Efficient Gap Repair in Drosophila melanogaster Requires a Maximum of 31 Nucleotides of Homologous Sequence at the Searching Ends

Abstract

Double-strand breaks (DSB) were generated in the Drosophila melanogaster white gene by excision of the P-w hd element. An ectopic P-element vector carrying a modified white gene was used as a template for DSB repair. All template-dependent repair events were examined, and four different classes of events were recovered. The two most common products observed were gene conversions external to the P-w hd element and gene conversions (targeted transpositions) internal to the P-w hd element. These two events were equally frequent. Similar numbers for both orientations of internal conversion events were recovered. The results suggest that P-element excision occurs by a staggered cut that leaves behind at least 33 nucleotides of single-stranded sequence. Our results further demonstrate that an efficient homology search is conducted by the broken end with less than 31 nucleotides. Double-strand break (DSB) repair is used for mating-type switching and meiotic recombination in yeast (34) and immunoglobulin V(D)J rearrangement in vertebrates (26). In addition, DSB repair has been exploited as a gene-targeting method in Drosophila melanogaster, yeast, Caenorhabditis elegans, and mice (6,19,38,44,49,59). Understanding this process of repair will improve in vivo genome manipulation techniques and may lead to an efficient gene-targeting system for higher eukaryotes.P elements in Drosophila transpose by a cut-and-paste mechanism, and their excision produces a DSB in the chromosome (12,19,32). The break can be repaired by copying homologous sequence from the sister strand, the homolog, an ectopic sequence, or a plasmid (12,13,19,31,33,38,39). Several lines of evidence suggest that P excision occurs by a staggered cut at the P ends, leaving behind a single-strand sequence that includes part or all of the terminal 31-bp inverted repeat (31,40,55).A number of models have been developed to explain the repair of DSBs in a genome. The single-strand annealing model proposes that the DNA ends at a break are rendered single stranded and that homologous sequences anneal (36,57). Repair synthesis and ligation seal the break. This model explains the formation of repair events with deletions.The DSB repair model (45,59) proposes that the break in the chromosome is enlarged to a gap by exonucleases and that this gap is repaired by copying homologous sequence from elsewhere in the genome. The Holliday junctions that are formed in this model are resolved by nucleolytic cleavage, thus leading to the homologous recombination products.Studies on DSB repair in mitotically dividing Drosophila cells have revealed information about the break repair pathway in this organism (3,12,13,19,31,33,38,39,55). Many of the gap repair products contain stretches of homologous DNA copied from a template. None of the products involve exchange of flanking markers but, instead, are the result of gene conversion (12,19,58). The synthesis-dependent strand-annealing (SDSA) model was proposed to account for these observations (16,38). This model predicts that each side o...

Drosophila P-element transposase is a novel site-specific endonuclease

“…At a low frequency, both 18 and 19 nucleotides from each end have been observed (Staveley et al 1995). In addition, it was found that a polymorphism located 33 nucleotides within the donor site P element was retained following excision and template-dependent repair from ectopically located P elements, suggesting that cleavage occurred at least 33 nucleotides within the P-element ends (Keeler and Gloor 1997). We propose that these products can either arise from alternative transposaseinduced cleavage products similar to those observed in our primer extension analysis (Fig.…”

Expression pattern of Gal4 enhancer trap insertions into the bric à brac locus generated by P element replacement

“…We used P element replacement to make bab P[Gal4] enhancer trap lines. This method is based on the finding that after a P element excises from a chromosome, a P element from a second site frequently transposes into the site of the excised P element (Gonzy-Treboul et al, 1995;Keeler and Gloor, 1997;Sepp and Auld, 1999). bab P and bab A128 are well characterized bab enhancer trap lines that express ␤-gal in patterns very similar to the endogenous bab expression patterns (Couderc et al, 2002;Godt et al, 1993;Godt and Laski, 1995).…”

Three-partner conversion induced by the P-element transposase in Drosophila melanogaster

“…As we (Gonzy-Tre boul et al 1995) and other authors (Keeler and Gloor 1997) have previously suggested, the conversion mechanism derives from the synthesisdependent strand annealing (SDSA) type of repair that takes place after excision of a P-element (Nassif et al 1994). We previously obtained six conversions at the BRC locus and proposed a mechanism involving three partners: after excision of the P-element ± which produces a double-strand break ± the broken 3¢ ends are repaired using ®rst the homologous template on the sister chromatid and second an ectopic template, i.e.…”

“…In some cases, it has been possible to introduce targeted mutations (Nassif et al 1994;Hogga and Karch 1995;Keeler et al 1996) at a precise site. The replacement of one P-element by another has been reported by several authors (Salz et al 1987;Geyer et al 1988;Heslip and Hodgetts 1994;Staveley et al 1994;Gonzy-Tre boul et al 1995;Keeler and Gloor 1997;Sepp and Auld 1999;J.-M. Dura, personal communication). Several conversion mechanisms have been postulated (Gonzy-Tre boul et al 1995;Keeler and Gloor 1997).…”

“…This mechanism was strongly supported by the presence of a polymorphic site at position 33 of the P-element sequence, which allowed us to detect, among the six independent convertants obtained, chimeric sequences composed of parts of both the original P-element and the enhancer-trap transposon. However, on the basis of an analysis of 24 cases of conversion, Keeler and Gloor (1997) concluded that the repair mechanism involves only two partners and proceeds by direct repair of the broken 3¢ ends using the ectopic template, without any requirement for the sister chromatid. Moreover, a third mechanism could not be excluded (Gonzy-Tre boul et al 1995): this is based on the capacity of P-elements to transpose within, or close to, pre-existing P-elements, thus creating double elements (Roiha et al 1988;Daniels and Chovnick 1993;Tower et al 1993;Delattre et al 1995).…”