The chaperone function of Nucleosome Assembly Protein 1

描述(申请人提供)：核小体组装蛋白1(NAP1)是一种组蛋白伴侣蛋白，通过与核心组蛋白和连接物组蛋白H1的相互作用参与组蛋白调控，并参与核小体形成和基因调控。在体外和体内，NAP1被认为通过阻止非核小体组蛋白-DNA相互作用来组装核小体[4]。NAP1是一种同源二聚体，由两个48 kDa的多肽组成，N-末端和C-末端无序，出现在穹顶状结构的底部[10]。初步数据显示，酸性的C-末端尾巴对于解决非核小体组蛋白-DNA相互作用是必不可少的。通过体外亲和力测定，结合电泳性迁移率改变分析(EMSA)，我们确定了野生型NAP1功能所必需的C末端尾巴。我们还确定它有助于屏蔽抑制的N末端尾巴。目前尚不清楚这种机制是否适用于其他NAP1家族成员，如人类NAP1(HNap1)和SET，这里将对此进行研究。研究表明，NAP1主要是一种H_2A/H_2B组蛋白伴侣[4]。由于NAP1主要与H_2A/H_2B一起发挥作用，我们的研究主要集中在确定其机制上。利用氢-氚交换-质谱仪(H/DX-MS)，我们的实验室最近表明，H_2A/H_2B迅速地以部分未折叠的状态采样(S.D‘Arcy，提交)。当与酵母NAP1(YNap1)结合时，H_2A/H_2B以折叠构象稳定(S.D‘Arcy，提交)。目前尚不清楚DNA是否也会像yNap1那样以折叠构象稳定H_2A/H_2B。同样，yNap1是否也稳定连接子组蛋白H1的折叠状态？通过使用H/DX，我们将能够回答这些问题。由于H/DX只是一种中等分辨率的技术，我们只能收集关于三级结构的信息。为了获得更高的分辨率，将使用X射线结晶学。它将提供分子相互作用的详细信息，从而增强我们对组蛋白伴侣蛋白在核小体动力学中所起作用的了解。

DESCRIPTION (provided by applicant): Nucleosome assembly protein 1 (Nap1) is a histone chaperone implicated in histone regulation through interactions with core histones and linker histone H1, as well as in nucleosome formation and gene regulation. Nap1 is thought to assemble nucleosomes through the prevention of non-nucleosomal histone-DNA interactions in vitro and in vivo [4]. Nap1 is a homo-dimer composed of two 48 kDa polypeptides with disordered N- and C-terminal tails that emerge at the underside of the dome-shaped structure [10]. Preliminary data shows the acidic C-terminal tail is essential for resolving non-nucleosomal histone-DNA interactions. Through affinity measurements in vitro, paired with Electrophoretic Mobility Shift Assays (EMSA), we have determined that the C-terminal tail is required for wild type Nap1 function. We also determined it aids in shielding the inhibitory N-terminal tail. It is unknown whether this mechanism applies to other Nap1-family members such as human Nap1 (hNap1) and SET, and this will be investigated here. It has been shown that Nap1 is primarily a H2A/H2B histone chaperone [4]. Since Nap1 primarily functions with H2A/H2B, our research has focused on determining the mechanism. Using hydrogen-deuterium exchange coupled to Mass Spectrometry (H/DX-MS), our laboratory has recently shown that H2A/H2B rapidly samples a partially unfolded state (S. D'Arcy, submitted). When bound to yeast Nap1 (yNap1), H2A/H2B is then stabilized in a folded conformation (S. D'Arcy, submitted). It is unknown whether DNA will also stabilize H2A/H2B in a folded conformation like yNap1 does. Likewise, does yNap1 also stabilize the folded state of linker histone H1? Through the use of H/DX we will be able to answer these questions. Since H/DX is only a medium resolution technique, we can only gather information on tertiary structure. To obtain greater resolution, x-ray crystallography will used. It will provide great detail of the molecular interactions that can enhance our knowledge of the role that histone chaperones have in nucleosome dynamics.