THE MECHANISM OF RECOMBINATION-MEDIATED LOSS OF HETEROZYGOSITY IN HUMAN

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The long-term goal of this study is to understand how recombination processes can result in loss of heterozygosity (LOH), a genetic alteration commonly found in tumors. LOH is a genotype in which the allele(s) of a locus are derived from only one parental chromosome, instead of from both parental chromosomes. One potential mechanism for LOH, inferred from karyotype analysis of individual tumors, involves recombination between homologous chromosomes, i.e. interhomolog recombination (Fig 1). To study how interhomolog recombination may result in the LOH events associated with cancer, we will develop systems in human cancer cells to characterize genetic loss resulting from recombination induced by a defined DNA lesion. Previously, we have studied interhomolog recombination and LOH in mouse embryonic stem (ES) cells. Specifically, reporters were introduced into cells that allow the selection of individual interhomolog recombination events induced by a chromosomal double-strand break (DSB). We found that the majority of recombinants exhibit LOH restricted to the site of the DSB and thus have not undergone significant amounts of genetic loss (20). Though, we also found a minor class with LOH of markers distal to the DSB, apparently extending to the end of the chromosome. This type of LOH is similar to that observed in some types of tumors, and is suggestive of mechanism involving recombination associated with crossing over (Figs. 1 and 3), as occurs during meiosis. Thus, these results indicate that homologous repair of a DSB can result in extensive LOH when the recombination events are resolved by crossing over. However, the low frequency of these events suggests that crossover resolution is either highly inefficient and/or is actively suppressed during DSB-induced interhomolog recombination. We hypothesize that identifying the factors important for the control of crossover resolution during interhomolog recombination will lead to an understanding of pathways that influence LOH formation during tumorigenesis. In testing this hypothesis, we suggest that further studies should be performed in human cancer cells rather than mouse ES cells, for a number of reasons. For one, ES and somatic cells have been observed to exhibit differences in the levels of spontaneous LOH and in their responses to DNA damage (7), suggesting that a broad understanding of LOH and recombination should include studies in additional cell types, such as human cancer cells. As well, since the long-term goal of this study is to understand LOH formation during human cancer development, mechanistic studies of human cancer cells may be more relevant to human biology than similar studies in mouse ES cells. Thus, we propose Aim 1. To characterize the frequency and extent of LOH resulting from break-induced interhomolog recombination in human cancer cells. Aim 2. To examine the effect of particular genetic deficiencies on recombination-mediated LOH, including deficiencies in MLH1, BRCA1, BRCA2, and BLM.