Department of Biomolecular Chemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
Genes Dev. 2010 Jul 1;24(13):1418-33. doi: 10.1101/gad.1906410.
The origin recognition complex (ORC) binds to the specific positions on chromosomes that serve as DNA replication origins. Although ORC is conserved from yeast to humans, the DNA sequence elements that specify ORC binding are not. In particular, metazoan ORC shows no obvious DNA sequence specificity, whereas yeast ORC binds to a specific DNA sequence within all yeast origins. Thus, whereas chromatin must play an important role in metazoan ORC's ability to recognize origins, it is unclear whether chromatin plays a role in yeast ORC's recognition of origins. This study focused on the role of the conserved N-terminal bromo-adjacent homology domain of yeast Orc1 (Orc1BAH). Recent studies indicate that BAH domains are chromatin-binding modules. We show that the Orc1BAH domain was necessary for ORC's stable association with yeast chromosomes, and was physiologically relevant to DNA replication in vivo. This replication role was separable from the Orc1BAH domain's previously defined role in transcriptional silencing. Genome-wide analyses of ORC binding in ORC1 and orc1bahDelta cells revealed that the Orc1BAH domain contributed to ORC's association with most yeast origins, including a class of origins highly dependent on the Orc1BAH domain for ORC association (orc1bahDelta-sensitive origins). Orc1bahDelta-sensitive origins required the Orc1BAH domain for normal activity on chromosomes and plasmids, and were associated with a distinct local nucleosome structure. These data provide molecular insights into how the Orc1BAH domain contributes to ORC's selection of replication origins, as well as new tools for examining conserved mechanisms governing ORC's selection of origins within eukaryotic chromosomes.
起始识别复合物（ORC）结合到作为 DNA 复制起点的染色体的特定位置。尽管 ORC 从酵母到人类都是保守的，但指定 ORC 结合的 DNA 序列元件却不是。特别是，真核生物的 ORC 没有明显的 DNA 序列特异性，而酵母 ORC 结合到所有酵母起点内的特定 DNA 序列。因此，尽管染色质在真核生物 ORC 识别起点的能力中必须发挥重要作用，但尚不清楚染色质是否在酵母 ORC 识别起点中发挥作用。本研究集中于酵母 Orc1（Orc1BAH）的保守 N 端溴邻位同源结构域的作用。最近的研究表明，BAH 结构域是染色质结合模块。我们表明，Orc1BAH 结构域对于 ORC 与酵母染色体的稳定结合是必需的，并且在体内 DNA 复制中具有生理相关性。该复制作用与 Orc1BAH 结构域先前在转录沉默中的定义作用是可分离的。在 ORC1 和 orc1bahDelta 细胞中对 ORC 结合的全基因组分析表明，Orc1BAH 结构域有助于 ORC 与大多数酵母起点的结合，包括一类高度依赖 Orc1BAH 结构域与 ORC 结合的起点（orc1bahDelta 敏感起点）。orc1bahDelta 敏感起点在染色体和质粒上的正常活性需要 Orc1BAH 结构域，并且与独特的局部核小体结构相关联。这些数据为 Orc1BAH 结构域如何有助于 ORC 选择复制起点提供了分子见解，以及研究真核染色体中 ORC 选择起点的保守机制的新工具。
