Efficient copying of nonhomologous sequences from ectopic sites via P-element-induced gap repair.

Abstract

P-element-induced gap repair was used to copy nonhomologous DNA into the Drosophila white locus. We found that nearly 8,000 bp of nonhomologous sequence could be copied from an ectopic template at essentially the same rate as a single-base substitution at the same location. An in vitro-constructed deletion was also copied into white at high frequencies. This procedure can be applied to the study of gene expression in Drosophila melanogaster, especially for genes too large to be manipulated in other ways. We also observed several types of more complex events in which the copied template sequences were rearranged such that the breakpoints occurred at direct duplications. Most of these can be explained by a model of double strand break repair in which each terminus of the break invades a template independently and serves as a primer for DNA synthesis from it, yielding two overlapping single-stranded sequences. These single strands then pair, and synthesis is completed by each using the other as a template. This synthesis-dependent strand annealing (SDSA) model as a possible general mechanism in complex organisms is discussed.We used a transposable P-element insertion allele of the white gene to study the repair of a double-stranded DNA break in Drosophila melanogaster. Unrepaired double-stranded DNA breaks can be cell lethal after mitosis. Consequently, organisms have developed efficient methods for their repair. Double strand break repair is thought to occur by a process in which the broken ends search for a homologous sequence, invade it, and serve as primers for DNA synthesis to reconstitute the broken chromosome (29, 32, 39).P elements are thought to transpose by a cut-and-paste process in which excision of the element breaks both DNA strands of the chromosome (6,12,19). Experiments have shown that the break is usually repaired by copying the corresponding sequences from the sister strand (6), thus restoring a copy of the P element to the break site. Alternatively, sequence may be copied from a template on the homolog (18,25) or one inserted in the genome at an ectopic site (12). The DNA sequence flanking the P excision site is usually converted as a continuous block with an average tract length of about 1,400 nucleotides. Recent results have also shown that small insertions and deletions from the homolog can be converted into the excision site (18).These observations suggested to us that the gap repair process could be adapted for use as an efficient gene targeting method in D. melanogaster (Fig. 1). We tested this possibility by using a P-element insertion in the white gene, whd (whd80KJ7), which is known to excise at high frequencies in the presence of P transposase. We then recovered gap repair products that occurred in the presence of one of five ectopi- cally located templates. In four of these templates, the white gene had been altered by the addition of unrelated sequences of lengths up to approximately 7,970 bp. We found that each of the nonhomologous sequences could be copied into the white locus almo...

Targeted gene alteration in Caenorhabditis elegans by gene conversion

“…In a general sense, however, any desired mutation could be created in vitro and then converted to the corresponding genetic locus in vivo, such as activating, dominantnegative, misexpression and other alleles with point mutations, specific substitutions or similar alterations in their genes. With regard to deletions, our finding that the 1.5-kb and 0.85-kb deletions are converted with roughly equal frequencies suggests that the homology scanning window may require that only one end of the dsDNA break be located and secured, as has been suggested for D. melanogaster 28 . To our knowledge, the 1.5-kb deletion is larger than any deletion so far reported in D. melanogaster.…”

“…Last, deletions and insertions might be converted more easily than point mutations. This last possibility seems less likely, considering that, first, evidence from P elementinduced gene conversion in D. melanogaster suggests that deletions are converted at a lower rate than are point mutations and insertions 27,28 ; and second, point mutations in a conversion template should contain more contiguous homologous sequence near the transposon insertion site than should deletions, and the nearer a polymorphism in the homologous sequence is to the strand break site, the more likely it is to be converted 20 .…”

DrosophilaP element: Transposition, regulation and evolution

“…Recently, analysis of conversion tracts from partially heterologous templates led Nassif et al (1994) to propose a synthesis-dependent strand annealing model (SDSA) similar to the one studied by Formosa and Alberts (1986). It involves the release of the growing strand behind a migration bubble.…”

Abortive Gap Repair: Underlying Mechanism for Ds element Formation

“…A mechanism responsible for the formation of nonautonomous elements has been proposed for the Drosophila P element. Upon P excision, gap repair was shown to be initiated from the donor site, leading to the replacement of the excised element at its original locus (11,43). Based on these findings, internally deleted elements were suggested to originate from premature interruption of gap repair (10) and insertions were proposed to occur during gap repair as a result of template switch to an ectopic site by the synthesisdependent strand-annealing (SDSA) gap repair pathway (43).…”

“…Upon P excision, gap repair was shown to be initiated from the donor site, leading to the replacement of the excised element at its original locus (11,43). Based on these findings, internally deleted elements were suggested to originate from premature interruption of gap repair (10) and insertions were proposed to occur during gap repair as a result of template switch to an ectopic site by the synthesisdependent strand-annealing (SDSA) gap repair pathway (43). In plants, a series of observations with the maize Mutator (Mu) elements suggest that abortive gap repair (AGR) might also be involved in the formation of nonautonomous Mu elements (21,32), but a detailed study of AGR has not been carried out for any species.…”