Domain architecture and biochemical characterization of vertebrate Mcm10
Domain architecture and biochemical characterization of vertebrate Mcm10
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DOI：
10.1074/jbc.m706267200
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发表时间：
2008-02-08
影响因子：
4.8
通讯作者：
Eichman, Brandt F.
中科院分区：
文献类型：
--
作者：
Robertson, Patrick D.;Warren, Eric M.;Eichman, Brandt F.
Mcm 10在真核细胞染色体DNA复制的起始和延伸中起关键作用。作为第一步，以更好地了解脊椎动物Mcm 10的结构和功能，我们已经确定了非洲爪蟾Mcm 10（xMcm 10）的结构体系结构，并表征每个域的生化。有限的全长蛋白质的蛋白水解消化显示N-末端，内部（ID），和C-末端（CTD）结构域。超离心分析表明，xMcm 10自我协会和N-末端结构域形成同源二聚体组件。将xMcm 10的DNA结合活性映射到ID和CTD，每个ID和CTD以低微摩尔亲和力结合单链和双链DNA。xMcm 10-ID和CTD的结构完整性取决于结合锌的存在，这通过原子吸收光谱法和蛋白水解保护测定进行了实验验证。ID和CTD还独立地结合DNA聚合酶α-引发酶的p180亚基的N-末端323个残基。我们建议，模块化的蛋白质结构，与离散域的二聚化和结合DNA和DNA聚合酶α-引发酶，提供了一种有效的手段，用于协调复制体内的Mcm 10的生化活性。
Mcm10 plays a key role in initiation and elongation of eukaryotic chromosomal DNA replication. As a first step to better understand the structure and function of vertebrate Mcm10, we have determined the structural architecture of Xenopus laevis Mcm10 (xMcm10) and characterized each domain biochemically. Limited proteolytic digestion of the full-length protein revealed N-terminal-, internal (ID)-, and C-terminal (CTD)-structured domains. Analytical ultracentrifugation revealed that xMcm10 self-associates and that the N-terminal domain forms homodimeric assemblies. DNA binding activity of xMcm10 was mapped to the ID and CTD, each of which binds to single- and double-stranded DNA with low micromolar affinity. The structural integrity of xMcm10-ID and CTD is dependent on the presence of bound zinc, which was experimentally verified by atomic absorption spectroscopy and proteolysis protection assays. The ID and CTD also bind independently to the N-terminal 323 residues of the p180 subunit of DNA polymerase alpha-primase. We propose that the modularity of the protein architecture, with discrete domains for dimerization and for binding to DNA and DNA polymerase alpha-primase, provides an effective means for coordinating the biochemical activities of Mcm10 within the replisome.