Biochemical characterization of Cdc6/Orc1 binding to the replication origin of the euryarchaeon Methanothermobacter thermoautotrophicus
Biochemical characterization of Cdc6/Orc1 binding to the replication origin of the euryarchaeon Methanothermobacter thermoautotrophicus
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DOI：
10.1093/nar/gkh819
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发表时间：
2004-01-01
影响因子：
14.9
通讯作者：
Berger, JM
中科院分区：
文献类型：
--
作者：
Capaldi, SA;Berger, JM
古细菌细胞分裂周期蛋白6 (Cdc6)/起源复制复合体亚单位1 （Orc1）蛋白序列与真核生物DNA复制起始因子具有同源性，但在结构上也与细菌启动因子DNA相似。为了更好地了解Cdc6/Orc1是否以真核或细菌的方式发挥作用，我们表征了两个Cdc6/Orc1类似物（mthCdc6-1和mthCdc6-2）与热自养甲烷杆菌复制起源的相互作用。我们发现，虽然这两种蛋白对随机序列的小dsDNA具有低亲和力，但mthCdc6-1与一个短双工紧密结合，该双工包含一个13bp序列的单拷贝，在整个起源中重复。令人惊讶的是，序列比较表明，这个13bp序列是在许多euryarchaeo起源中发现的起源识别盒元素的最小化版本。对mthCdc6-1突变体的分析表明，翼状螺旋结构域的螺旋-转-螺旋基序介导了与该序列的相互作用。两个mthCdc6/Orc1类似物与包含最小起源识别盒的双链的结合符合独立的结合位点模型，但它们与较长的DNA配体的相互作用是合作的。总之，我们的数据提供了古细菌DNA复制启动物与其起源的关联的第一个详细的生物物理特征。我们的观察还表明，Cdc6/Orc1蛋白的起源结合特性与细菌dna非常相似。
Archaeal cell division cycle protein 6 (Cdc6)/Origin Replication Complex subunit 1 (Orc1) proteins share sequence homology with eukaryotic DNA replication initiation factors but are also structurally similar to the bacterial initiator DnaA. To better understand whether Cdc6/Orc1 functions in an eukaryotic or bacterial-like manner, we have characterized the interaction of two Cdc6/Orc1 paralogs (mthCdc6-1 and mthCdc6-2) with the replication origin from Methanothermobacter thermoautotrophicus. We show that while both proteins display a low affinity for a small dsDNA of random sequence, mthCdc6-1 binds tightly to a short duplex containing a single copy of a 13 bp sequence that is repeated throughout the origin. Surprisingly, sequence comparisons show that this 13 bp sequence is a minimized version of the Origin Recognition Box element found in many euryarchaeotal origins. Analysis of mthCdc6-1 mutants demonstrates that the helix-turn-helix motif in the winged-helix domain mediates the interaction with this sequence. Association of both mthCdc6/Orc1 paralogs with the duplex containing the minimized Origin Recognition Box fits to an independent binding sites model, but their interaction with longer DNA ligands is cooperative. Together, our data provide the first detailed biophysical characterization of the association of an archaeal DNA replication initiator with its origin. Our observations also indicate that the origin-binding properties of Cdc6/Orc1 proteins closely resemble those of bacterial DnaA.