Mutational analysis of an archaeal minichromosome maintenance protein exterior hairpin reveals critical residues for helicase activity and DNA binding.
Mutational analysis of an archaeal minichromosome maintenance protein exterior hairpin reveals critical residues for helicase activity and DNA binding.
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DOI：
10.1186/1471-2199-11-62
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发表时间：
2010-08-18
影响因子：
--
通讯作者：
Chen XS
中科院分区：
文献类型：
--
作者：
Brewster AS;Slaymaker IM;Afif SA;Chen XS
微染色体维持蛋白（MCM）复合体是古细菌和真核生物DNA复制必不可少的复制解旋酶。真核复合体由6个同源蛋白（MCM2-7）组成，而古细菌solfataricus Sulfolobus solfataricus只有一个MCM蛋白（ssoMCM），其6个亚基形成同六聚体。我们最近报道了近乎全长的ssoMCM的4.35Å晶体结构。该结构揭示了每个亚基共有4个β-发夹，其中3个位于基于n端热自养甲烷菌（mtMCM）结构对称生成的ssoMCM六聚体模型的主通道或侧通道内。然而，第四个β发夹位于六聚体的外部，靠近假定的侧通道出口，靠近ATP结合袋。为了更好地了解这种发夹在DNA结合和解旋酶活性中的作用，我们对这种外部β-发夹（EXT-hp）上的9个残基进行了详细的突变和生化分析。我们检测了与解旋酶功能相关的突变体的活性，包括六聚体化、atp酶、DNA结合和解旋酶活性。实验表明，该EXT-hp上的一些残基对DNA结合和解旋酶活性起作用。这些结果暗示了目前几种关于解旋酶活性的理论。由于数据表明EXT-hp参与DNA结合，本文报道的结果表明，位于侧通道外部出口附近的EXT-hp可能以影响DNA解绕的方式与DNA底物接触。
The mini-chromosome maintenance protein (MCM) complex is an essential replicative helicase for DNA replication in Archaea and Eukaryotes. While the eukaryotic complex consists of six homologous proteins (MCM2-7), the archaeon Sulfolobus solfataricus has only one MCM protein (ssoMCM), six subunits of which form a homohexamer. We have recently reported a 4.35Å crystal structure of the near full-length ssoMCM. The structure reveals a total of four β-hairpins per subunit, three of which are located within the main channel or side channels of the ssoMCM hexamer model generated based on the symmetry of the N-terminal Methanothermobacter thermautotrophicus (mtMCM) structure. The fourth β-hairpin, however, is located on the exterior of the hexamer, near the exit of the putative side channels and next to the ATP binding pocket. In order to better understand this hairpin's role in DNA binding and helicase activity, we performed a detailed mutational and biochemical analysis of nine residues on this exterior β-hairpin (EXT-hp). We examined the activities of the mutants related to their helicase function, including hexamerization, ATPase, DNA binding and helicase activities. The assays showed that some of the residues on this EXT-hp play a role for DNA binding as well as for helicase activity. These results implicate several current theories regarding helicase activity by this critical hexameric enzyme. As the data suggest that EXT-hp is involved in DNA binding, the results reported here imply that the EXT-hp located near the exterior exit of the side channels may play a role in contacting DNA substrate in a manner that affects DNA unwinding.
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影响因子：
14.9
作者：
Liu W;Pucci B;Rossi M;Pisani FM;Ladenstein R
通讯作者：
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DOI：
10.1073/pnas.030539597
发表时间：
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10.1073/pnas.0808037105
发表时间：
2008-12-23
影响因子：
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作者：
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影响因子：
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作者：
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