A Strand Invasion 3′ Polymerization Intermediate of Mammalian Homologous Recombination

Abstract

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

Nascent DNA Synthesis During Homologous Recombination Is Synergistically Promoted by the Rad51 Recombinase and DNA Homology

“…Influence of homology length on the efficiency of new DNA synthesis Previously, we described a novel assay that detects the new DNA synthesis that accompanies the early strand invasion step of homologous recombination in vivo (Si et al 2010). In this assay, recipient mouse hybridoma cells are electroporated with a gene-targeting vector in which BstEII digestion creates a 1.2-kb double-stranded gap (DSG) within the region of homology to the single-copy hybridoma chromosomal immunoglobulin m-gene ( Figure 1A).…”

“…The various BstEII-linearized vectors were electroporated separately into recipient igm482 hybridoma cells. After 6 hr of incubation [shown previously to result in maximum 39 extension (Si et al 2010)], plasmid DNA was extracted and PCR-amplified with primers neoF209/CmR1 (to measure 39 extension) and neoR+6/neoF-1 (to measure vector backbone recovery). As shown in the polynomial regression in Figure 1C (R 2 = 0.99), the frequency of 39 extension/ vector backbone displays an approximate linear response for homology lengths .…”

“…All Rad51 constructs were verified by DNA sequencing. To generate transformants, 50 mg of XmnI-linearized plasmid DNA was electroporated into 2 3 10 7 viable igm482 cells as described in Si et al (2010). The number of viable cells was enumerated by trypan blue exclusion using a Bright-Line Hemacytometer (Hausser Scientific).…”

“…The formation of nascent DNA that follows the strand invasion event of homologous recombination (39 extension) was measured by PCR as described previously by Si et al (2010). The intensity of specific PCR bands was quantified by densitometric analysis using BioRad Gel Doc instrumentation and Quantity One imaging software (Version 4.4.6, BioRad).…”

“…While mammalian cells contain functional analogs of yeast recombination proteins, their role in HR, especially in the early steps of strand invasion and new DNA synthesis, is not well understood. Previously, we transfected mammalian cells with linearized plasmid DNA bearing homology to a chromosomal target gene and detected the nascent DNA formed by polymerizing 39 ends (39 extension) in vivo (Si et al 2010). In the present study, we investigated the requirement for homology and the role of Rad51 and Brca2 proteins in the process of 39 extension.…”