Biochemical characterization of the Methanothermobacter thermautotrophicus minichromosome maintenance (MCM) helicase N-terminal domains
Biochemical characterization of the Methanothermobacter thermautotrophicus minichromosome maintenance (MCM) helicase N-terminal domains
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DOI：
10.1074/jbc.m403202200
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发表时间：
2004-07-02
影响因子：
4.8
通讯作者：
Kelman, Z
中科院分区：
文献类型：
--
作者：
Kasiviswanathan, R;Shin, JH;Kelman, Z
微型染色体维持解旋酶是环形复合物，通过分离染色体 DNA 的两条链，为复制聚合酶提供单链底物，在古菌和真核 DNA 复制中发挥重要作用。对于古细菌蛋白，研究表明该蛋白的 N 末端部分由结构域 A、B 和 C 组成，参与多聚体形成和单链 DNA 结合，也可能在调节解旋酶活性中发挥作用。在此，描述了嗜热甲烷嗜热杆菌微型染色体维持解旋酶 N 末端区域的详细生化特征。使用生化和生物物理分析表明，位于解旋酶催化结构域附近的 N 端部分的结构域 C 是蛋白质多聚化所必需的，而结构域 B 是与单链 DNA 的主要接触区域。还表明，虽然寡聚化对于单链 DNA 结合和 ATP 酶活性不是必需的，但结构域 C 的存在对于解旋酶活性是必需的。
Minichromosome maintenance helicases are ring-shaped complexes that play an essential role in archaeal and eukaryal DNA replication by separating the two strands of chromosomal DNA to provide the single-stranded substrate for the replicative polymerases. For the archaeal protein it was shown that the N-terminal portion of the protein, which is composed of domains A, B, and C, is involved in multimer formation and single-stranded DNA binding and may also play a role in regulating the helicase activity. Here, a detailed biochemical characterization of the N-terminal region of the Methanothermobacter thermautotrophicus minichromosome maintenance helicase is described. Using biochemical and biophysical analyses it is shown that domain C of the N-terminal portion, located adjacent to the helicase catalytic domains, is required for protein multimerization and that domain B is the main contact region with single-stranded DNA. It is also shown that although oligomerization is not essential for single-stranded DNA binding and ATPase activity, the presence of domain C is essential for helicase activity.