A genetic screen for DNA double-strand break repair mutations in Drosophila

Abstract

The study of DNA double-strand break (DSB) repair has been greatly facilitated by the use of rare-cutting endonucleases, which induce a break precisely at their cut sites that can be strategically placed in the genome. We previously established such a system in Drosophila and showed that the yeast I-SceI enzyme cuts efficiently in Drosophila cells and those breaks are effectively repaired by conserved mechanisms. In this study, we determined the genetic requirements for the repair of this I-SceI-induced DSB in the germline. We show that Drosophila Rad51 and Rad54 are both required for homologous repair by gene conversion, but are dispensable for single-strand annealing repair. We provided evidence suggesting that Rad51 is more stringently required than Rad54 for intersister gene conversion. We uncovered a significant role of DNA ligase IV in nonhomologous end joining. We conducted a screen for candidate mutations affecting DSB repair and discovered novel mutations in genes that include mutagen sensitive 206, single-strand annealing reducer, and others. In addition, we demonstrated an intricate balance among different repair pathways in which the cell differentially utilizes repair mechanisms in response to both changes in the genomic environment surrounding the break and deficiencies in one or the other repair pathways.

Figure 1.—

The hemizygous assay. The wIw insertion has two w genes: the copy to the left of the I-SceI cut site (red box) is nonfunctional (shorter arrow), and the copy to the right is a functional mini-w (longer arrow). The shading helps illustrate the part of w that is repeated. Four possible repair mechanisms are given below the I-SceI-generated DSB (middle), with the names of the mechanism on top and the phenotypic classifications at the bottom of the diagrams that depict the molecular structures of the different repair products. The blue box represents a mutated I-SceI cut site due to imperfect NHEJ. The ovals represent eyes with different degrees of pigmentation. A mosaic eye has both white and red areas.

Figure 2.—

The homozygous assays. (A) The assay with [wIw]8z as a template for interhomolog GC. The red box represents a normal I-SceI cut site. The green box represents the mutated cut site in [wIw]8z. The four possible outcomes of this assay are given at the bottom. The phenotypic classification of the progeny was done the same way as it was done for the hemizygous assay, except that the products of interhomolog GC were distinguished from those of imperfect NHEJ by an allelic-specific PCR reaction. (B) The assay with [wIw]yellow as the GC template. The [wIw]yellow chromosome has half of an I-SceI cut site remaining (the narrower red box), and a 333-bp deletion (dotted line), which includes part of the mini-w gene (see main text). The interhomolog GC products can be scored directly by the presence of yellow-colored eyes (yellow oval).

Figure 3.—

Two possible mechanisms for interhomolog GC with [wIw]yellow. Horizontal lines represents DNA single strands. The region marked by vertical lines represents the 333-bp region that is not present on the [wIw]yellow chromosome. To the left is the traditional “Double Holliday Junction (DHJ)” model for GC. First, the DSB is enlarged to a double-strand gap removing heterology between the homologs (area marked by vertical lines). Both ends invade the template and initiate DNA synthesis with the direction indicated by the arrow. DHJ formation and resolution complete GC repair. To the right is the SDSA model for GC. Following single-strand resection, only the left end of the DSB invades the template and initiates DNA synthesis. Once synthesis has passed the heterologous region marked by vertical lines, the invading strand detaches from the template and anneals with the right end of the DSB. Other mechanisms might also be possible.