THE MECHANISM OF RECOMBINATION-MEDIATED LOSS OF HETEROZYGOSITY IN HUMAN

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。这项研究的长期目标是了解重组过程如何导致杂合性缺失（LOH），这是一种在肿瘤中常见的遗传改变。LOH是一种基因型，其中一个位点的等位基因仅来自亲本染色体的一条，而不是来自亲本染色体的两条。从单个肿瘤的核型分析推断，LOH的一种潜在机制涉及同源染色体之间的重组，即同源间重组（图1）。为了研究同源间重组如何导致与癌症相关的LOH事件，我们将在人类癌细胞中开发系统，以表征由特定DNA损伤诱导的重组导致的遗传损失。此前，我们研究了小鼠胚胎干细胞的同源间重组和LOH。具体来说，报告基因被引入细胞，允许选择由染色体双链断裂（DSB）诱导的单个同源间重组事件。我们发现，大多数重组体表现出局限于DSB位点的LOH，因此没有经历大量的遗传损失（20）。尽管如此，我们也发现了一小类在DSB远端具有LOH的标记，显然延伸到染色体的末端。这种类型的LOH与在某些类型的肿瘤中观察到的LOH相似，并且提示了与交叉相关的重组机制（图1和3），正如减数分裂期间发生的那样。因此，这些结果表明，当重组事件通过交叉解决时，DSB的同源修复可以导致广泛的LOH。然而，这些事件的低频率表明，在dsb诱导的同源间重组期间，交叉分辨率要么效率极低，要么被主动抑制。我们假设，确定同源间重组过程中控制交叉分辨率的重要因素将有助于理解肿瘤发生过程中影响LOH形成的途径。为了验证这一假设，我们建议在人类癌细胞而不是小鼠胚胎干细胞中进行进一步的研究，原因有很多。首先，已经观察到胚胎干细胞和体细胞在自发LOH水平和对DNA损伤的反应上表现出差异(7)，这表明对LOH和重组的广泛理解应该包括对其他细胞类型的研究，如人类癌细胞。此外，由于本研究的长期目标是了解人类癌症发展过程中LOH的形成，因此对人类癌细胞的机制研究可能比对小鼠ES细胞的类似研究更与人类生物学相关。因此，我们提出目标1。表征断裂诱导的人类癌细胞同源间重组引起LOH的频率和程度。目标2。研究特定基因缺陷对重组介导的LOH的影响，包括MLH1、BRCA1、BRCA2和BLM缺陷。

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.