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Initial events in double-strand break repair by homologous recombination in vivo involve homology searching, 39 strand invasion, and new DNA synthesis. While studies in yeast have contributed much to our knowledge of these processes, in comparison, little is known of the early events in the integrated mammalian system. In this study, a sensitive PCR procedure was developed to detect the new DNA synthesis that accompanies mammalian homologous recombination. The test system exploits a well-characterized gene targeting assay in which the transfected vector bears a gap in the region of homology to the single-copy chromosomal immunoglobulin m heavy chain gene in mouse hybridoma cells. New DNA synthesis primed by invading 39 vector ends copies chromosomal m-gene template sequences excluded by the vector-borne double-stranded gap. Following electroporation, specific 39 extension products from each vector end are detected with rapid kinetics: they appear after 0.5 hr, peak at 3-6 hr, and then decline, likely as a result of the combined effects of susceptibility to degradation and cell division. New DNA synthesis from each vector 39 end extends at least $1000 nucleotides into the gapped region, but the efficiency declines markedly within the first $200 nucleotides. Over this short distance, an average frequency of 39 extension for the two invading vector ends is $0.007 events/vector backbone. DNA sequencing reveals precise copying of the cognate chromosomal m-gene template. In unsynchronized cells, 39 extension is sensitive to aphidicolin supporting involvement of a replicative polymerase. Analysis suggests that the vast majority of 39 extensions reside on linear plasmid molecules. Homologous recombination is an important pathway of repairing DSBs and its proper function is integral to cell survival (Wyman and Kanaar 2006). The repair of a DSB by homologous recombination involves 59-to-39 resection of the DNA ends, homology searching, strand invasion of the homologous template, and new DNA synthesis. Later steps in the recombination process can involve unwinding of the newly synthesized strand from the repair template to yield a noncrossover product or formation of a joint molecule bearing Holliday junctions that may be resolved to yield either crossover or noncrossover products (Pâques and Haber 1999;Li and Heyer 2008).Studies of homologous recombination in yeast, mammalian cells, and other organisms have benefitted from analyzing repair of DSBs generated at specific sites in vivo by the endonucleases HO and I-SceI (Haber 2000;Johnson and Jasin 2001;Wei and Rong 2007). Current insight into the early initiation events of homolSupporting information is available online at

In this study, we exploited a plasmid-based assay that detects the new DNA synthesis (39 extension) that accompanies Rad51-mediated homology searching and strand invasion steps of homologous recombination to investigate the interplay between Rad51 concentration and homology length. Mouse hybridoma cells that express endogenous levels of Rad51 display an approximate linear increase in the frequency of 39 extension for homology lengths of 500 bp to 2 kb. At values below 500 bp, the frequency of 39 extension declines markedly, suggesting that this might represent the minimal efficient processing segment for 39 extension. Overexpression of wild-type Rad51 stimulated the frequency of 39 extension by 3-fold for homology lengths ,900 bp, but when homology was .2 kb, 39 extension frequency increased by as much as 10-fold. Excess wild-type Rad51 did not increase the average 39 extension tract length. Analysis of cell lines expressing N-terminally FLAG-tagged Rad51 polymerization mutants F86E, A89E, or F86E/A89E established that the 39 extension process requires Rad51 polymerization activity. Mouse hybridoma cells that have reduced Brca2 (Breast cancer susceptibility 2) due to stable expression of small interfering RNA show a significant reduction in 39 extension efficiency; expression of wild-type human BRCA2, but not a BRCA2 variant devoid of BRC repeats 1-8, rescues the 39 extension defect in these cells. Our results suggest that increased Rad51 concentration and homology length interact synergistically to promote 39 extension, presumably as a result of enhanced Brca2-mediated Rad51 polymerization. Homologous recombination is a complex, multi-step process (reviewed in Brugmans et al. 2007). In eukaryotes, the central step in the repair of a DNA DSB involves the binding of multiple monomers of the RAD51 protein (a functional homolog of the Escherichia coli RecA recombinase) to 39-ending single-stranded DNA overhangs created by nucleolytic resection (Van Den Bosch et al. 2003). The resulting RAD51 nucleoprotein filament promotes formation of a joint molecule between the processed broken DNA and the homologous repair template by the orchestrated steps of homology searching and DNA strand invasion and exchange. Joint molecule formation is followed by new DNA synthesis, which replaces nucleotides lost through DSB formation and 39-end resection (Pâques and Haber 1999;Symington 2002;Li and Heyer 2008). Depending on the subpathway of HR, subsequent steps may involve unwinding (dissolution) of the DNA strand containing the newly synthesized DNA and ligation to the processed second end or the formation of a stably joined Holliday junction intermediate that can be processed further by structure-specific endonucleases yielding recombinant products (Li and Heyer 2008).At the heart of the HR process is the Rad51 (or RecA) nucleoprotein filament, whose formation requires the initial association of four to five monomers with 39-ending singlestranded DNA in the step known as nucleation (Galletto