Analysis of One-Sided Marker Segregation Patterns Resulting from Mammalian Gene Targeting

By McCulloch, Richard D.; Baker, Mark D. | Genetics, March 2006 | Go to article overview

Analysis of One-Sided Marker Segregation Patterns Resulting from Mammalian Gene Targeting


McCulloch, Richard D., Baker, Mark D., Genetics


ABSTRACT

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.

THE double-strand break repair (DSBR) model (ORR-WEAVER et al 1981; SZOSTAK et al 1983), as revised by SUN et al. (1991), 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 explains homologous recombination between a linearized gene-targeting vector and the chromosome (Figure 1). The events involve resection on the two sides of the DSB, invasion by one 3'-end, which primes DNA synthesis leading to the capture of the second 3'-end, and eventually, formation of the double Holliday junction intermediate. If the recombining sequences differ, the initial events of strand invasion and annealing generate asymmetric heteroduplex DNA (hDNA) tracts on opposite strands on the two sides of the DSB. Outward branch migration of the double Holliday junction intermediate will result in asymmetric hDNA being flanked by symmetric hDNA on one or both sides of the DSB. Alternate sense cleavage of the double Holliday junction intermediate in either the 2,4 or 1,3 mode generates the crossover products in Figure 1, F and G, respectively, with the regions undergoing recombination being preserved as 5' and 3' homologous repeats on the chromosome. Nicks at positions 2,2' and 4,4' in the crossover product in Figure IF and those at positions 1,1' and 3,3' in the crossover product in Figure IG correspond to the DNA cuts required to resolve Hollidayjunctions previously located to the left and right of the DSB, respectively. In the crossover products, the positions of gene conversion tracts toward the chromosomal sequence (GCC), asymmetric hDNA (A), and symmetric hDNA (S) in the 5' and 3' homology regions are indicated. The crossover products in Figure 1, F and G, differ with respect to the arrangement of asymmetric hDNA (A) and gene conversion (GCC) tracts about the DSB in the two homology regions. Although the canonical DSBR model depicts formation of both crossover products, a bias yielding products predominantly of the 2,4 resolution type (Figure IF) is observed (Foss et al. 1999; BAKER and BIRMINGHAM 2001; MERKER et al. 2003; JESSOP et al. 2005). To account for the bias in recovery of type 2,4 resolution products, Foss et al. (1999) propose that each Holliday junction bears a structural asymmetry resulting from new DNA, or its synthesis, that biases the pair of like strands that are cleaved at each junction: with respect to Figure 1, crossed strands in the Holliday junction to the right of the DSB and noncrossed strands in the Holliday junction to the left of the DSB. …

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Analysis of One-Sided Marker Segregation Patterns Resulting from Mammalian Gene Targeting
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