A dimer of Escherichia coli UvrD is the active form of the helicase in vitro
A dimer of Escherichia coli UvrD is the active form of the helicase in vitro
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DOI：
10.1016/s0022-2836(02)01277-9
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发表时间：
2003-01-31
影响因子：
5.6
通讯作者：
Lohman, TM
中科院分区：
文献类型：
--
作者：
Maluf, NK;Fischer, CJ;Lohman, TM
大肠杆菌UvrD蛋白是一种3'至5' SF 1 DNA解旋酶，参与DNA的甲基指导错配修复和核苷酸切除修复。我们已经在体外表征了一系列18个双链体DNA底物的UVrD催化解旋，所述双链体DNA底物具有3'单链DNA（ssDNA）尾部，长度范围从2到40 nt。使用化学淬灭流动方法进行单周转DNA解旋实验，作为在UvrD-DNA结合是化学计量的条件下的[UvrD]和[DNA]两者的函数。尽管如果3' ssDNA尾至少为4 nt，则单个UvrD单体紧密结合于单链/双链DNA（dsDNA）连接处，但对于尾长度小于或等于8 nt的DNA底物，即使在高[UvrD]/[DNA]比下也没有观察到解旋。对于3' ssDNA尾长大于或等于12 nt的DNA底物，观察到解旋，解旋幅度与[UvrD（tot）] / [DNA（tot）]呈S形关系。对这些数据的定量分析表明，结合在任何DNA底物的ssDNA/dsDNA连接处的单个UvrD单体，不依赖于3' ssDNA尾长，不能完全解旋甚至短的18 bp双链DNA，并且两个UvrD单体必须结合DNA底物以形成能够在体外解旋短DNA底物的复合物。其他蛋白质，包括没有ATP酶活性的突变体UvrD以及结构上同源的E.大肠杆菌Rep解旋酶不能取代第二个UvrD单体，这表明两个UvrD单体之间存在特异性相互作用，并且两者都必须能够水解ATP。DNA解旋的起始-在体外似乎需要二聚体UvrD复合物，其中一个亚基与ssDNA/dsDNA连接点结合，而第二个亚基与3' ssDNA尾结合。(C)2003爱思唯尔科技有限公司版权所有。
The Escherichia coli UvrD protein is a 3' to 5' SF1 DNA helicase involved in methyl-directed mismatch repair and nucleotide excision repair of DNA. We have characterized in vitro UvrD-catalyzed unwinding of a series of 18 by duplex DNA substrates with 3' single-stranded DNA (ssDNA) tails ranging in length from two to 40 nt. Single turnover DNA-unwinding experiments were performed using chemical quenched flow methods, as a function of both [UvrD] and [DNA] under conditions such that UvrD-DNA binding is stoichiometric. Although a single UvrD monomer binds tightly to the single-stranded/double-stranded DNA (dsDNA) junction if the 3' ssDNA tail is at least four nt, no unwinding was observed for DNA substrates with tail-lengths less than or equal to8 nt, even at high [UvrD]/[DNA] ratios. Unwinding is observed for DNA substrates with 3' ssDNA tail lengths greater than or equal to12 nt, and the unwinding amplitude displays a sigmoidal dependence on [UvrD(tot)] / [DNA(tot)]. Quantitative analysis of these data indicates that a single UvrD monomer bound at the ssDNA/dsDNA junction of any DNA substrate, independent of 3' ssDNA tail length, is not competent to fully unwind even a short 18 bp duplex DNA, and that two UvrD monomers must bind the DNA substrate in order to form a complex that is able to unwind short DNA substrates in vitro. Other proteins, including a mutant UvrD with no ATPase activity as well as a monomer of the structurally homologous E. coli Rep helicase, cannot substitute for the second UvrD monomer, suggesting a specific interaction between two UvrD monomers and that both must be able to hydrolyze ATP Initiation of DNA unwinding -in vitro appears to require a dimeric UvrD complex in which one subunit is bound to the ssDNA/dsDNA junction, while the second subunit is bound to the 3' ssDNA tail. (C) 2003 Elsevier Science Ltd. All rights reserved.