The chaperone function of Nucleosome Assembly Protein 1

描述（由申请人提供）：核小体组装蛋白1 （Nap1）是一种组蛋白伴侣，通过与核心组蛋白和连接组蛋白H1的相互作用参与组蛋白调控，也参与核小体的形成和基因调控。在体外和体内，Nap1被认为是通过阻止非核小体组蛋白- dna相互作用来组装核小体的。Nap1是一种同源二聚体，由两个48 kDa的多肽组成，它们的N端和c端尾部是无序的，出现在圆顶结构[10]的底部。初步数据显示，酸性c端尾对于解决非核体组蛋白- dna相互作用至关重要。通过体外亲和力测量，结合EMSA (electrophotic Mobility Shift Assays)，我们确定了野生型Nap1的功能需要c端尾部。我们还确定它有助于屏蔽抑制n端尾部。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，本文将对此进行研究。研究表明，Nap1主要是H2A/H2B组蛋白伴侣[4]。由于Nap1主要与H2A/H2B一起发挥作用，我们的研究重点是确定其机制。使用氢-氘交换耦合质谱（H/DX-MS），我们的实验室最近发现H2A/H2B快速取样部分未展开状态（S. D'Arcy，提交）。当与酵母Nap1 （yNap1）结合时，H2A/H2B随后稳定在折叠构象中（S. D'Arcy，提交）。目前尚不清楚DNA是否也会像yNap1一样将H2A/H2B稳定在折叠构象中。同样，yNap1是否也稳定了连接蛋白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只是一种中分辨率技术，我们只能收集三级结构的信息。为了获得更高的分辨率，将使用x射线晶体学。它将提供分子相互作用的大量细节，可以增强我们对组蛋白伴侣在核小体动力学中的作用的认识。

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.