A region near the C-terminal end of Escherichia coli DNA helicase II is required for single-stranded DNA binding
A region near the C-terminal end of Escherichia coli DNA helicase II is required for single-stranded DNA binding
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DOI：
10.1128/jb.181.8.2519-2526.1999
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发表时间：
1999-04-01
影响因子：
3.2
通讯作者：
Matson, SW
中科院分区：
文献类型：
--
作者：
Mechanic, LE;Latta, NE;Matson, SW
使用三种突变体研究了大肠杆菌 DNA 解旋酶 II (UvrD) C 末端（保守解旋酶基序之外的区域）的作用：UvrD Delta 107C（删除最后 107 个 C 末端氨基酸）、UvrD Delta 102C 和 UvrD Delta 40C。该区域与其他解旋酶缺乏序列相似性，可能会根据其特定的体内作用来调整 UvrD。遗传互补分析表明，突变蛋白 UvrD Delta 107C 和 UvrD Delta 102C 未能在甲基定向错配修复和核苷酸切除修复中替代野生型蛋白。 UvrD Delta 40C 蛋白完全补充了两条修复途径中解旋酶 II 的损失。 UvrD Delta 102C 和 UvrD Delta 40C 纯化至表观同质性并进行生化表征。与野生型蛋白相比，UvrD Delta 102C 无法结合单链 DNA，并且表现出大大降低的单链 DNA 刺激的 ATP 酶活性（k(cat) 为野生型水平的 0.01%）。 UvrD Delta 40C 在 DNA 结合方面存在轻微缺陷，并且在测量单链 DNA 刺激的 ATP 水解和解旋酶活性时，与野生型 UvrD 基本上无法区分。这些结果表明解旋酶 II C 末端附近的区域在与单链 DNA 的结合中发挥着作用。
The role of the C terminus of Escherichia coli DNA helicase II (UvrD), a region outside the conserved helicase motifs, was investigated by using three mutants: UvrD Delta 107C (deletion of the last 107 C-terminal amino acids), UvrD Delta 102C, and UvrD Delta 40C. This region, which lacks sequence similarity with other helicases, may function to tailor UvrD for its specific in vivo roles. Genetic complementation assays demonstrated that mutant proteins UvrD Delta 107C and UvrD Delta 102C failed to substitute for the wild-type protein in methyl-directed mismatch repair and nucleotide excision repair. UvrD Delta 40C protein fully complemented the loss of helicase II in both repair pathways. UvrD Delta 102C and UvrD Delta 40C were purified to apparent homogeneity and characterized biochemically. UvrD Delta 102C was unable to bind single-stranded DNA and exhibited a greatly reduced single-stranded DNA-stimulated ATPase activity in comparison to the wild-type protein (k(cat) 0.01% of the wild-type level). UvrD Delta 40C was slightly defective for DNA binding and was essentially indistinguishable from wild-type UvrD when single-stranded DNA-stimulated ATP hydrolysis and helicase activities were measured. These results suggest a role for a region near the C terminus of helicase II in binding to single-stranded DNA.