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Genetics, Vol. 172, 1767-1781, March 2006, Copyright © 2006
doi:10.1534/genetics.105.051680
Analysis of One-Sided Marker Segregation Patterns Resulting From Mammalian Gene Targeting
Richard D. McCulloch and Mark D. Baker1
Department of Molecular and Cellular Biology, College of Biological Sciences, University of Guelph, Guelph, Ontario N1G 2W1, Canada
1 Corresponding author: Department of Molecular and Cellular Biology, College of Biological Science, University of Guelph, Guelph, ON N1G 2W1, Canada.
E-mail: mdbaker{at}uoguelph.ca
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.