Mechanisms of double-strand-break repair during gene targeting in mammalian cells

In the present study, the mechanism of double-strand-break (DSB) repair dur ing gene targeting at the chromosomal immunoglobulin CL-locus in a murine h ybridoma was examined. The gene-targeting assay utilized specially designed insertion vectors genetically marked in the region of homology to the chro mosomal mu-locus by six diagnostic restriction enzyme site markers. The res triction enzyme markers permitted the contribution of vector-borne and chro mosomal mu-sequences in the recombinant product to be determined. The use o f the insertion vectors in conjunction with a Plating procedure in which in dividual integrative homologous recombination events were retained fur anal ysis revealed several important features about the mammalian DSB repair pro cess: 1. The presence of the markers within tl-le region of shared homology did n ot affect the efficiency of gene targeting. 2. In the majority of recombinants, the vector-borne marker proximal to the DSB was absent, being replaced with the corresponding chromosomal restrict ion enzyme site. This result is consistent with either formation and repair of a vector-borne gap or an "end" bias in mismatch repair of heteroduplex DNA (hDNA) that favored the chromosomal sequence. 3. Formation of hDNA was frequently associated with gene targeting and, in most cases, began similar to 645 bp from the DSB and could encompass a dist ance of at least 1469 bp. 4. The hDNA was efficiently repaired prior to DNA replication. 5. The repair of adjacent mismatches in hDNA occurred predominantly on the same strand, suggesting the involvement of a long-patch repair mechanism.