Department of Gene Vectors, Helmholtz Zentrum München, German Research Center for Environmental Health, Munich, Germany.
Cell Mol Life Sci. 2011 Nov;68(22):3741-56. doi: 10.1007/s00018-011-0675-9. Epub 2011 Apr 2.
In eukaryotes, binding of the six-subunit origin recognition complex (ORC) to DNA provides an interactive platform for the sequential assembly of pre-replicative complexes. This process licenses replication origins competent for the subsequent initiation step. Here, we analyze the contribution of human Orc6, the smallest subunit of ORC, to DNA binding and pre-replicative complex formation. We show that Orc6 not only interacts with Orc1-Orc5 but also with the initiation factor Cdc6. Biochemical and imaging experiments reveal that this interaction is required for licensing DNA replication competent. Furthermore, we demonstrate that Orc6 contributes to the interaction of ORC with the chaperone protein HMGA1a (high mobility group protein A1a). Binding of human ORC to replication origins is not specified at the level of DNA sequence and the functional organization of origins is poorly understood. We have identified HMGA1a as one factor that might direct ORC to AT-rich heterochromatic regions. The systematic analysis of the interaction between ORC and HMGA1a revealed that Orc6 interacts with the acidic C-terminus of HMGA1a and also with its AT-hooks. Both domains support autonomous replication if targeted to DNA templates. As such, Orc6 functions at different stages of the replication initiation process. Orc6 can interact with ORC chaperone proteins such as HMGA1a to facilitate chromatin binding of ORC and is also an essential factor for pre-RC formation.
在真核生物中，六聚体起始识别复合物（ORC）与 DNA 的结合为复制前复合物的顺序组装提供了一个相互作用平台。这个过程使复制起点具有随后起始步骤的能力。在这里，我们分析了人类 ORC 的最小亚基 Orc6 对 DNA 结合和复制前复合物形成的贡献。我们表明，Orc6 不仅与 Orc1-Orc5 相互作用，还与起始因子 Cdc6 相互作用。生化和成像实验表明，这种相互作用对于复制起点的能力至关重要。此外，我们证明 Orc6 有助于 ORC 与伴侣蛋白 HMGA1a（高迁移率族蛋白 A1a）的相互作用。人类 ORC 与复制起点的结合不是在 DNA 序列水平上特异性的，并且起点的功能组织理解甚少。我们已经确定 HMGA1a 是一种可能将 ORC 导向富含 AT 的异染色质区域的因素。对 ORC 和 HMGA1a 之间相互作用的系统分析表明，Orc6 与 HMGA1a 的酸性 C 末端以及其 AT 钩相互作用。如果将这两个结构域靶向 DNA 模板，它们都支持自主复制。因此，Orc6 在复制起始过程的不同阶段发挥作用。Orc6 可以与 ORC 伴侣蛋白（如 HMGA1a）相互作用，以促进 ORC 与染色质的结合，并且也是预 RC 形成的必需因素。
