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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Studies of the properties of human origin recognition complex and its Walker A motif mutants.
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Studies of the properties of human origin recognition complex and its Walker A motif mutants.

机译：研究人类起源识别复合物及其Walker A基序突变体的特性。

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The eukaryotic six-subunit origin recognition complex (ORC) governs the initiation site of DNA replication and formation of the prereplication complex. In this report we describe the isolation of the wild-type Homo sapiens (Hs)ORC and variants containing a Walker A motif mutation in the Orc1, Orc4, or Orc5 subunit using the baculovirus-expression system. Coexpression of all six HsORC subunits yielded a stable complex containing HsOrc subunits 1-5 (HsORC1-5) with virtually no Orc6 protein (Orc6p). We examined the ATPase, DNA-binding, and replication activities of these complexes. Similar to other eukaryotic ORCs, wild-type HsORC1-5 possesses ATPase activity that is stimulated only 2-fold by single-stranded DNA. HsORC1-5 with a mutated Walker A motif in Orc1p contains no ATPase activity, whereas a similar mutation of either the Orc4 or Orc5 subunit did not affect this activity. The DNA-binding activity of HsORC1-5, using lamin B2 DNA as substrate, is stimulated by ATP 3- to 5-fold. Mutations in the Walker A motif of Orc1p, Orc4p, or Orc5p reduced the binding efficiency of HsORC1-5 modestly (2- to 5-fold). Xenopus laevis ORC-depleted extracts supplemented with HsORC1-5 supported prereplication complex formation and X. laevis sperm DNA replication, whereas the complex with a mutation in the Walker A motif of the Orc1, Orc4, or Orc5 subunit did not. These studies indicate that the ATP-binding motifs of Orc1, Orc4, and Orc5 are all essential for the replication activity associated with HsORC.
机译：真核六亚基起源识别复合物（ORC）控制DNA复制的起始位点和复制前复合物的形成。在此报告中，我们描述了使用杆状病毒表达系统分离野生型智人（Hs）ORC及其在Orc1，Orc4或Orc5亚基中包含Walker A基序突变的变体。所有六个HsORC亚基的共表达产生了一个稳定的复合物，其中含有HsOrc 1-5（HsORC1-5），而实际上没有Orc6蛋白（Orc6p）。我们检查了这些复合物的ATPase，DNA结合和复制活性。与其他真核ORC相似，野生型HsORC1-5具有的ATPase活性仅被单链DNA刺激2倍。在Orc1p中具有Walker A突变基序的HsORC1-5不包含ATPase活性，而Orc4或Orc5亚基的类似突变均不影响该活性。以层粘连蛋白B2 DNA为底物的HsORC1-5的DNA结合活性被ATP刺激了3-5倍。 Orc1p，Orc4p或Orc5p的Walker A基序中的突变适度降低了HsORC1-5的结合效率（2至5倍）。补充了HsORC1-5的非洲爪蟾ORC缺失提取物支持复制前复合物的形成和X. laevis精​​子DNA复制，而在Orc1，Orc4或Orc5亚基的Walker A基序中具有突变的复合物则不支持。这些研究表明，Orc1，Orc4和Orc5的ATP结合基序对于与HsORC相关的复制活性都是必不可少的。

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