic expression of PSI in the female germ line is sufficient to repress splicing of an IVS3 reporter transgene. We also show that IVS3 splicing is activated in somatic embryonic cells in the presence of an antisense PSI ribozyme. These results support the model that PSI is a tissue-specific regulator of IVS3 splicing in vivo.

There has been a long history of innovation and development of tools for gene discovery and genetic analysis in Drosophila melanogaster. This includes methods to induce mutations and to screen for those mutations that disrupt specific processes, methods to map mutations genetically and physically, and methods to clone and characterize genes at the molecular level. Modern genetics also requires techniques to do the reverse to disrupt the functions of specific genes, the sequences of which are already known. This is the process referred to as reverse genetics. During recent years, some valuable new methods for conducting reverse genetics in Drosophila have been developed.

Paget's disease of bone (PDB) is a common disorder characterized by focal areas of increased and disorganized osteoclastic bone resorption, leading to bone pain, deformity, pathological fracture, and an increased risk of osteosarcoma. Genetic factors play an important role in the pathogenesis of Paget's disease. In some families, the disease has been found to be linked to a susceptibility locus on chromosome 18q21-22, which also contains the gene responsible for familial expansile osteolysis (FEO)--a rare bone dysplasia with many similarities to Paget's disease. Insertion mutations of the TNFRSF11A gene encoding Receptor Activator of NF kappa B (RANK) have recently been found to be responsible for FEO and rare cases of early onset familial Paget's disease. Loss of heterozygosity (LOH) affecting the PDB/FEO critical region has also been described in osteosarcomas suggesting that TNFRSF11A might also be involved in the development of osteosarcoma. In order to investigate the possible role of TNFRSF11A in the pathogenesis of Paget's disease and osteosarcoma, we conducted mutation screening of the TNFRSF11A gene in patients with familial and sporadic Paget's disease as well as DNA extracted from Pagetic bone lesions, an osteosarcoma arising in Pagetic bone and six osteosarcoma cell lines. No specific abnormalities of the TNFRSF11A gene were identified in a Pagetic osteosarcoma, the osteosarcoma cell lines, DNA extracted from Pagetic bone lesions, or DNA extracted from peripheral blood in patients with familial or sporadic Paget's disease including several individuals with early onset Paget's disease. These data indicate that TNFRSF11A mutations contribute neither to the vast majority of cases of sporadic or familial PDB, nor to the development of osteosarcoma.