染色质携带表观遗传信息和DNA蕴含的基因组信息保持互动，DNA复制时染色质也同步复制，其中关键的一步是DNA复制耦联（RC）的核小体组装。相对于清晰的DNA的半保留复制机制，染色质复制的机制有太多的未知有待探索，其中之一是组蛋白在已完成复制的DNA子链的分配机制。目前发现参与DNA复制耦联的核小体组装的组蛋白一半是来源于复制叉前“旧”组蛋白，另一半是来源于S期合成的“新”组蛋白，而且“新”“旧”组蛋白各自被分子伴侣呈递到复制叉参与核小体组装。由此而产生的问题是“新”“旧”组蛋白在DNA子链的分配机制，包括在同一条子链上的排列规律以及前导链和滞后链间的分配机制。此问题困扰了该领域几十年，本项目将使用eSPAN技术研究带有特异标记H3K56Ac的“新”组蛋白在DNA子链上的分配规律，揭示组蛋白分子伴侣及DNA复制机器在这个过程的调节功能，理解染色质复制的机理。

Successful assembly replicating DNA into nucleosomes using both newly synthesized and parental histones during S phase is critical for the inheritance of epigenetic information as well as maintenance of genome integrity. However, how newly synthesized histone and recycled parental histones segregate on daughter strands remains one of the most enigmatic, yet fundamental questions in the epigenetic and chromatin field. Accumulated data showed that histone modifications and histone chaperones are critical to regulate nucleosome assembly coupled with DNA synthesis. For instance, in the budding yeast Saccharomyces cerevisiae, acetylation on H3 lysine 56 (H3K56Ac) is new born histone marker and regulates newly synthesized histone H3-H4 to deposit on replicating DNA mediated by histone chaperone CAF-1 and Rtt106. Besides CAF-1 and Rtt106, our recent study also showed that FACT complex also plays a direct role in depositing H3K56Ac marked histone onto replicating DNA. Enrichment and sequencing of protein-associated nascent DNA (eSPAN) is a recently developed method using BrdU incorporation into replicating DNA in combination with NGS sequencing to detect proteins with strand specificity at replication forks. In this proposal, we will use eSPAN methods to map the H3K56Ac marked histone on replication forks in wild type and a serial of FACT mutants. We expect that successful completion of this project will provide novel insight into histone segregation during chromatin replication and the inheritance of epigenetic information.