Genome-Wide Single-Molecule Analysis of Human Replication Kinetics

项目总结 DNA复制的时间是细胞生长的关键参数。它与 转录调节、染色质修饰、染色体结构和 基因组进化。此外，复制时间随着细胞的分化而改变，并且 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的关系。一个 理解人类基因组中复制时间的调节的主要障碍是 缺乏可靠的分析方法来确定人类复制的位置和激发时间 起源。目前的方法存在信噪比低、一致性差的问题 在独立的实验室之间。此外，合奏技术无法探测到 起源射击的协调，是该领域的一个重大兴趣的主题，因为它已经 建议将其作为复制时机和效率的关键因素。我们建议应用两个新的 我们开发的高通量单分子方法--光学复制 映射和SMRT复制序列-以映射复制起点和复制分叉进程 在人类基因组中。我们将使用我们获得的复制配置文件来开发 关于基因组生物学基本方面的假设，例如复制和 如果复制终止位点的位置受到调控，则转录是协调的 叉子如何导航难以复制的序列。这项工作的圆满完成将 阐明整个人类基因组复制时间的调节，允许 表征序列和起源功能的表观遗传决定因素，并提供 强大的源图和复制配置文件，供其他人使用。此外，传播这一点 技术将改变生物学家能够提出的关于DNA调控的问题 复制时机及其在不同领域的影响，如发育、染色质 生物学和表观遗传学。

PROJECT SUMMARY The timing of DNA replication is a critical parameter of cellular growth. It correlates with patterns 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. A major impediment to understand the regulation of replication timing in the human genome has been the lack of robust assays for identifying the location and firing times of human replication origins. Current approaches suffer from low signal-to-noise ratios and poor concordance between independent laboratories. Moreover, ensemble techniques are unable to probe the coordination of origin firing, a subject significant interest in the field, because it has been proposed as a key factor in replication timing and efficiency. We propose to apply two new high-throughput single-molecule approaches that we have developed—Optical Replication Mapping and SMRT Repli-seq—to map replication origins and replication fork progression across the human genome. We will use replication profiles that we obtain to develop hypotheses about fundamental aspects of genome biology, such as such as how replication and transcription are coordinated, if the location of replication termination sites are regulated and how forks navigate difficult-to-replicate sequences. Successful completion of this work will elucidate the regulation of replication timing across the human genome, allow for the characterization of the sequence and epigenetic determinants for origin function, and provide robust origin maps and replication profiles for others to use. Moreover, dissemination of this technology will change the questions that biologists are able to ask about the regulation of DNA replication timing and its repercussions in diverse fields, such as development, chromatin biology, and epigenetics.