We have studied intrachromosomal gene conversion in mouse Ltk-cells with a substrate designed to provide genetic evidence for heteroduplex DNA. Our recombination substrate consists of two defective chicken thymidine kinase genes arranged so as to favor the selection of gene conversion products. The When uncorrected mispairs arise in duplex DNA, semiconservative DNA replication of the resultant heteroduplex segregates genetically distinct daughter molecules. Since one source for the generation of heteroduplex DNA is the process of genetic recombination, the two strands of a single DNA molecule can emerge from meiosis as nonidentical daughter molecules. Whereas four pairs of genes synapse during meiosis (4:4), fungal meiosis occasionally produces uneven aberrant segregations such as 5:3 or 3:5. Such postmeiotic segregations (PMS) are thought to result from the mitotic replication of heteroduplex DNA produced during genetic recombination.Most current models for genetic recombination derive from a model proposed by Holliday (15), who interpreted PMS as nonrepair of heteroduplex DNA (hDNA) and suggested that gene conversion resulted from correction of mismatches in hDNA. Although current models for meiotic recombination invoke different initiation mechanisms for gene conversion, such as single-strand invasion (22) or double-strand gap formation (30), these models generally suggest that heteroduplex formation may accompany gene conversion. In fungi, the most compelling genetic evidence for hDNA is the observation of PMS during meiotic recombination (16, 26) or of sectored colonies during mitotic recombination (8,11,28,36). Such recombinants most likely result from the failure to repair hDNA intermediates prior to DNA replication.Evidence for the existence of heteroduplex DNA in mammalian cells is primarily indirect. Results of both in vivo and in vitro studies indicate that mammalian cells do possess the ability to process preformed mismatched DNA, hDNA, to the homoduplex forms following transfection (1,2,6,9,10,13,32,33
We have studied intrachromosomal gene conversion in mouse Ltk- cells with a substrate designed to provide genetic evidence for heteroduplex DNA. Our recombination substrate consists of two defective chicken thymidine kinase genes arranged so as to favor the selection of gene conversion products. The gene intended to serve as the recipient in gene conversion differs from the donor sequence by virtue of a palindromic insertion that creates silent restriction site polymorphisms between the two genes. While selection for gene conversion at a XhoI linker insertion within the recipient gene results in coconversion of the nearby palindromic site in more than half of the convertants, 4% of convertant colonies show both parental and nonparental genotypes at the polymorphic site. We consider these mixed colonies to be the result of genotypic sectoring and interpret this sectoring to be a consequence of unrepaired heteroduplex DNA at the polymorphic palindromic site. DNA replication through the heteroduplex recombination intermediate generates genetically distinct daughter cells that comprise a single colony. We believe that the data provide the first compelling genetic evidence for the presence of heteroduplex DNA during chromosomal gene conversion in mammalian cells.
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