The E. coli UvrD protein is a nonhexameric DNA helicase that belongs to superfamily I and plays a crucial role in both nucleotide excision repair and methyl-directed mismatch repair. Previous data suggested that wild-type UvrD has optimal activity in its oligomeric form. However, crystal structures of the UvrD-DNA complex were only resolved for monomeric UvrD, using a UvrD mutant lacking the C-terminal 40 amino acids (UvrDΔ40C). However, biochemical findings performed using UvrDΔ40C indicated that this mutant failed to dimerize, although its DNA-unwinding activity was comparable to that of wild-type UvrD. Although the C-terminus plays essential roles in nucleic acid binding for many proteins with helicase and dimerization activities, the exact function of the C-terminus is poorly understood. Thus, to understand the function of the C-terminal amino acids of UvrD, we performed single-molecule direct visualization. Photobleaching of dye-labeled UvrDΔ40C molecules revealed that two or three UvrDΔ40C molecules could bind simultaneously to an 18-bp double-stranded DNA with a 20-nucleotide, 3' single-stranded DNA tail in the absence of ATP. Simultaneous visualization of association/dissociation of the mutant with/from DNA and the DNA-unwinding dynamics of the mutant in the presence of ATP demonstrated that, as with wild-type UvrD, two or three UvrDΔ40C molecules were primarily responsible for DNA unwinding. The determined association/dissociation rate constants for the second bound monomer were ∼2.5-fold larger than that of wild-type UvrD. The involvement of multiple UvrDΔ40C molecules in DNA unwinding was also observed under a physiological salt concentration (200 mM NaCl). These results suggest that multiple UvrDΔ40C molecules, which may form an oligomer, play an active role in DNA unwinding in vivo and that deleting the C-terminal 40 residues altered the interaction of the second UvrD monomer with DNA without affecting the interaction with the first bound UvrD monomer.

中文翻译：

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缺乏 C 端 40 个氨基酸的大肠杆菌 UvrD 的 DNA 解旋动力学

大肠杆菌 UvrD 蛋白是一种非六聚体 DNA 解旋酶，属于超家族 I，在核苷酸切除修复和甲基定向错配修复中都起着至关重要的作用。以前的数据表明，野生型 UvrD 以其寡聚形式具有最佳活性。然而，UvrD-DNA 复合物的晶体结构仅适用于单体 UvrD，使用缺乏 C 端 40 个氨基酸 (UvrDΔ40C) 的 UvrD 突变体。然而，使用 UvrDΔ40C 进行的生化发现表明该突变体未能二聚化，尽管其 DNA 解旋活性与野生型 UvrD 相当。尽管 C 端在许多具有解旋酶和二聚化活性的蛋白质的核酸结合中起重要作用，但对 C 端的确切功能知之甚少。因此，为了了解 UvrD C 端氨基酸的功能，我们进行了单分子直接可视化。染料标记的 UvrDΔ40C 分子的光漂白表明，在没有 ATP 的情况下，两个或三个 UvrDΔ40C 分子可以同时结合到具有 20 个核苷酸的 3' 单链 DNA 尾的 18 bp 双链 DNA。在 ATP 存在下突变体与 DNA 的结合/解离和突变体的 DNA 解旋动力学的同时可视化表明，与野生型 UvrD 一样，两个或三个 UvrDΔ40C 分子主要负责 DNA 解旋。确定的第二个结合单体的结合/解离速率常数比野生型 UvrD 大 2.5 倍。在生理盐浓度（200 mM NaCl）下也观察到多个 UvrDΔ40C 分子参与 DNA 解旋。这些结果表明，多个 UvrDΔ40C 分子可能形成寡聚体，在体内 DNA 解旋中发挥积极作用，删除 C 端 40 个残基改变了第二个 UvrD 单体与 DNA 的相互作用，而不影响与第一个结合的相互作用。 UvrD 单体。