Histone chaperones are a group of histone-binding proteins that facilitate the assembly of nucleosomes, the fundamental structural units of chromatin in eukaryotes. In nucleosome assembly, deposition of a histone H3-H4 tetramer onto DNA is the first and critical step, which is mediated by the histone chaperones HIRA and CAF-1. HIRA and CAF-1 are reportedly involved in DNA replication independent (RI) and replication coupled nucleosome assembly, respectively. However, the mechanisms by which they mediate histone deposition remain unclear. In this study, we focused on the mechanism by which HIRA induces RI-nucleosome assembly. We looked for HIRA domains that are required for nucleosome assembly and its localization to chromatin. We used cell-free extracts from Xenopus eggs that carry out RI-nucleosome assembly of plasmid DNA. We confirmed that HIRA formed stable complexes with Asf1, another histone H3-H4 chaperone, and the HIRA-Asf1 complex was solely responsible for RI-nucleosome assembly in egg extracts. We further demonstrated that the HIRA N-terminus containing the WD40 domain, which comprises seven WD40 repeats, and the B domain, to which Asf1 binds, were essential for RI-nucleosome assembly; the three WD40 repeats from the N-terminus were especially critical. Using egg extracts that reproduce nuclear formation accompanying the duplication of chromatin, we also demonstrated that the Hir domain was indispensable for the binding of HIRA to chromatin. Thus, the WD40 and B domains are the core elements for inducing RI-nucleosome assembly. Hir domain regulates the binding to chromatin. Based on these findings, similarities and differences between HIRA and CAF-1 are discussed.

中文翻译：

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HIRA的WD40结构域对于非洲爪蟾卵提取物中的RI-核小体组装至关重要。

组蛋白伴侣是一组组蛋白结合蛋白，可促进核小体的组装，核小体是真核生物中染色质的基本结构单位。在核小体装配中，组蛋白H3-H4四聚体在DNA上的沉积是第一步，也是至关重要的一步，这是由组蛋白伴侣HIRA和CAF-1介导的。据报道，HIRA和CAF-1分别参与DNA复制独立（RI）和复制偶联核小体装配。但是，它们介导组蛋白沉积的机制仍不清楚。在这项研究中，我们集中于HIRA诱导RI-核小体装配的机制。我们寻找了核小体组装及其定位到染色质所需的HIRA结构域。我们使用非洲爪蟾卵的无细胞提取物进行质粒DNA的RI-核小体组装。我们证实，HIRA与另一个组蛋白H3-H4分子伴侣Asf1形成了稳定的复合物，而HIRA-Asf1复合物仅负责卵提取物中的RI-核小体组装。我们进一步证明，含有WD40结构域的HIRA N末端（包含七个WD40重复序列）以及与Asf1结合的B结构域对于RI-核小体装配是必不可少的。N末端的三个WD40重复序列尤其重要。使用卵提取物复制伴随染色质复制的核形成，我们还证明了Hir域对于HIRA与染色质的结合是必不可少的。因此，WD40和B结构域是诱导RI-核小体装配的核心元件。Hir结构域调节与染色质的结合。根据这些发现，