Genetic Analysis of MCM Protein Function in S. pombe

描述(由申请人提供)：本研究计划的主要目标是了解模式生物--裂殖酵母的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的稳定性和恶性程度密切相关。基因组完整性的进行性丧失会导致或加剧癌症。分裂酵母提供了一个强大的模型系统，具有简单的遗传学，以及大的染色体和复制来源，使人想起后生动物细胞中的那些。最近完成的S.pombe基因组序列提供了新的工具，可以快速鉴定和描述体内蛋白质的功能，做出预测，然后在人类细胞中进行测试。在所有真核生物中，保守的MCM蛋白在DNA合成的起始和延伸阶段都是必需的。体内MCM功能的分析为它们在S阶段的作用提供了必要的细胞背景。这一建议提供了证据，将MCMS与在DNA代谢中具有多种作用的蛋白质联系起来。第一个目标将调查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。我们还将在人体细胞中研究MCMS和重组机制之间的相互作用。第二个目标将通过对预测起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表征野生型和突变蛋白在复制染色质上的定位。这一目标还将分析在正常细胞周期和扰乱S时相中MCM蛋白的修饰。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，研究先前发现的组蛋白修饰酶和组蛋白修饰酶之间的相互作用，并确定组蛋白修饰酶如何在染色模板中获得DNA，来研究染色质结构与MCM功能之间的联系。所有这些蛋白质都存在于人类体内，都与癌症有关，但在组织培养中，以遗传为导向的体内方法是不可能的。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直接关系到了解这些因素在人类细胞分裂中的作用。

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.