In mammalian cells, several features of the way homologous recombination occurs between transferred and chromosomal DNA are consistent with the double-strand-break repair (DSBR) model of recombination. In this study, we examined the segregation patterns of small palindrome markers, which frequently escape mismatch repair when encompassed within heteroduplex DNA formed in vivo during mammalian homologous recombination, to test predictions of the DSBR model, in particular as they relate to the mechanism of crossover resolution. According to the canonical DSBR model, crossover between the vector and chromosome results from cleavage of the joint molecule in two alternate sense modes. The two crossover modes lead to different predicted marker configurations in the recombinants, and assuming no bias in the mode of Holliday junction cleavage, the two types of recombinants are expected in equal frequency. However, we propose a revision to the canonical model, as our results suggest that the mode of crossover resolution is biased in favor of cutting the DNA strands upon which DNA synthesis is occurring during formation of the joint molecule. The bias in junction resolution permitted us to examine the potential consequences of mismatch repair acting on the DNA breaks generated by junction cutting. The combination of biased junction resolution with both early and late rounds of mismatch repair can explain the marker patterns in the recombinants.In the yeast Saccharomyces cerevisiae, transferred DNA undergoes homologous recombination with cognate chromosomal sequences (gene targeting) according to the doublestrand-break repair (DSBR) model of recombination (34,37,44). Features of meiotic recombination in S. cerevisiae are also consistent with repair of chromosomal double-strand breaks (DSBs) by this model (15,33,35,39,40,44). According to the canonical DSBR model (34, 37, 44) and its later revision (42), as illustrated in Fig. 1 for a typical gene targeting reaction, recombination is initiated by a DSB in the vector-borne region of homology to the chromosome. The DSB undergoes 5Ј33Ј resection (Fig. 1A) resulting in the formation of two 3Ј-ending single strands which invade cognate chromosomal sequences (Fig. 1B). The invading 3Ј ends prime DNA synthesis, finally generating two Holliday junctions (Fig. 1C). Opposite-sense cleavage of the Holliday junctions in the joint molecule (Fig. 1D) results in crossover, integrating the vector into the chromosome and duplicating the region of shared homology ( Fig. 1E and F).Our laboratory has been investigating mechanisms of homologous recombination in mammalian somatic cells using a gene targeting assay as one approach. By examining the segregation patterns of small palindromic insertions, which frequently escape mismatch repair (MMR) when encompassed within heteroduplex DNA (hDNA) formed in vivo during homologous recombination, we have shown that (i) hDNA is formed on each side of the vector-borne DSB and (ii) palindrome markers in hDNA formed in each homology region reside in a tr...

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In yeast, four-stranded, biparental "joint molecules" containing a pair of Holliday junctions are demonstrated intermediates in the repair of meiotic double-strand breaks (DSBs). Genetic and physical evidence suggests that when joint molecules are resolved by the cutting of each of the two Holliday junctions, crossover products result at least most of the time. The double-strand break repair (DSBR) model is currently accepted as a paradigm for acts of DSB repair that lead to crossing over. In this study, a welldefined mammalian gene-targeting assay was used to test predictions that the DSBR model makes about the frequency and position of hDNA in recombinants generated by crossing over. The DSBR model predicts that hDNA will frequently form on opposite sides of the DSB in the two homologous sequences undergoing recombination [half conversion (HC); 5:3, 5:3 segregation]. By examining the segregation patterns of poorly repairable small palindrome genetic markers, we show that this configuration of hDNA is rare. Instead, in a large number of recombinants, full conversion (FC) events in the direction of the unbroken chromosomal sequence (6:2 segregation) were observed on one side of the DSB. A conspicuous fraction of the unidirectional FC events was associated with normal 4:4 marker segregation on the other side of the DSB. In addition, a large number of recombinants displayed evidence of hDNA formation. In several, hDNA was symmetrical on one side of the DSB, suggesting that the two homologous regions undergoing recombination swapped single strands of the same polarity. These data are considered within the context of modified versions of the DSBR model.