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。它与转录调控模式、染色质修饰、染色体结构和基因组进化相关。此外，随着细胞分化，复制时间发生变化，并且复制时间的中断与基因组不稳定性相关，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，调节复制时间的机制在很大程度上仍然是神秘的。我们已经开发出，并建议测试，一个详细的，普遍适用的模型的机制复制的时间。我们的模型假定随机调节的起源射击，其中每个起源有一个特征概率的射击，和起源射击的平均时间由该orgin射击概率调节。我们建议，起源射击的概率是由MCM复合物的数量调节-复制解旋酶，它建立了一个起点作为复制起始的网站-在G1加载。装载有更多MCM的原点更有可能击发，因此平均而言，击发更早。此外，我们提出，MCM加载的数量是由ORC -MCM加载器-结合原点的亲和力调节的。更高的亲和力来源结合ORC以获得更大比例的G1，从而允许装载更多的MCM复合物。最后，我们提出，异染色质提供了第二个层次的监管上的MCM为基础的机制的起源定时调节，这样，在异染色质区域的起源射击延迟在一个或多个基本步骤：ORC结合，MCM加载或MCM激活。我们将使用基于深度测序的方法，通过映射ORC结合、MCM结合和跨越出芽和分裂酵母基因组的起源时间来测试我们的模型。这些进化上遥远的酵母的互补优势允许对我们的模型进行更严格的测试。此外，两者之间保守的任何机制都是真核生物学一般原理的良好候选者。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，从专注于试图发现复制时机的机制，能够直接测试MCM加载如何调节以控制后生动物基因组中的复制时机。此外，关于复制时机机制的准确信息对于理解复制时机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其在维持基因组稳定性和预防癌症中的作用至关重要。 公共卫生相关性： 许多导致癌症的遗传变化是由DNA复制过程中的错误引起的。DNA复制的正确组织对于防止此类错误至关重要。拟议的研究将阐明组织复制的机制，从而确定新的治疗靶点和诊断工具，用于治疗和预防人类癌症。

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. PUBLIC HEALTH RELEVANCE: Many of the genetic changes that lead to cancer are caused by errors during DNA replication. Proper organization of DNA replication is essential to prevent such errors. The proposed research will elucidate the mechanisms that organize replication, allowing for the identification o new therapeutic targets and diagnostic tools for the treatment and prevention of human cancer.