Control of Hexamerization, Assembly, and Excluded Strand Specificity for the Sulfolobus solfataricus MCM Helicase
Control of Hexamerization, Assembly, and Excluded Strand Specificity for the Sulfolobus solfataricus MCM Helicase
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DOI：
10.1021/acs.biochem.8b00766
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发表时间：
2018-10-02
期刊：
影响因子：
2.9
通讯作者：
Trakselis, Michael A.
中科院分区：
文献类型：
--
作者：
Graham, Brian W.;Bougoulias, Michael E.;Trakselis, Michael A.
越来越多的证据支持DNA解绕的空间排除和包裹模型，其中六聚体解旋酶与被排除的单链DNA （ssDNA）相互作用。与被排除的ssDNA的相互作用已被证明主要是通过静电相互作用介导的，但与表面暴露的酪氨酸残基的碱基堆叠是另一种假设。在这里，我们沿着排除链结合的路径突变了来自全长solfataricus Sulfolobus MCM的几个外部酪氨酸和带正电的残基，并评估了它们对DNA解绕的影响。5个酪氨酸残基中有4个的解绕水平显著降低，其中一个位于c端结构域α / β - α连接区域的Y519A由于六聚化的破坏而受到最严重的扰动。Y519突变体表现出增强和稳定的二级结构，可以通过温度调节，以更高的表观亲和力结合DNA，并提示六聚体组装途径。采用氢/氘交换耦合质谱法绘制了野生型和Y519A载子结构之间的氘吸收差异，突出了与第四纪结构改变相一致的溶剂可达区域的全球差异。五个静电突变体中的两个DNA解绕水平显著降低，与先前的突变体结合可以更好地定义外部结合路径。通过使用morpholino DNA底物证实了静电排除链相互作用的重要性，该底物显示出类似的降低解绕速率。这些结果更好地定义了六聚体组装和被排除的链相互作用在控制DNA解绕中被古菌MCM复合物的影响。
A growing body of evidence supports a steric exclusion and wrapping model for DNA unwinding in which hexameric helicases interact with the excluded single-stranded DNA (ssDNA) in addition to the encircled strand. Interactions with the excluded ssDNA have been shown to be mediated primarily by electrostatic interactions, but base stacking with surface-exposed tyrosine residues is an alternative hypothesis. Here, we mutated several external tyrosine and positively charged residues from full-length Sulfolobus solfataricus MCM along the proposed path of excluded strand binding and assessed their impact on DNA unwinding. Four of the five tyrosine residues had significant decreases in their level of unwinding, and one, Y519A, located within the alpha/beta-alpha linker region of the C-terminal domain, had the most severe perturbation attributed to the disruption of hexamerization. The Y519 mutant exhibits an enhanced and stabilized secondary structure that is modulated by temperature, binding DNA with a higher apparent affinity and suggesting a pathway for hexameric assembly. Hydrogen/deuterium exchange coupled to mass spectrometry was used to map deuterium uptake differences between wild-type and Y519A apo structures highlighting global differences in solvent accessible areas consistent with altered quaternary structure. Two of the five electrostatic mutants had significantly reduced levels of DNA unwinding and combined with previous mutations better define the exterior binding path. The importance of the electrostatic excluded strand interaction was confirmed by use of morpholino DNA substrates that showed analogous reduced unwinding rates. These results better define the hexameric assembly and influence of the excluded strand interactions in controlling DNA unwinding by the archaeal MCM complex.