Lig4 and rad54 are required for repair of DNA double-strand breaks induced by P-element excision in Drosophila - PubMed (original) (raw)

Comparative Study

Lig4 and rad54 are required for repair of DNA double-strand breaks induced by P-element excision in Drosophila

Ron J Romeijn et al. Genetics. 2005 Feb.

Abstract

Site-specific double-strand breaks (DSBs) were generated in the white gene located on the X chromosome of Drosophila by excision of the w(hd) P-element. To investigate the role of nonhomologous end joining (NHEJ) and homologous recombination (HR) in the repair of these breaks, the w(hd) P-element was mobilized in flies carrying mutant alleles of either lig4 or rad54. The survival of both lig4- and rad54-deficient males was reduced to 25% in comparison to the wild type, indicating that both NHEJ and HR are involved in the repair P-induced gaps in males. Survival of lig4-deficient females was not affected at all, implying that HR using the homologous chromosome as a template can partially compensate for the impaired NHEJ pathway. In rad54 mutant females survival was reduced to 70% after w(hd) excision. PCR analysis indicated that the undamaged homologous chromosome may compensate for the potential loss of the broken chromosome in rad54 mutant females after excision. Molecular analysis of the repair junctions revealed microhomology (2-8 bp)-dependent DSB repair in most products. In the absence of Lig4, the 8-bp target site duplication is used more frequently for repair. Our data indicate the presence of efficient alternative end-joining mechanisms, which partly depend on the presence of microhomology but do not require Lig4.

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Figures

F<sc>igure</sc> 1.—

Figure 1.—

Structure and excision of the _whd P_-element. (A) The _whd P_-element is inserted into the sixth exon of the white locus on the X chromosome (O'H

are

and R

ubin

1983). The analysis of the repair junctions following the mobilization of the _whd P_-element was carried out using p7 and p9 primers (see

materials and methods

). (B) A DNA double-strand gap is generated after excision of the _whd P_-element by the _P_-element-specific endonuclease. This endonuclease is encoded by the P[_ry+_ Δ_2-3_](99B) _P_-element abbreviated here as Δ2-3. The endonucleolytic cleavage creates two staggered DSBs containing 17-bp 3′ overhangs within the 31-bp inverted repeats. TSD, target site duplication.

F<sc>igure</sc> 2.—

Figure 2.—

Scheme of the genetic crosses performed to mobilize the _whd P_-element. (A) To study the contribution of Lig4 to the repair of _whd P_-element-induced DSBs, y whd lig457/y whd lig457 females were crossed to y2 wa lig457/Y; Sb P[ry+ Δ_2-3_]/TM3, Ser males. In the next generation, both F1 males and females containing Δ2-3 transposase can be recognized on the basis of the Sb marker and were either directly analyzed by PCR and sequencing or mated to C(1)DX y w f females and M5 males, respectively. F2 males emerging from these crosses were analyzed as described for F1 progeny (see

materials and methods

). (B) The contribution of Rad54 to the repair of _P_-induced DSBs was studied by crossing y whd/y whd; JS17/SM5, Cy females to y2 wa/Y; A17-11/SM5, Cy; Sb P[ry+ Δ_2-3_]/TM3, Ser males. Both male and female F1 progenies containing Δ2-3 transposase were used for PCR and sequence analysis. In the control cross (not shown), y whd/y whd females were crossed to y2 wa; Sb P[ry+ Δ_2-3_]/TM3, Ser males and the offspring was analyzed as described above.

F<sc>igure</sc> 3.—

Figure 3.—

Mosaic eye phenotypes of _lig4-_proficient (middle) and _lig4_-mutant (right) females after the repair of _whd P_-element-induced DSBs. Use of the _wa_-containing chromosome as a template for recombination leads to restoration of the wild-type sequence of the white gene. Clonal outgrowth results in red (wild-type) spots against an apricot (wa) background. For comparison, the apricot eye of a wa, lig457 female not carrying the Δ2-3 transposase is shown on the left.

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