Copyright 2009 by the Genetics Society of America DOI 10 1534 genetics 109 101329 Genetic Analysis of Zinc Finger Nuclease Induced Gene Targeting in Drosophila Ana Bozas 1 Kelly J Beumer Jonathan K Trautman and Dana Carroll2 Department of Biochemistry University of Utah School of Medicine Salt Lake City Utah 84112 5650 Manuscript received February 10 2009 Accepted for publication April 12 2009 ABSTRACT Using zinc finger nucleases ZFNs to cleave the chromosomal target we have achieved high frequencies of gene targeting in the Drosophila germline Both local mutagenesis through nonhomologous end joining NHEJ and gene replacement via homologous recombination HR are stimulated 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 for mutations in spnA Rad51 or okr Rad54 two components of the invasion mediated synthesis dependent strand annealing SDSA pathway When single strand annealing SSA was also blocked by the use of a circular donor DNA HR was completely abolished This indicates that the majority of HR proceeds via SDSA with a minority mediated by SSA In flies deficient in lig4 DNA ligase IV a component of the major NHEJ pathway the proportion of HR products rose significantly This indicates that most NHEJ products are produced in a lig4 dependent process When both spnA and lig4 were mutated and a circular donor was provided the frequency of ry mutations was still high and no HR products were recovered The local mutations produced in these circumstances must have arisen through an alternative lig4independent end joining mechanism These results show what repair pathways operate on double strand breaks in this gene targeting system They also demonstrate that the outcome can be biased toward gene replacement by disabling the major NHEJ pathway and toward simple mutagenesis by interfering with the major HR process E XPERIMENTAL gene targeting relies on cellular DNA repair activities When a donor DNA carrying the desired sequence modifications is introduced into cells or organisms successful gene replacement depends on cellular capabilities for homologous recombination HR We have developed a very efficient gene targeting procedure for Drosophila based on target cleavage by designed zinc finger nucleases ZFNs Bibikova et al 2002 2003 Beumer et al 2006 Because the DNAbinding domain consists of Cys2His2 zinc fingers these hybrid proteins are very flexible in their recognition capabilities Each finger makes contact primarily with 3 bp of DNA and arrays of three to four fingers provide sufficient affinity for in vivo binding Since two ZFNs are required to cleave any single target a pair of threefinger proteins provides adequate specificity in principle to attack a unique genomic sequence Supporting information is available online at http www genetics org cgi content full genetics 109 101329 DC1 1 Present address Boston Biomedical Research Institute 64 Grove St Watertown MA 02472 2 Corresponding author University of Utah School of Medicine Department of Biochemistry 15 N Medical Dr East Room 4100 Salt Lake City UT 84112 5650 E mail dana biochem utah edu Genetics 182 641 651 July 2009 When a double strand break DSB is created at a specific site in the genome DNA sequence changes result either from HR with a marked donor DNA or from inaccurate nonhomologous end joining NHEJ In this study we set out to determine which cellular activities support each of these processes and to learn whether the repair outcome could be biased by elimination of one or another pathway Earlier studies showed that Drosophila uses DSB repair mechanisms that are very similar to other eukaryotic organisms Wyman and Kanaar 2006 In the realm of HR homologs of the Rad51 spnA and Rad54 okr proteins are required for the break initiated meiotic recombination events needed for proper chromosome segregation in females Kooistra et al 1997 1999 Ghabrial et al 1998 Staeva Vieira et al 2003 Mutations in both these genes sensitize somatic cells in early developmental stages to ionizing radiation IR and to other DNA damaging agents In yeast mutations in the RAD51 gene sensitize cells to IR and lead to severe sporulation defects Symington 2002 Mutations in RAD54 also confer sensitivity to DNA damaging agents but are less severely affected in meiosis In mice absence of the Rad51 protein is lethal in early embryonic development Lim and Hasty 1996 Tsuzuki et al 1996 Absence of Rad54 is tolerable but confers sensitivity to IR and other agents Essers et al 1997 642 A Bozas et al TABLE 1 Repair mutations Gene spnA Rad51 okr Rad54 mei W68 Spo11 lig4 Allele Mutation Reference spnA057 spnA093A okrAA okrAG mei W681 mei W68k05603 lig4169 Null Null Null Null Null Hypomorph Null Staeva Vieira et al 2003 Staeva Vieira et al 2003 Ghabrial et al 1998 Ghabrial et al 1998 McKim and Hayashi Hagihara 1998 McKim and Hayashi Hagihara 1998 McVey et al 2004c Sources spnA057 Yikang Rong National Institutes of Health spnA093A and lig4169 Jeff Sekelsky University of North Carolina okr stocks Trudi Schupback Princeton University mei W68 stocks Drosophila Stock Center Bloomington IN The Drosophila genome encodes components of the major NHEJ pathway including DNA ligase IV lig4 Xrcc4 and the Ku proteins ku70 ku80 Loss of Lig4 sensitizes early developmental stages to ionizing radiation and this effect is more severe in the absence of Rad54 Gorski et al 2003 In other assays a considerable amount of end joining still occurs in lig4 mutants McVey et al 2004c Romeijn et al 2005 suggesting a secondary or backup pathway as has been observed in other organisms Nussenzweig and Nussenzweig 2007 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 by elimination of p53 Frank et al 2000 The molecular process of DSB repair by HR has been studied in Drosophila by introducing a single break at a unique target either by P element excision or by I SceI cleavage The evidence strongly points to an invasion and copying mechanism called synthesis dependent strand annealing SDSA see below Kurkulos et al 1994 Nassif et al 1994 McVey et al 2004a These events are largely dependent on spnA McVey et al 2004a Johnson Schlitz et al 2007 Wei and Rong 2007 okr Johnson Schlitz et al 2007 Wei and Rong 2007 and
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