Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee, USA.
PLoS One. 2013 Jul 23;8(7):e70518. doi: 10.1371/journal.pone.0070518. Print 2013.
Minichromosome maintenance protein 10 (Mcm10) is an essential eukaryotic DNA-binding replication factor thought to serve as a scaffold to coordinate enzymatic activities within the replisome. Mcm10 appears to function as an oligomer rather than in its monomeric form (or rather than as a monomer). However, various orthologs have been found to contain 1, 2, 3, 4, or 6 subunits and thus, this issue has remained controversial. Here, we show that self-association of Xenopus laevis Mcm10 is mediated by a conserved coiled-coil (CC) motif within the N-terminal domain (NTD). Crystallographic analysis of the CC at 2.4 Å resolution revealed a three-helix bundle, consistent with the formation of both dimeric and trimeric Mcm10 CCs in solution. Mutation of the side chains at the subunit interface disrupted in vitro dimerization of both the CC and the NTD as monitored by analytical ultracentrifugation. In addition, the same mutations also impeded self-interaction of the full-length protein in vivo, as measured by yeast-two hybrid assays. We conclude that Mcm10 likely forms dimers or trimers to promote its diverse functions during DNA replication.
微小染色体维持蛋白 10（Mcm10）是一种必需的真核 DNA 结合复制因子，被认为作为复制体中酶活性的支架。Mcm10 似乎作为一个寡聚体而不是单体（或者不是单体）发挥作用。然而，已经发现各种同源物含有 1、2、3、4 或 6 个亚基，因此，这个问题一直存在争议。在这里，我们表明非洲爪蟾 Mcm10 的自组装是由 N 端结构域（NTD）内保守的卷曲螺旋（CC）基序介导的。CC 的晶体结构分析分辨率为 2.4 Å，揭示了一个三螺旋束，与溶液中形成二聚体和三聚体 Mcm10 CC 一致。通过分析超速离心监测，突变亚基界面的侧链破坏了 CC 和 NTD 的体外二聚化。此外，相同的突变也阻碍了全长蛋白在体内的自我相互作用，如酵母双杂交测定所示。我们得出结论，Mcm10 可能形成二聚体或三聚体，以促进其在 DNA 复制过程中的多种功能。
