参与核小体组装的人源CAF-1与组蛋白H3.1-H4复合物的结构研究

Chromatin assembly factor 1 (CAF-1) is one of the histone chaperones involved in nucleosome assembly during DNA replication and repair. Newly synthesized H3.1-H4 first binds to the antisilencing function 1(ASF1) as a dimer, and ASF1 transfers H3.1-H4 dimer to the downstream chaperone CAF-1. CAF-1 targets H3.1-H4 and deposits them to newly synthesized DNA, which finished the first step of nucleosome assembly. To date, how CAF-1 specific recognizing H3.1 and which oligomerization state of H3.1-H4 binding to CAF-1 are not known. In this project, we plan to determine the cyrstal structure of human CAF-1 in complex with histones H3.1-H4 by X-ray crystallography. The structure of the CAF-1-H3.1-H4 complex will uncover how CAF-1 binding with histones H3.1-H4. Based on the structure, we will design the mutagenesis of CAF-1. By a combination of biochemical methods such as pull down and Co-IP, and cell-based fluorescent co-localization technique, we are trying to investigate the molecular mechanisms of CAF-1 specific recognizing H3.1.This work will be useful for delineating the CAF-1 mediated H3.1 deposition pathway in nucleosome assembly and facilitating strcuture-based drug design for CAF-1 associated diseases.

染色质装配因子1(CAF-1)是一种在DNA复制及修复过程中参与核小体组装的组蛋白伴侣。反沉默功能蛋白1(ASF1)将新合成的组蛋白H3.1-H4二聚体传递给下游的CAF-1，再由CAF-1将组蛋白(H3.1-H4)2四聚体存储到新生DNA链上，以实现核小体组装的第一步反应。但是，CAF-1如何特异识别组蛋白H3.1及CAF-1结合何种聚合状态的组蛋白H3.1-H4，目前仍不清楚。本项目计划运用X射线晶体学测定人源CAF-1与组蛋白H3.1-H4复合物的晶体结构，揭示CAF-1与H3.1-H4的结合方式。以结构为基础设计突变实验，综合运用Pull down和Co-IP生化手段以及细胞荧光共定位技术，研究CAF-1特异识别H3.1的分子机理。该项工作将为我们了解CAF-1参与H3.1的核小体组装途径提供精确的结构基础，并为开展与CAF-1相关疾病的药物设计提供结构支持。

染色质装配因子1(CAF-1)是一种在DNA复制及修复过程中参与核小体组装的组蛋白伴侣。反沉默功能蛋白1(ASF1)将新合成的组蛋白H3.1-H4二聚体传递给下游的CAF-1，再由CAF-1 将组蛋白(H3.1-H4)2 四聚体存储到新生DNA链上，以实现核小体组装的第一步反应。本项目计划运用X射线晶体学测定CAF-1与组蛋白H3.1-H4复合物的晶体结构，并结合生化手段研究CAF-1结合组蛋白H3.1-H4的模式以及特异识别H3.1的分子机理。经过三年的研究，我们成功的获得了CAF-1复合物的高纯度蛋白，生长出可衍射的复合物晶体，并进行了初步X射线晶体学分析。为了揭示CAF-1结合组蛋白H3.1-H4的模式，我们成功的组装了CAF-1与组蛋白H3.1-H4五元复合物，并进行了大量的晶体筛选，并通过生化手段验证CAF-1对H3.1的识别特异性。更进一步地，我们成功的组装了ASF1和CAF-1-H3.1-H4六元复合物。这些数据将对进一步研究CAF-1的识别和装配机制提供详实的基础，并有助于了解整个核小体组装的第一步反应过程。