Homologous recombination (HR) is critical for the repair of double strand breaks and broken replication forks. Although HR is mostly error free, inherent or environmental conditions that either suppress or induce HR cause genomic instability. Despite its importance in carcinogenesis, due to limitations in our ability to detect HR in vivo, little is known about HR in mammalian tissues. Here, we describe a mouse model in which a direct repeat HR substrate is targeted to the ubiquitously expressed Rosa26 locus. In the Rosa26 Direct Repeat-GFP (RaDR-GFP) mice, HR between two truncated EGFP expression cassettes can yield a fluorescent signal. In-house image analysis software provides a rapid method for quantifying recombination events within intact tissues, and the frequency of recombinant cells can be evaluated by flow cytometry. A comparison among 11 tissues shows that the frequency of recombinant cells varies by more than two orders of magnitude among tissues, wherein HR in the brain is the lowest. Additionally, de novo recombination events accumulate with age in the colon, showing that this mouse model can be used to study the impact of chronic exposures on genomic stability. Exposure to N-methyl-N-nitrosourea, an alkylating agent similar to the cancer chemotherapeutic temozolomide, shows that the colon, liver and pancreas are susceptible to DNA damage-induced HR. Finally, histological analysis of the underlying cell types reveals that pancreatic acinar cells and liver hepatocytes undergo HR and also that HR can be specifically detected in colonic somatic stem cells. Taken together, the RaDR-GFP mouse model provides new understanding of how tissue and age impact susceptibility to HR, and enables future studies of genetic, environmental and physiological factors that modulate HR in mammals.

A transgenic mouse has been created that provides a powerful tool for revealing genetic and environmental factors that modulate mitotic homologous recombination. The fluorescent yellow directrepeat (FYDR) mice described here carry two different copies of expression cassettes for truncated coding sequences of the enhanced yellow fluorescent protein (EYFP), arranged in tandem. Homologous recombination between these repeated elements can restore full-length EYFP coding sequence to yield a fluorescent phenotype, and the resulting fluorescent recombinant cells are rapidly quantifiable by flow cytometry. Analysis of genomic DNA from recombined FYDR cells shows that this mouse model detects gene conversions, and based on the arrangement of the integrated recombination substrate, unequal sister-chromatid exchanges and repair of collapsed replication forks are also expected to reconstitute EYFP coding sequence. The rate of spontaneous recombination in primary fibroblasts derived from adult ear tissue is 1.3 ؎ 0.1 per 10 6 cell divisions. Interestingly, the rate is Ϸ10-fold greater in fibroblasts derived from embryonic tissue. We observe an Ϸ15-fold increase in the frequency of recombinant cells in cultures of ear fibroblasts when exposed to mitomycin C, which is consistent with the ability of interstrand crosslinks to induce homologous recombination. In addition to studies of recombination in cultured primary cells, the frequency of recombinant cells present in skin was also measured by direct analysis of disaggregated cells. Thus, the FYDR mouse model can be used for studies of mitotic homologous recombination both in vitro and in vivo.H uman cells incur Ϸ10 6 base lesions per day (1), many of which inhibit DNA replication and͞or induce DNA strand breaks. Homology-directed repair provides an important strategy for preventing toxicity caused by such DNA lesions. More specifically, when the replication machinery stalls, recombination between sister chromatids can replace the damaged template with an undamaged copy (2). In addition, should a replication fork collapse to form a double-strand break (e.g., because of an encounter with a single-strand break in the template DNA), the fork can be repaired by homology-directed reinsertion of the broken end (3-5). Thus, the frequency of recombination reflects the levels of certain types of DNA damage.Repair and lesion-avoidance pathways that involve homology searching are integral to DNA replication (3-7). It is estimated that Ϸ10 double-strand breaks are formed each time the mammalian genome is replicated (3), and proteins that are essential for homologous recombination (e.g., Rad51) are also essential for life (8-10). Most of the time, sequences are aligned perfectly, and flanking sequences are not exchanged. However, misalignments may result in deletions, and exchanges between homologous chromosomes may lead to loss of heterozygosity, events that are known to promote cancer (11)(12)(13)(14).A useful approach to studying recombination is to engineer two mutant expression cassett...

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Inflammation is an important risk factor for cancer. During inflammation, macrophages secrete nitric oxide (NO*), which reacts with superoxide or oxygen to create ONOO- or N2O3, respectively. Although homologous recombination causes DNA sequence rearrangements that promote cancer, little was known about the ability of ONOO- and N2O3 to induce recombination in mammalian cells. Here, we show that ONOO- is a potent inducer of homologous recombination at an integrated direct repeat substrate, whereas N2O3 is relatively weakly recombinogenic. Furthermore, on a per lesion basis, ONOO(-)-induced oxidative base lesions and single-strand breaks are significantly more recombinogenic than N2O3-induced base deamination products, which did not induce detectable recombination between plasmids. Similar results were observed in mammalian cells from two different species. These results suggest that ONOO(-)-induced recombination may be an important mechanism underlying inflammation-induced cancer.

Homologous recombination can induce tumorigenic sequence rearrangements. Here, we show that persistent hyper-recombination can be induced following exposure to a bifunctional alkylating agent, mitomycin C (MMC), and that the progeny of exposed cells induce a hyperrecombination phenotype in unexposed neighboring cells. Residual damage cannot be the cause of delayed recombination events, since recombination is observed after drug and template damage are diluted over a millionfold. Furthermore, not only do progeny of MMC-exposed cells induce recombination in unexposed cells (bystanders), but these bystanders can in turn induce recombination in their unexposed neighbors. Thus, a signal to induce homologous recombination can be passed from cell to cell. Although the underlying molecular mechanism is not yet known, these studies reveal that cells suffer consequences of damage long after exposure, and that can signal unexposed neighboring cells to respond similarly. Thus, a single acute exposure to a chemotherapeutic agent can cause long-term changes in genomic stability. If the results of these studies of mouse embryonic stem (ES) cells are generally applicable to many cell types, these results suggest that a relatively small number of cells could potentially induce a tissue-wide increase in the risk of de novo homologous recombination events.