Mechanisms of double-strand-break repair during gene targeting in mammalian cells.
In the present study, the mechanism of double-strand-break (DSB) repair during gene targeting at the chromosomal immunoglobulin mu-locus in a murine hybridoma was examined. The gene-targeting assay utilized specially designed insertion vectors genetically marked in the region of homology to the chromosomal mu-locus by six diagnostic restriction enzyme site markers. The restriction enzyme markers permitted the contribution of vector-borne and chromosomal mu-sequences in the recombinant product to be determined. The use of the insertion vectors in conjunction with a plating procedure in which individual integrative homologous recombination events were retained for analysis revealed several important features about the mammalian DSB repair process:The presence of the markers within the region of shared homology did not affect the efficiency of gene targeting. In the majority of recombinants, the vector-borne marker proximal to the DSB was absent, being replaced with the corresponding chromosomal restriction 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. Formation of hDNA was frequently associated with gene targeting and, in most cases, began approximately 645 bp from the DSB and could encompass a distance of at least 1469 bp. The hDNA was efficiently repaired prior to DNA replication. The repair of adjacent mismatches in hDNA occurred predominantly on the same strand, suggesting the involvement of a long-patch repair mechanism.
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Department of Molecular Biology and Genetics, University of Guelph, Guelph, Ontario, Canada N1G 2W1.
Baker
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D000818:Animals; D004260:DNA Repair / Q000235:genetics*; D004262:DNA Restriction Enzymes / Q000235:genetics; D018274:Electroporation; D004742:Enhancer Elements, Genetic; D018390:Gene Targeting; D005819:Genetic Markers; D005822:Genetic Vectors; D006825:Hybridomas / Q000378:metabolism; D007148:Immunoglobulin mu-Chains / Q000235:genetics; D051379:Mice; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D011995:Recombination, Genetic; D015183:Restriction Mapping; D014170:Transformation, Genetic
D005819:Genetic Markers; D007148:Immunoglobulin mu-Chains; D004262:DNA Restriction Enzymes