THE MECHANISM OF RECOMBINATION-MEDIATED LOSS OF HETEROZYGOSITY IN HUMAN

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。本研究的长期目标是了解重组过程如何导致杂合性丢失（洛），这是一种常见于肿瘤的遗传改变。洛是基因座的等位基因仅来源于一条亲本染色体而不是两条亲本染色体的基因型。从个体肿瘤的核型分析推断，洛缺失的一个潜在机制涉及同源染色体之间的重组，即同源物间重组（图1）。为了研究同源重组如何导致与癌症相关的洛缺失事件，我们将在人类癌细胞中开发系统，以表征由确定的DNA损伤诱导的重组导致的遗传丢失。在此之前，我们研究了小鼠胚胎干细胞中的同源重组和洛缺失。具体地，将报告基因引入允许选择由染色体双链断裂（DSB）诱导的个体同源物间重组事件的细胞中。我们发现，大多数重组体表现出局限于DSB位点的洛缺失，因此没有发生显著的遗传丢失（20）。然而，我们也发现了一个小类的洛标记远端的DSB，显然延伸到染色体的末端。这种类型的洛与在某些类型的肿瘤中观察到的相似，并且提示了涉及与交换相关的重组的机制（图1A和1B）。1和3），如在减数分裂期间发生的。因此，这些结果表明，当重组事件通过交换解决时，DSB的同源修复可导致广泛的洛。然而，这些事件的低频率表明，交叉分辨率是非常低效的和/或在DSB诱导的同源物间重组过程中被积极抑制。我们假设，确定重要的因素，在同源重组过程中的交叉分辨率的控制将导致理解的途径，影响洛形成在肿瘤发生。为了验证这一假设，我们建议进一步的研究应该在人类癌细胞而不是小鼠ES细胞中进行，原因有很多。一方面，已经观察到ES和体细胞在自发性洛水平和它们对DNA损伤的反应方面表现出差异（7），这表明对洛和重组的广泛理解应该包括在其他细胞类型（例如人类癌细胞）中的研究。同样，由于本研究的长期目标是了解人类癌症发展过程中的洛缺失形成，因此人类癌细胞的机制研究可能比小鼠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。描述人类癌细胞中断裂诱导的同源物间重组导致的洛缺失的频率和程度。目标2.检测特定遗传缺陷对重组介导的洛缺失的影响，包括MLH 1、BRCA 1、BRCA 2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.