Chen Yi-Hsing, Kocherginskaya Svetlana A, Lin Yuyen, Sriratana Binjon, Lagunas Angelica M, Robbins Justin B, Mackie Roderick I, Cann Isaac K O
Department of Animal Sciences, University of Illinois at Urbana-Champaign, 61801, USA.
J Biol Chem. 2005 Dec 23;280(51):41852-63. doi: 10.1074/jbc.M508684200. Epub 2005 Oct 27.
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. Mac-RFCS1 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.
夹子装载机协调从分布式到连续式DNA合成的转换。它们在细胞过程中的重要性通过在所有生命形式中的保守性得到强调。在这里，我们描述了一种来自嗜乙酸甲烷八叠球菌古菌的新型夹子装载机。与先前描述的由一个小亚基和一个大亚基组成的古菌夹子装载机不同，嗜乙酸甲烷八叠球菌夹子装载机由两个相似的小亚基（嗜乙酸甲烷八叠球菌复制因子C小亚基（MacRFCS））和一个大亚基（MacRFCL）组成。古菌和真核生物夹子装载机（由四个相似的小亚基和一个大亚基组成）的相关性表明，嗜乙酸甲烷八叠球菌夹子装载机可能是古菌/真核生物姐妹谱系中的一种中间形式。由MacRFCS1、MacRFCS2和MacRFCL这三个亚基重构的夹子装载机复合物在其滑动夹存在的情况下刺激同源DNA聚合酶进行DNA合成。我们在沃克A和SRC基序中使用定点诱变来检查每个亚基对嗜乙酸甲烷八叠球菌夹子装载机功能的贡献。虽然MacRFCL和MacRFCS2中的突变不会损害夹子装载活性，但任何在MacRFCS1中带有突变的突变夹子装载机都没有夹子装载特性。因此，Mac-RFCS1对嗜乙酸甲烷八叠球菌夹子装载机的夹子装载活性至关重要。我们预计，随着更复杂的生物体在古菌/真核生物姐妹谱系中进化，这种独特的复制因子C同源物的发现将为从更简单的夹子装载机进化出更复杂的夹子装载机提供关键见解。
