Chen Yi-Hsing, Lin Yuyen, Yoshinaga Aya, Chhotani Benazir, Lorenzini Jenna L, Crofts Alexander A, Mei Shou, Mackie Roderick I, Ishino Yoshizumi, Cann Isaac K O
Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
J Bacteriol. 2009 Nov;191(21):6539-49. doi: 10.1128/JB.00414-09. Epub 2009 Aug 28.
Chromosomal DNA replication is dependent on processive DNA synthesis. Across the three domains of life and in certain viruses, a toroidal sliding clamp confers processivity to replicative DNA polymerases by encircling the DNA and engaging the polymerase in protein/protein interactions. Sliding clamps are ring-shaped; therefore, they have cognate clamp loaders that open and load them onto DNA. Here we use biochemical and mutational analyses to study the structure/function of the Methanosarcina acetivorans clamp loader or replication factor C (RFC) homolog. M. acetivorans RFC (RFC(Ma)), which represents an intermediate between the common archaeal RFC and the eukaryotic RFC, comprises two different small subunits (RFCS1 and RFCS2) and a large subunit (RFCL). Size exclusion chromatography suggested that RFCS1 exists in oligomeric states depending on protein concentration, while RFCS2 exists as a monomer. Protein complexes of RFCS1/RFCS2 formed in solution; however, they failed to stimulate DNA synthesis by a cognate DNA polymerase in the presence of its clamp. Determination of the subunit composition and previous mutational analysis allowed the prediction of the spatial distribution of subunits in this new member of the clamp loader family. Three RFCS1 subunits are flanked by an RFCS2 and an RFCL. The spatial distribution is, therefore, reminiscent of the minimal Escherichia coli clamp loader that exists in space as three gamma-subunits (motor) flanked by the delta' (stator) and the delta (wrench) subunits. Mutational analysis, however, suggested that the similarity between the two clamp loaders does not translate into the complete conservation of the functions of individual subunits within the RFC(Ma) complex.
染色体DNA复制依赖于持续性DNA合成。在生命的三个域以及某些病毒中，一种环形滑动夹通过环绕DNA并使聚合酶参与蛋白质/蛋白质相互作用，赋予复制性DNA聚合酶持续性。滑动夹呈环形；因此，它们有同源的夹装载器，可将其打开并装载到DNA上。在这里，我们使用生化和突变分析来研究嗜乙酸甲烷八叠球菌夹装载器或复制因子C（RFC）同源物的结构/功能。嗜乙酸甲烷八叠球菌RFC（RFC(Ma)）代表普通古菌RFC和真核生物RFC之间的中间体，由两个不同的小亚基（RFCS1和RFCS2）和一个大亚基（RFCL）组成。尺寸排阻色谱表明，RFCS1根据蛋白质浓度以寡聚状态存在，而RFCS2以单体形式存在。RFCS1/RFCS2的蛋白质复合物在溶液中形成；然而，在其夹存在的情况下，它们未能刺激同源DNA聚合酶进行DNA合成。亚基组成的确定和先前的突变分析使得能够预测夹装载器家族这个新成员中亚基的空间分布。三个RFCS1亚基两侧分别是一个RFCS2和一个RFCL。因此，这种空间分布让人联想到最小的大肠杆菌夹装载器，它在空间中以三个γ亚基（马达）为侧翼，两侧分别是δ'（定子）和δ（扳手）亚基。然而，突变分析表明，这两种夹装载器之间的相似性并没有转化为RFC(Ma)复合物中各个亚基功能的完全保守。
