The Regulation of DNA Replication Kinetics

描述(由申请人提供)： DNA复制的时间是细胞生长的关键参数。它与转录调控、染色质修饰、染色体结构和基因组进化的模式有关。此外，随着细胞的分化，复制时间会发生变化，而复制时间的中断与基因组的不稳定性相关，这表明复制时间与染色体代谢的其他重要方面之间存在密切关系。然而，调控复制时机的机制在很大程度上仍然是个谜。我们已经开发并打算测试一个详细的、普遍适用的复制计时机制模型。我们的模型假设了起射的随机规则，其中每个起射都有一个特征的发射概率，平均起射时间由该起射概率来调节。我们认为，原点激发的可能性是由G1期间加载的MCM复合体的数量调节的--MCM复合体是一种复制解旋酶，它建立了一个起始点作为复制起始位置。装载了更多MCM的来源更有可能开火，因此，平均而言，开火更早。此外，我们建议，加载的MCM的数量由ORC(MCM加载器)与原点绑定的亲和力调节。亲和力较高的起始点与ORC结合的G1部分更大，从而允许加载更多的MCM复合体。最后，我们认为，异染色质在基于MCM的起源定时调节机制之上提供了第二水平的调节，这样在异染色区，起始激发在一个或多个基本步骤中被延迟：ORC结合、MCM加载或MCM激活。我们将使用基于深度测序的方法，通过绘制芽酵母和分裂酵母基因组中的ORC结合、MCM结合和起源时间来测试我们的模型。这些进化上相距遥远的酵母的优势使我们的模型得到了更严格的测试。此外，在两者之间保守的任何机制都是真核生物学一般原理的很好候选者。如果我们的模型得到证实，它将改变人们对复制时机的看法。此外，它将改变该领域的方向，从试图发现复制计时的机制，到能够直接测试MCM加载是如何调节的，以控制后生动物基因组中的复制计时。此外，关于复制时间机制的准确信息对于理解复制时间如何影响干细胞维持和细胞分化所需的基因组重编程以及它在维持基因组稳定和预防癌症中的作用至关重要。

DESCRIPTION (provided by applicant): The timing of DNA replication is a critical parameter of cellular growth. It correlates with patters of transcriptional regulation, chromatin modification, chromosome structure and genome evolution. Furthermore, replication timing changes as cells differentiate, and disruption of replication timing correlates with genome instability, suggesting an intimate relation between replication timing and other important aspects of chromosome metabolism. However, the mechanisms that regulate replication timing are still largely mysterious. We have developed, and proposes to test, a detailed, generally-applicable model for the mechanism of replication timing. Our model posits stochastic regulation of origin firing, in which each origin has a characteristic probability of firing, and the average time of origin firing is regulated by that orgin firing probability. We propose that the probability of origin firing is regulated by the number of MCM complexes - the replicative helicase which establishes an origin as a site of replication initiation - loaded during G1. Origins with more MCMs loaded are more likely to fire and thus, on average, fire earlier. Further, we propose that the number of MCMs loaded is regulated by the affinity with which ORC - the MCM loader - binds the origin. Higher affinity origins bind ORC for a greater fraction of G1, thus allowing more MCM complexes to be loaded. Finally, we propose that heterochromatin provides a second level of regulation on top of the MCM-based mechanism of origin timing regulation, such that in heterochromatic regions origin firing is delayed at one or more of the basic steps: ORC binding, MCM loading or MCM activation. We will test our model by mapping ORC binding, MCM binding and origin timing across both the budding and fission yeast genomes using deep-sequencing-based approaches. The complimentary strengths of these evolutionarily distant yeasts allow for a more rigorous test of our model. Furthermore, any mechanisms that are conserved between the two are good candidates for general principles of eukaryotic biology. If our model is confirmed, it will change the way people think about replication timing. Moreover, it will change the direction of the field from a focus on trying to discover the mechanisms of replication timing, to being able to directly test how MCM loading is regulated to control replication timing in metazoan genomes. Furthermore, accurate information about the mechanism of replication timing is essential to understand how replication timing influences the genome reprogramming required for stem-cell maintenance and cellular differentiation, as well as its role in maintaining genome stability and preventing cancer.