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, 3' 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 mu heavy chain gene in mouse hybridoma cells. New DNA synthesis primed by invading 3' vector ends copies chromosomal mu-gene template sequences excluded by the vector-borne double-stranded gap. Following electroporation, specific 3' 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 3' end extends at least approximately 1000 nucleotides into the gapped region, but the efficiency declines markedly within the first approximately 200 nucleotides. Over this short distance, an average frequency of 3' extension for the two invading vector ends is approximately 0.007 events/vector backbone. DNA sequencing reveals precise copying of the cognate chromosomal mu-gene template. In unsynchronized cells, 3' extension is sensitive to aphidicolin supporting involvement of a replicative polymerase. Analysis suggests that the vast majority of 3' extensions reside on linear plasmid molecules.