Crossovers have rarely been observed in specific association with interchromosomal gene conversion in mammalian cells. In this investigation two isogenic human B-lymphoblastoid cell lines, TI-112 and TSCER2, were used to select for I-SceI-induced gene conversions that restored function at the selectable thymidine kinase locus. Additionally, a haplotype linkage analysis methodology enabled the rigorous detection of all crossover-associated convertants, whether or not they exhibited loss of heterozygosity. This methodology also permitted characterization of conversion tract length and structure. In TI-112, gene conversion tracts were required to be complex in tract structure and at least 7.0 kb in order to be selectable. The results demonstrated that 85% (39/46) of TI-112 convertants extended more than 11.2 kb and 48% also exhibited a crossover, suggesting a mechanistic link between long tracts and crossover. In contrast, continuous tracts as short as 98 bp are selectable in TSCER2, although selectable gene conversion tracts could include a wide range of lengths. Indeed, only 16% (14/95) of TSCER2 convertants were crossover associated, further suggesting a link between long tracts and crossover. Overall, these results demonstrate that gene conversion tracts can be long in human cells and that crossovers are observable when long tracts are recoverable.In mammalian cells, gene conversion without associated crossover has been widely reported for both intrachromosomal and interchromosomal recombining substrates (13,51,62,80,87). However, most studies to date have not succeeded in associating interchromosomal crossovers with specific conversion tracts (37,67,71,80,83); reports of crossovers are rare (7,72). Nevertheless, presumptive interchromosomal crossovers, in the form of chromosome-scale loss of heterozygosity (LOH), are commonly observed in mammalian cells. LOH is the major contributor to the mutational spectrum in mice and humans (31,33,47,49,78), notably at tumor suppressor loci (3,11,15,25,34,35,40,68,88,89). Crossover products have also been associated with intrachromosomal recombination between repeat sequences in mammalian cells (12,30,52,75), and similar outcomes are observed during the integration of plasmids into host cell chromosomes (9,48,90). Additional evidence for crossovers in mammalian cells is provided by the relatively frequent occurrence of sister chromatid exchange (94), although these products may also result from other mechanisms (56). Since the occurrence of crossovers in mammalian cells has been clearly demonstrated, the absence of crossovers associated with specific interchromosomal gene conversion tracts requires further explanation.Several mechanisms have been proposed to explain the preference for gene conversion without associated crossover in eukaryotic organisms. Crossover is believed to require the formation of Holliday intermediates (17,36,44,76,77) and their resolution through structure-specific endonuclease scissions (79). Theoretically, half of Holliday junctions that are resol...

To examine the relationship between gene conversion and reciprocal exchange at an endogenous chromosomal locus, we developed a reversion assay in a thymidine kinase deficient mutant, TX545, derived from the human lymphoblastoid cell line TK6. Selectable revertants of TX545 can be generated through interchromosomal gene conversion at the site of inactivating mutations on each tk allele or by reciprocal exchange that alters the linkage relationships of inactivating polymorphisms within the tk locus. Analysis of loss of heterozygosity (LOH) at intragenic polymorphisms and flanking microsatellite markers was used to initially evaluate allelotypes in TK+ revertants for patterns associated with either gene conversion or crossing over. The linkage pattern in a subset of convertants was then unambiguously established, even in the event of prereplicative recombinational exchanges, by haplotype analysis of flanking microsatellite loci in tk-/- LOH mutants collected from the tk+/- parental convertant. Some (7/38; 18%) revertants were attributable to easily discriminated nonrecombinational mechanisms, including suppressor mutations within the tk coding sequence. However, all revertants classified as a recombinational event (28/38; 74%) were attributed to localized gene conversion, representing a highly significant preference (P < 0.0001) over gene conversion with associated reciprocal exchange, which was never observed.