Mutations in subdomain B of the minichromosome maintenance (MCM) helicase affect DNA binding and modulate conformational transitions.
Mutations in subdomain B of the minichromosome maintenance (MCM) helicase affect DNA binding and modulate conformational transitions.
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DOI：
10.1074/jbc.m806973200
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发表时间：
2009-02-27
期刊：
影响因子：
--
通讯作者：
Chong JP
中科院分区：
文献类型：
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作者：
Jenkinson ER;Costa A;Leech AP;Patwardhan A;Onesti S;Chong JP
微染色体维持蛋白（MCM）被认为在真核生物和古细菌中提供复制解旋酶活性。来自嗜热甲烷热杆菌（Methanothermobacter thermautotrophicus，MthMCM）的单一MCM直向同源物已被广泛表征为真核异六聚体MCM复合物的模型。MthMCM在溶液中形成与十二聚体一致的高分子量复合物。通过电子显微镜观察该复合物表明可以形成单和双七聚体或六聚体环。我们已经突变了MthMCM的N-末端亚结构域B中的两个精氨酸残基（R137，R160），基于它们形成环间氢键的表观潜力。单R137 A和双RR 137，160 AA突变体的特征在于通过生物物理，生物化学和电子显微镜（EM）技术的组合。结合EM研究的生物物理分析表明，R137 A突变体形成一个双七聚体环，而RR 137，160 AA蛋白组装成一个单一的七聚体。它们都显示出DNA结合缺陷和伴随的亚结构域A的构象变化，双突变体也显示出解旋酶活性的显著缺陷。我们提出了一个模型，其中MCM加载和随后的激活的解旋酶活性涉及的构象转变，连接到一个DNA结合事件。
Minichromosome maintenance (MCM) proteins are believed to provide the replicative helicase activity in eukaryotes and archaea. The single MCM orthologue from Methanothermobacter thermautotrophicus (MthMCM) has been extensively characterised as a model of the eukaryotic heterohexameric MCM complex. MthMCM forms high molecular weight complexes in solution consistent with a dodecamer. Visualization of this complex by electron microscopy suggests that single and double heptameric or hexameric rings can form. We have mutated two arginine residues (R137, R160) in the N-terminal sub-domain B of MthMCM based on their apparent potential to form inter-ring hydrogen bonds. Both the single R137A and the double RR137,160AA mutants were characterised by a combination of biophysical, biochemical and electron microscopy (EM) techniques. Biophysical analysis coupled with EM studies shows that the R137A mutant forms a double heptameric ring, whereas the RR137,160AA protein assembles as a single heptamer. They both show a defect in DNA binding and a concomitant conformational change in sub-domain A, with the double mutant displaying significant defects in helicase activity as well. We propose a model in which MCM loading and the subsequent activation of the helicase activity involves a conformational transition that is connected to a DNA binding event.