Biochemical and mutational analyses of a unique clamp loader complex in the archaeon Methanosarcina acetivorans
Biochemical and mutational analyses of a unique clamp loader complex in the archaeon Methanosarcina acetivorans
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DOI：
10.1074/jbc.m508684200
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发表时间：
2005-12-23
影响因子：
4.8
通讯作者：
Cann, IKO
中科院分区：
文献类型：
--
作者：
Chen, YH;Kocherginskaya, SA;Cann, IKO
钳装载器协调从分配到进行性DNA合成的转换。它们在细胞过程中的重要性因其在所有生命形式中的保守性而得到强调。在这里，我们描述了一种新形式的钳装载机从古菌甲烷八叠球菌醋酸。与先前描述的由一个小亚基和一个大亚基组成的古细菌钳形装载器不同，M。acetivorans clamp loader包括两个相似的小亚基（M.乙酰化酵母复制因子C小亚基（MacRFCS））和一个大亚基（MacRFCL）。古细菌和真核细胞的钳装载器（由四个相似的小亚基和一个大亚基组成）的相关性表明M. acetivorans clamp loader可能是古细菌/真核生物姐妹谱系中的中间形式。由三个亚基MacRFCS 1、MacRFCS 2和MacRFCL重构的钳加载复合物在其滑动钳存在下通过同源DNA聚合酶刺激DNA合成。我们在步行者A和SRC基序中使用定点突变来检查每个亚基对M的功能的贡献。acetivorans夹具装载机。尽管MacRFCL和MacRFCS 2中的突变不损害夹加载活性，但任何在MacRFCS 1中携带突变的突变夹加载器都缺乏夹加载特性。因此，MacRFCS 1对M. acetivorans夹具装载机。这是我们的预期，这种独特的复制因子C同源物的发现将导致更复杂的钳装载机从简单的进化的关键见解，因为更复杂的生物体进化的古/真核姐妹谱系。
Clamp loaders orchestrate the switch from distributive to processive DNA synthesis. Their importance in cellular processes is underscored by their conservation across all forms of life. Here, we describe a new form of clamp loader from the archaeon Methanosarcina acetivorans. Unlike previously described archaeal clamp loaders, which are composed of one small subunit and one large subunit, the M. acetivorans clamp loader comprises two similar small subunits (M. acetivorans replication factor C small subunit (MacRFCS)) and one large subunit (MacRFCL). The relatedness of the archaeal and eukaryotic clamp loaders (which are made up of four similar small subunits and one large subunit) suggests that the M. acetivorans clamp loader may be an intermediate form in the archaeal/eukaryotic sister lineages. The clamp loader complex reconstituted from the three subunits MacRFCS1, MacRFCS2, and MacRFCL stimulated DNA synthesis by a cognate DNA polymerase in the presence of its sliding clamp. We used site-directed mutagenesis in the Walker A and SRC motifs to examine the contribution of each subunit to the function of the M. acetivorans clamp loader. Although mutations in MacRFCL and MacRFCS2 did not impair clamp loading activity, any mutant clamp loader harboring a mutation in MacRFCS1 was devoid of the clamp loading property. MacRFCS1 is therefore critical to the clamp loading activity of the M. acetivorans clamp loader. It is our anticipation that the discovery of this unique replication factor C homolog will lead to critical insights into the evolution of more complex clamp loaders from simpler ones as more complex organisms evolved in the archaeal/eukaryotic sister lineages.