Structure and Function of Histone Chaperones

描述（由申请人提供）：DNA的适当包装有助于防止遗传损伤，并为基因调控提供平台。因此，在复制和修复过程中需要组蛋白及其分子伴侣。更专门的伴侣蛋白在卵母细胞、胚胎和体细胞中发挥重要作用，在那里它们形成组蛋白储存复合物，使精子染色质解聚并将组蛋白转移到DNA上。 为了研究这些特殊分子伴侣的功能，我们正在对Np和N1-家族进行结构和生化调查。因此，我们已经表明NP-家族成员形成五聚体和十聚体。此外，Np、NO38、dNLP和NO29在与核心组蛋白重构时各自形成超分子复合物。在我们的模型中，这些复合物包含一个伴侣十聚体和5个组蛋白八聚体。当N1加入到NP-组蛋白复合物中时，形成了一个显著的超复合物。因此，Np和N1可能作为辅助分子伴侣促进组蛋白在卵母细胞中的储存，而Np将组蛋白转移到精子染色质。此外，Np和N1可以将储存的组蛋白传递给NAP 1和CAF 1，或者将组蛋白直接转移到胚胎中的DNA上。我们正在使用X射线晶体学和生物化学的方法来研究组蛋白结合这些分子伴侣的机制。为此，我们将确定与组蛋白复合的NP家族成员的晶体结构，并获得NO29和Mp 62在基态的晶体结构。在平行实验中，我们将表征组蛋白结合N1及其结构域。然后，我们将研究单独的N1和N1-C及其同源组蛋白复合物的结构。NP家族的体细胞成员是核仁蛋白（NO38，B23，ANO 39和Mp 62），它们可能有助于在核糖体生物发生期间重塑r-染色质。这些分子伴侣通过与DNA结合的C末端H结构域靶向核仁。为了理解这个靶向过程，我们将描述NO38 H结构域的DNA结合偏好并确定其结构。

DESCRIPTION (provided by applicant): The proper packaging of DNA helps prevent genetic damage and provides a platform for gene regulation. Thus, histones and their chaperones are required during replication and repair. More specialized chaperones play significant roles in oocytes, embryos and somatic cells, where they form histone storage complexes, decondense sperm chromatin and transfer histones onto DNA. To investigate the function of these specialized chaperones, we are doing a structural and biochemical survey of the Np and N1-families. Thus, we have shown that Np-family members form pentamers and decamers. In addition, Np, NO38, dNLP and NO29 each form supramolecular complexes when they are reconstituted with core histones. In our model these complexes contain a chaperone decamer and 5 histone octamers. When N1 is added to the Np-histone complex, a remarkable super-complex is formed. Thus, Np and N1 may function as co-chaperones to promote histone storage in oocytes, while Np transfers histones to sperm chromatin. Moreover, Np and N1 may pass stored histones to NAP1 and CAF1 or transfer histones directly onto DNA in the embryo. We are using X-ray crystallography and biochemical approaches to study the mechanism of histone binding by these chaperones. To this end, we will determine crystal structures for Np-family members complexed with histones and obtain crystal structures for NO29 and Mp62 in the ground state. In parallel experiments, we will characterize histone binding by N1 and its domains. We will then investigate the structures of N1 and N1-C alone and with their cognate histone complexes. Somatic members of the Np-family are nucleolar proteins (NO38, B23, ANO39 and Mp62) which may help remodel r-chromatin during ribosome biogenesis. These chaperones are targeted to the nucleolus by a C-terminal H-domain which binds to DNA. To understand this targeting process, we will characterize the DNA binding preferences of the NO38 H-domain and determine its structure.