Academic journal article Genetics

A Genetic Screen for DNA Double-Strand Break Repair Mutations in Drosophila

Academic journal article Genetics

A Genetic Screen for DNA Double-Strand Break Repair Mutations in Drosophila

Article excerpt

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.

Aeukaryotic cellemploys a variety of conservedmechanisms torepairdouble-strandbreaks(DSBs),which threaten the integrity of its genome. These mechanisms can be grossly grouped into two pathways: homologous recombinational (HR) repair and nonhomologous end joining (NHEJ). Gene conversion (GC) is a common outcome of HR both in mitotic and in meiotic cells (reviewed in Paques andHaber 1999). In GC, theDSBis repaired by DNA synthesis templated from a homologous segment. GC is generally conservative, resulting in no net loss of DNA sequences. If the template for GC is located on the sister chromatid, such repair precisely restores the original sequence at the break. Many factors play important roles in regulatingGC, notably the Rad52 epistasis group in budding yeast and their homologs in other organisms (Symington 2002). These include Rad50, Rad51, Rad52, Rad54, Rad59, Mre11, and others. Single-strand annealing (SSA) repair is commonly used to repair DSBs that occur between direct repeats (Paques and Haber 1999). SSA is nonconservative, resulting in the loss of one of the repeats as well as the segment between the repeats. The budding yeast Rad52 and Rad59 proteins are essential for SSA (Ivanov et al. 1996; Sugawara et al. 2000; Davis and Symington 2001), but Drosophila homologs for neither protein can be identified by sequence homology searches. The identification of their functional homologs in flies would have important implications since a similar situation exists for both Caenorhabditis elegans and Arabidopsis.

In NHEJ, the two ends of the DSB are ligated with little or no homology requirement between them. NHEJ is intrinsically mutagenic in that it can lead to sequence alteration at the site of DSB. On the other hand, precise end joining can be a predominant pathway if the ends have complementary single-stranded overhangs (Boulton and Jackson 1996). Several conserved proteins have been shown to regulate NHEJ, which include the Ku70-Ku80 heterodimer andDNAligase IV(reviewed inDaley et al. 2005). However, recent studies in Drosophila shed doubts on the importance of ligase IV in regulating end joining (Bi et al. 2004; McVey et al. 2004a; Romeijn et al. 2005).

Our understanding of repair mechanisms has been greatly enhanced by studies using site-specific endonucleases, especially rare cutters. The advantages of being able to induce site-specific DSBs on demand are manyfold. One can control the timing and severity of DSB generation by manipulating endonuclease production. One can control the number and genomic location of the DSB by strategically placing the enzyme cut site. …

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