Analysis of one-sided marker segregation patterns resulting from mammalian gene targeting.
The double-strand break repair (DSBR) model is currently accepted as the paradigm for acts of double-strand break (DSB) repair that lead to crossing over between homologous sequences. The DSBR model predicts that asymmetric heteroduplex DNA (hDNA) will form on both sides of the DSB (two-sided events; 5:3/5:3 segregation). In contrast, in yeast and mammalian cells, a considerable fraction of recombinants are one sided: they display full conversion (6:2 segregation) or half-conversion (5:3 segregation) on one side of the DSB together with normal 4:4 segregation on the other side of the DSB. Two mechanisms have been proposed to account for these observations: (i) hDNA formation is restricted to one side of the DSB or the other, and (ii) recombination is initially two sided, but hDNA repair directed by Holliday junction cuts restores normal 4:4 segregation on that side of the DSB in which the mismatch is closest to the cut junction initiating repair. In this study, we exploited a well-characterized gene-targeting assay to test the predictions that these mechanisms make with respect to the frequency of recombinants displaying 4:4 marker segregation on one side of the DSB. Unexpectedly, the results do not support the predictions of either mechanism. We propose a derivation of mechanism (ii) in which the nicks arising from Holliday junction cleavage are not equivalent with respect to directing repair of adjacent hDNA, possibly as a result of asynchronous cleavage of the DSBR intermediate.
McCulloch
Richard D
RD
Department of Molecular and Cellular Biology, College of Biological Sciences, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
Baker
Mark D
MD
D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D020090:Chromosome Segregation; D004260:DNA Repair / Q000235:genetics; D045566:DNA, Cruciform / Q000235:genetics; D018390:Gene Targeting; D005819:Genetic Markers; D005822:Genetic Vectors; D006825:Hybridomas; D051379:Mice; D008957:Models, Genetic; D011995:Recombination, Genetic
D045566:DNA, Cruciform; D005819:Genetic Markers