A common genetic alteration in tumor cells is loss of heterozygosity (LOH), in which genetic information at a chromosomal locus is derived from only one parental chromosome rather than from both parental chromosomes (18
). LOH can involve large segments of chromosomes and therefore lead to a substantial loss of allele-specific genetic information. LOH is one of the genetic alterations commonly observed in sporadic tumors, and it is also presumed to be an obligate step in tumorigenesis in several familial cancer syndromes involving tumor suppressor genes. For example, individuals carrying one mutated RB
allele in the germ line are at high risk of developing retinoblastoma, with tumors displaying loss of the wild-type RB
Potential mechanisms of LOH can be inferred from combined analyses of tumor karyotypes and markers that are polymorphic between parental chromosomes. Researchers analyzing human cancers have inferred that LOH arises from several pathways, including chromosomal deletion, mitotic nondisjunction, and recombination between homologous chromosomes (18
). In retinoblastoma, recombination and nondisjunction appear to be common pathways (42 and 51% of LOH events, respectively, in one large study) (15
); however, a recent study of colorectal cancers found that deletions associated with chromosome structural aberrations are more common (52
). Each of these pathways has also been implicated in generating LOH in mouse models, with mitotic recombination predominating in many of these cases (18
). Despite the implication of several pathways in the generation of LOH, little is understood about the molecular events that lead to LOH, including those involving mitotic recombination pathways.
Chromosomal double-strand breaks (DSBs) are extremely potent inducers of homologous recombination in mammalian cells (17
), raising the issue of whether they are the causative lesions for LOH by allelic recombination. During meiosis, DSBs generated by the Spo11 protein induce a high frequency of recombination between homologous chromosomes (20
). Although Spo11 is expressed primarily during meiosis (21
), DSBs can be generated in nonmeiotic cells in several ways, for example, by oxygen free radicals, topoisomerase failure, radiation treatment, and DNA replication. Repair of these mitotic DSBs by homologous recombination involves strand invasion of a broken end into a homologous sequence which templates repair by gene conversion (17
) and thus can be termed homology-directed repair (HDR).
In mitotically dividing mammalian cells, the identical-sister chromatid is the most frequently used template for HDR (19
). HDR involving the sister restores the intact chromatid without loss of sequence information, making this a very precise type of repair. High-fidelity repair is not guaranteed, however, when the template for HDR is the allele on the homologous chromosome, since maternal and paternal chromosomes are not identical in individuals. Such events can lead to LOH of large chromosome segments or even whole chromosome arms when recombination between the homologs is associated with either crossing over or extensive gene conversion without crossing over. DSBs have previously been shown to induce HDR between homologous chromosomes in mammalian cells at a recombination reporter, resulting in LOH at the reporter (35
). However, in that study the full extent of LOH could not be determined because the two chromosomes were isogenic outside of the reporter substrate.
In this report, we sought to test whether HDR in mammalian cells leads to extensive LOH by using a reporter for DSB-induced allelic recombination. The reporter was introduced into a mouse embryonic stem (ES) cell line derived from an F1
hybrid of two different inbred mouse strains, BALB/c and 129/Sv, such that the maternal and paternal chromosomes contain polymorphic markers. Overall, the heterology between the two strains is estimated at approximately one single-nucleotide polymorphism per 1,050 bp (27
), which is within the range of diversity in the human population (estimated at one single-nucleotide polymorphism per 1,000 to 2,000 bp) (45
). We find that although spontaneous recombination at the allelic positions is not detectable, DSBs are potent inducers of allelic recombination. Examination of a number of markers indicates that the majority of recombinant clones have LOH restricted to the site of the DSB, although 2% of recombinants exhibit LOH extending to a marker 6 kb from the DSB. Importantly, we find no evidence of LOH extending to polymorphic markers 1 centimorgan (cM) or more from the DSB nor do we find evidence for crossing over between alleles. These results indicate that extensive LOH is suppressed during HDR of a DSB in mammalian cells. Thus, either DSBs are not the causative lesions for extensive LOH events arising from recombination between homologous chromosomes or the suppression of extensive LOH can be relieved under some circumstances.