Formation and repair of heteroduplex DNA on both sides of the double-strand break during mammalian gene targeting.
In this study, we examined homologous recombination in mammalian cells using a gene targeting assay in which the introduction of a double-strand-break (DSB) in the vector-borne region of homology to the chromosome resulted in targeted vector integration. The vector-borne DSB was flanked with small palindromic insertions that, when encompassed within heteroduplex DNA (hDNA) formed during targeted vector integration, were capable of avoiding the activity of the mismatch repair (MMR) system. When used in conjunction with an isolation procedure in which the product(s) of each targeted vector integration event were retained for molecular analysis, information about recombination mechanisms was obtained. The examination of marker segregation patterns in independent recombinants revealed the following, (i) hDNA tracts could form simultaneously on each side of the DSB and in both participating homologous regions. Clonal analysis of sectored recombinants revealed that, in the homologous repeats generated by the recombination event, vector-borne palindrome and chromosomal markers were linked in the expected way in each strand of the hDNA intermediate, (ii) hDNA tracts were subject to MMR processing that occurred on opposite sides of the DSB, and (iii) in the majority of recombinants, the vector-borne marker was replaced with the corresponding marker from the chromosome. Bidirectional hDNA formation and MMR processing of both sides of the DSB are consistent with the double-strand-break repair (DSBR) model of recombination.
Li
J
J
Department of Molecular Biology, University of Guelph, Guelph, Ontario, N1G 2W1, Canada.
Baker
M D
MD
D001483:Base Sequence; D015139:Blotting, Southern; D004249:DNA Damage / Q000235:genetics*; D017931:DNA Primers; D004260:DNA Repair / Q000235:genetics*; D018390:Gene Targeting; D005819:Genetic Markers; D005822:Genetic Vectors; D006825:Hybridomas; D009692:Nucleic Acid Heteroduplexes; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic
D017931:DNA Primers; D005819:Genetic Markers; D009692:Nucleic Acid Heteroduplexes