Mechanisms of Mitotic Recombination

描述（由申请人提供）： 有丝分裂的交换是有害的，因为它会导致杂合性丢失或染色体重排，这两者都与癌症有关。有丝分裂交叉的危险在布卢姆综合征患者中是明显的，布卢姆综合征是一种罕见的遗传性疾病，其特征是发病率高，早期发生广泛的恶性肿瘤。来自这些患者的细胞的主要特征是姐妹染色单体、同源染色体和异源染色体之间的有丝分裂交换增加。对编码RecQ解旋酶的BLM的研究为细胞用于防止有丝分裂交叉的机制提供了重要的见解。我们利用果蝇作为模型后生动物的BLM解旋酶的细胞功能的遗传和分子研究的独特优势，这些见解作出了重大贡献。我们已经开发了一个详细的模型，果蝇DmBLM在双链断裂修复的功能，我们现在将测试这个模型的预测，以及其他人提出的模型，使用已建立的和新的分子遗传学检测。虽然双链断裂的修复可能导致交叉，但大多数自发的有丝分裂交叉被认为是由复制叉的问题引起的，包括叉进展的障碍，断裂的叉和一些叉收敛。基于我们对DmBLM和三种结构特异性DNA内切酶中的任何一种都不存在时发生的致死表型的研究，我们修改了模型来解释DmBLM在复制叉修复中的作用;我们将使用创新方法来测试这些模型的关键预测。我们还将结合联合收割机在体内和体外的研究，以揭示这三种核酸内切酶（MUS 81-MMS 4，GEN和MUS 312-SLX 1）的功能，其中每一种都涉及霍利迪连接中间体的分辨率。重要的是，我们发现，GEN，这似乎只是一个次要的作用，在芽殖酵母和不存在的裂殖酵母，有几个重要的功能，在果蝇。因此，我们的研究将导致更好地了解这种酶和其他假定的分解酶的细胞功能。最后，我们将通过候选基因的研究和物理相互作用实验来确定参与预防或促进有丝分裂交换的其他基因/蛋白质。我们提出的研究结果将增强我们对有丝分裂交叉产生机制的理解，以及细胞如何利用肿瘤抑制蛋白BLM和其他蛋白质来防止有丝分裂交叉，以及假定的霍利迪连接消退酶如何发挥作用以促进交叉。 公共卫生相关性： 染色体之间遗传物质的不适当交换会导致癌症。我们正在研究这种交换发生的过程，以及保护基因组免受这些事件影响的遗传机制。

DESCRIPTION (provided by applicant): ABSTRACT Mitotic crossing over is detrimental because it can lead to loss of heterozygosity or chromosome rearrange- ment, both of which are associated with cancer. The dangers of mitotic crossing over are evident in persons with Bloom syndrome, a rare, hereditary disease characterized by a highly elevated incidence and early onset of a broad range of malignancies. A predominant feature of cells from these patients is elevated mitotic crossing over between sister chromatids, homologous chromosomes, and heterologous chromosomes. Studies of BLM, which encodes a RecQ helicase, are providing important insights into mechanisms cells use to prevent mitotic crossing over. We have made significant contributions to these insights by exploiting unique advantages of Drosophila as a model metazoan for genetic and molecular studies of the cellular functions of the BLM helicase. We have developed a detailed model for the function of Drosophila DmBLM in double-strand break repair; we will now test predictions of this model, as well as models proposed by others, using established and novel molecular genetic assays. Although repair of double-strand breaks may lead to crossing over, most spontaneous mitotic crossovers are thought to arise from problems at the replication fork, including blocks to fork progression, broken forks, and some fork convergences. Based on our studies of lethal phenotypes that occur when both DmBLM and any of three structure-specific DNA endonuclease is absent, we have modified models to explain roles of DmBLM in replication fork repair; we will use innovative methods to test key predictions of these models. We will also combine in vivo and in vitro studies to uncover functions of these three endonucleases (MUS81-MMS4, GEN, and MUS312-SLX1), each of which has been implicated in resolution of Holliday junction intermediates. Importantly, we find that GEN, which appears to play only a secondary role in budding yeast and is absent from fission yeast, has several important functions in Drosophila. Our studies will therefore lead to a greater understanding of the cellular functions of this enzyme and the other putative resolvases. Finally, we will identify additional genes/proteins involved in preventing or promoting mitotic crossovers through studies of candidate genes and through physical interaction experiments. The results from our proposed studies will enhance our understanding of mechanisms through which mitotic crossovers are generated and how cells employ the tumor suppressor protein BLM and other proteins to prevent mitotic crossing over, and how putative Holliday junction resolvases function to promote crossing over. PUBLIC HEALTH RELEVANCE: NARRATIVE Inappropriate exchange of genetic material between chromosomes can lead to cancer. We are studying processes through which such exchange occurs, and the genetic mechanisms that safeguard the genome from these events.