Repair of P element-induced DNA double-strand breaks

DNA transposons are clearly delineated segments of DNA that have the ability to move about within a genome in a process known as transposition. Non-replicatioe, cutand-paste transposons are mobilized by the complete excision of the transposon from the genomic DNA, thus creating a DNA double-strand break at the transposon donor site. The P element transposon of Drosophila melanogaster transposes by a cut-and-paste mechanism, thereby providing a rich experimental system to study the repair of DNA double-strand breaks at a specific location in the genome.; P element excision from the white gene results in DNA breaks that are most often repaired by recombination with the sister chromatid, thereby suggesting that P element excision is regulated to occur after DNA replication, in G2 of the cell cycle. We tested this hypothesis by arresting Drosophila tissue culture cells with cyclin-specific dsRNA to induce G1 or G2 cell cycle arrest and by exogenous decapo (p21) expression to induce G1 cell cycle arrest. P element transposase activity was determined in arrested and asynchronous cells using an in vivo P element excision assay that measures the repair of P element donor sites by non-homologous end joining (NHEJ) and therefore indicates both P element excision and NHEJ repair. Our results indicate that transposase is active in both G1- and G2-arrested cells. In addition, repair by NHEJ occurred in both G1- and G2-arrested cells, indicating that this pathway of double-strand break repair is not restricted to G1. These data cast considerable doubt on the hypothesis that P element transposase activity is cell cycle-regulated and provide supporting evidence for the continued activity of NHEJ after DNA replication.; The Drosophila mus309 gene product is homologous to the Bloom's syndrome helicase. Bloom's syndrome is a rare genetic disorder that is marked by cancer predisposition, genome instability, and hyper recombination. Genetic analysis of mus309 mutants in Drosophila has provided valuable insights into the role of the Bloom's helicase in DNA repair, as a complementary approach we used biochemical methods to study Drosophila Bloom's (DmBLM) in vitro. DmBLM was purified as an N-terminal polyoma epitope-tagged fusion protein from Drosophila tissue culture cells. Purified DmBLM exists exclusively as a high molecular weight (∼1.17MDa) species, is a DNA-dependent ATPase, has 3'-5' DNA helicase activity, prefers forked substrate DNAs, and anneals complementary DNAs. High-affinity DNA binding is ATP-dependent and low-affinity ATP-independent interactions contribute to forked DNA substrate binding and drive strand annealing. DmBLM combines DNA strand displacement with DNA strand annealing in a coordinated reaction where the displacement of one DNA strand is coupled to the annealing of another complementary DNA strand.; The Bloom's syndrome helicase and RecQ-family helicases in general have poor processivity shown by an inability to unwind long duplex substrates. DmBLM helicase activity was analyzed with a 644 base pair (bp) duplex substrate. Unwinding requires single-stranded binding protein (SSB) and appears to occur at ∼30bp/s with a processivity of several hundred base pairs. Analysis of substrate binding shows that DNA binding is rapid in the presence of ATP and occurs slowly in the presence of ATPgammaS. Binding in the presence of ATPgammaS traps the DmBLM-DNA interaction and results in accumulation of DmBLM-DNA complexes. Single molecule analysis of DNA duplex unwinding by DmBLM indicates a processivity of ∼500bp and a rate of 50-100bp/s, thus supporting observations made in bulk reactions.; In Drosophila, P element mobilization in the female germline during hybrid dysgenesis results in germ cell death and sterility. We were interested in determining the role of the DNA damage checkpoint response in mitigating or contributing to female sterility following a dysgenic cross. Hybrid dysgenesis-induced and radiation-induced germ...