Copyright Ó 2009 by the Genetics Society of AmericaDOI: 10.1534/genetics.109.101329Genetic Analysis of Zinc-Finger Nuclease-Induced GeneTargeting in DrosophilaAna Bozas,1Kelly J. Beumer, Jonathan K. Trautman and Dana Carroll2Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, Utah 84112-5650Manuscript received February 10, 2009Accepted for publication April 12, 2009ABSTRACTUsing zinc-finger nucleases (ZFNs) to cleave the chromosomal target, we have achieved highfrequencies of gene targeting in the Drosophila germline. Both local mutagenesis through non-homologous end joining (NHEJ) and gene replacement via homologous recombination (HR) arestimulated by target cleavage. In this study we investigated the mechanisms that underlie these processes,using materials for the rosy (ry) locus. The frequency of HR dropped significantly in flies homozygous formutations in spnA (Rad51) or okr (Rad54), two components of the invasion-mediated synthesis-dependentstrand annealing (SDSA) pathway. When single-strand annealing (SSA) was also blocked by the use of acircular donor DNA, HR was completely abolished. This indicates that the majority of HR proceeds viaSDSA, with a minority mediated by SSA. In flies deficient in lig4 (DNA ligase IV), a component of themajor NHEJ pathway, the proportion of HR products rose significantly. This indicates that most NHEJproducts are produced in a lig4-dependent process. When both spnA and lig4 were mutated and a circulardonor was provided, the frequency of ry mutations was still high and no HR products were recovered. Thelocal mutations produced in these circumstances must have arisen through an alternative, lig4-independent end-joining mechanism. These results show what repair pathways operate on double-strandbreaks in this gene targeting system. They also demonstrate that the outcome can be biased toward genereplacement by disabling the major NHEJ pathway and toward simple mutagenesis by interfering with themajor HR process.EXPERIMENTAL gene targeting relies on cellularDNA repair activities. When a donor DNA carryingthe desired sequence modifications is introduced intocells or organisms, successful gene replacement dependson cellular capabilities for homologous recombination(HR).We have developed a very efficient gene targetingprocedure for Drosophila based on target cleavage bydesigned zinc-finger nucleases (ZFNs) (Bibikova et al.2002, 2003; Beumer et al. 2006). Because the DNA-binding domain consists of Cys2His2zinc fingers, thesehybrid proteins are very flexible in their recognitioncapabilities. Each finger makes contact primarily with3 bp of DNA, and arrays of three to four fingers providesufficient affinity for in vivo binding. Since two ZFNs arerequired to cleave any single target, a pair of three-finger proteins provides adequ ate specificity, in princi-ple, to attack a unique genomic sequence.When a double-strand break (DSB) is cre ated at aspecific site in the genome, DNA sequence changes resulteither from HR with a marked donor DNA or frominaccurate nonhomologous end joining (NHEJ). In thisstudy we set out to determine which cellular activitiessupport each of these processes and to learn whether therepair outcome could be biased by elimination of one oranother pathway.Earlier studies showed that Drosophila uses DSB repairmechanisms that are very similar to other eukaryoticorganisms (Wyman and Kanaar 2006). In the realm ofHR, homologs of the Rad51 (spnA) and Rad54 (okr)proteins are required for the break-initiated meioticrecombination events needed for proper chromosomesegregation in females (Kooistra et al. 1997, 1999;Ghabrial et al. 1998; Staeva-Vieira et al. 2003). Muta-tions in both these genes sensitize somatic cells in earlydevelopmental stages to ionizing radiation (IR) and toother DNA damaging agents. In yeast, mutations in theRAD51 gene sensitize cells to IR and lead to severesporulation defects (Symington 2002). Mutations inRAD54 also confer sensitivity to DNA damaging agents,but are less severely affected in meiosis. In mice absenceof the Rad51 protein is lethal in early embryonic de-velopment (Lim and Hasty 1996; Tsuzuki et al. 1996).Absence of Rad54 is tolerable, but confers sensitivity to IRand other agents (Essers et al. 1997).Supporting information is available online at http://www.genetic s.o rg/cgi/content/full/genetics.109.101329/DC1.1Present address: Boston Biomedical Research Institute, 64 Grove St.,Watertown, MA 02472.2Corresponding author: University of Utah School of Medicine, De-partment of Biochemistry , 15 N. Medical Dr. East, Room 4100, Salt LakeCity, UT 84112-5650. E-mail: [email protected] 182: 641–651 ( July 2009)The Drosophila genome encodes components of themajor NHEJ pathway, including DNA ligase IV (lig4),Xrcc4, and the Ku proteins (ku70, ku80). Loss of Lig4sensitizes early developmental stages to ionizing radia-tion, and this effect is more severe in the absence ofRad54 (Gorski et al. 2003). In other assays a consider-able amount of end joining still occurs in lig4 mutants(McVey et al. 2004c; R omeijn et al. 2005), suggesting asecondary or backup pathway, as has been observed inother organisms (Nussenzweig and Nussenzweig2007). Yeasts rely more heavily on HR for DSB repair,so lig4 mutations have little effect unless HR is impaired.In contrast, lig4/mice die early in embryogenesis(Barnes et al. 1998), although they can be rescued byelimination of p53 (Frank et al. 2000).The molecular pro cess of DSB repair by HR has beenstudied in Drosophila by introducing a single break at aunique target either by P -element excision or by I-SceIcleavage. The evidence strongly points to an invasionand copying mechanism called synthesis-dependentstrand annealing (SDS A) (see below) (Kurkulos et al.1994; Nassif et al. 1994; McVey et al. 2004a). Theseevents are largely dep endent on spnA (McVey et al.2004a; Johnson-Schlitz et al. 2007; Wei and Rong2007), okr (Johnson-Schlitz et al. 2007; Wei and Rong2007), and other factors, including mus309 (the Dro-sophila Bloom syndrome protein, DmBlm) (Adamset al. 2003; McVey et al. 2004b, 2007; Johnson-Schlitzand Engels 2006). When the break site is surroundedby direct repeats, repair proceeds efficiently by single-strand annealing (SSA) (Rong and Golic 2003; Prestonet al. 2006).The key difference between SDSA and SSA is themechanistic requirement for strand invasion in theformer. SSA has rather modest genetic dependenciesand is independent of
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