Genetic Analysis of MCM Protein Function in S. pombe

描述（由申请人提供）：本研究项目的主要目标是了解一种模式生物——裂糖酵母（Schizosaccharomyces pombe）中DNA复制和基因组完整性之间的联系。染色体复制和染色质组装过程与基因组稳定性和恶性肿瘤密切相关。基因组完整性的逐渐丧失会导致或加剧癌症。裂变酵母提供了一个强大的模型系统，具有简单的遗传，大的染色体和复制起源，使人想起后生动物细胞。最近完成的S. pombe基因组序列提供了新的工具，可以快速鉴定和表征蛋白质在体内的功能，做出预测，然后可以在人类细胞中进行测试。保守的MCM蛋白在所有真核生物DNA合成的起始和延伸阶段都是必需的。体内MCM功能的分析为其在S期的作用提供了必要的细胞背景。这一提议提供了将mcm与DNA代谢中多种作用的蛋白质联系起来的证据。第一个目标是调查MCM功能丧失导致DNA双链断裂的证据，并确定细胞如何使用同源重组机制的蛋白质来防止这种情况。mcm和重组机制之间的相互作用也将在人类细胞中进行研究。第二个目标将通过预测发挥重要结构作用的残基的突变分析来表征MCM的功能，并将使用新的可视化方法来表征野生型和突变蛋白在复制染色质上的定位。该目的还将分析MCM蛋白在正常细胞周期和受干扰的S期的修饰。第三个目标将通过检查起源时间和组蛋白修饰之间的联系来研究染色质结构如何与MCM功能联系起来，研究先前确定的组蛋白修饰酶和MCM之间的相互作用，并确定MCM如何在染色质化模板内获得DNA。所有这些蛋白质都存在于人类体内，并且都与癌症有关，但在体内，以基因为导向的方法在组织培养中是不可能的。然而，在酵母系统中产生的预测可以在人类细胞中进行测试。因此，对简单酵母细胞的研究对理解这些因素在人类细胞分裂中的作用有直接的意义。

DESCRIPTION (provided by applicant): The broad goal of this research program is to understand the link between DNA replication and genome integrity in a model organism, Schizosaccharomyces pombe. The processes of chromosome replication and chromatin assembly are intimately coupled with genome stability and malignancy. Progressive loss of genome integrity can cause or exacerbate cancer. Fission yeast offers a powerful model system with facile genetics, and large chromosomes and replication origins reminiscent of those in metazoan cells. Recent completion of the genome sequence of S. pombe provides new tools allowing rapid identification and characterization of protein function in vivo, making predictions that can then be tested in human cells. The conserved MCM proteins are required both for initiation and elongation stages of DNA synthesis in all eukaryotes. Analysis of MCM function in vivo provides the essential cellular context for their role during S phase. This proposal provides evidence linking MCMs to proteins with multiple roles in DNA metabolism. The first aim will investigate evidence that loss of MCM function leads to double strand breaks in the DNA, and determine how the cell uses proteins of the homologous recombination machinery to prevent this. Interactions between MCMs and recombination machinery will also be examined in human cells. The second aim will characterize MCM function by mutational analysis of residues predicted to play important structural roles, and will characterize the localization of wild type and mutant proteins on replicating chromatin using new methods of visualization. This aim will also analyze modifications of MCM proteins during normal cell cycle and perturbed S phase. The third aim will investigate how chromatin structure is linked to MCM function by examining the links between origin timing and histone modifications, investigating previously identified interactions between histone modifying enzymes and MCMs, and determine how MCMs gain access to DNA within a chromatinized template. All these proteins exist in humans and both have links to cancer, but the in vivo, genetically oriented approach is not possible in tissue culture. However, predictions generated in the yeast system can be tested in human cells. Thus, studies in simple yeast cells have direct relevance to understanding the role of these factors in human cell division.