Role of Walker motif A of RuvB protein in promoting branch migration of Holliday junctions - Walker motif A mutations affect ATP binding, ATP hydrolyzing, and DNA binding activities of RuvB
Role of Walker motif A of RuvB protein in promoting branch migration of Holliday junctions - Walker motif A mutations affect ATP binding, ATP hydrolyzing, and DNA binding activities of RuvB
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DOI：
10.1074/jbc.274.36.25335
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发表时间：
1999-09-03
影响因子：
4.8
通讯作者：
Shinagawa, H
中科院分区：
文献类型：
--
作者：
Hishida, T;Iwasaki, H;Shinagawa, H
大肠杆菌RuvB蛋白是一种依赖于ATP的六聚体DNA解旋酶，在同源重组和重组修复过程中与Ruva蛋白一起促进Holliday连接的分支迁移。为了阐明RuvB(GXGKT；X代表非保守残基)的Walker基序A在ATP水解酶和分支迁移活性中的作用，我们通过定点突变构建了四个ruvB突变基因，改变了高度保守的Lys(68)和Thr(69)K68R、K68A和T69A突变体，但T69S未能补充ruvB株对W敏感的表型。这三种突变蛋白在过度表达时，使野生型菌株对紫外线有不同程度的敏感。K68R、K68A和T69A在体外ATP水解性和分支迁移活性方面存在缺陷，在有镁离子存在的情况下，K68R对ATP的亲和力显著降低，而K68A和T69A仅表现出轻微的降低。K68A和T69A在Mg2+和ATP存在下能形成六聚体，而K68R不能形成六聚体，而是以较高的齐聚物形式存在，可能是十二聚体。与野生型RuvB相比，K68R、K68A和T69A本身在DNA结合上存在缺陷。然而，Ruva能促进K68A和T69A与DNA的结合，而不能促进R68R与DNA的结合，三个突变的RuvB都能与Ruva发生物理作用。这些结果表明，RuvB的Walker基序A的不变Lys(68)和Thr(69)残基直接参与了ATP的结合和ATP的水解，并表明这些残基在这些活性与RuvB的构象变化之间发挥了关键作用，而RuvB的构象变化是分支迁移活动所必需的。
Escherichia coli RuvB protein, an ATP-dependent hexameric DNA helicase, acts together with RuvA protein to promote branch migration of Holliday junctions during homologous recombination and recombinational repair. To elucidate the role of the Walker motif A of RuvB (GXGKT; X indicates a nonconserved residue) in ATP hydrolysis and branch migration activities, we constructed four ruvB mutant genes by site-directed mutagenesis, altering the highly conserved Lys(68) and Thr(69) K68R, K68A, and T69A mutants except T69S failed to complement W-sensitive phenotype of the ruvB strain. These three mutant proteins, when overexpressed, made the wild-type strain UV-sensitive to varying degrees. K68R, K68A, and T69A were defective in ATP hydrolysis and branch migration activities in vitro, In the presence of Mg2+, K68R showed markedly reduced affinity for ATP, while K68A and T69A showed only mild reduction. K68A and T69A could form hexamers in the presence of Mg2+ and ATP, while K68R failed to form hexamers and existed instead as a higher oligomer, probably a dodecamer. In contrast to wild-type RuvB, K68R, K68A, and T69A by themselves were defective in DNA binding. However, RuvA could facilitate binding of K68A and T69A to DNA, whereas it could not promote binding of R68R to DNA, All of the three mutant RuvBs could physically interact with RuvA. These results indicate the direct involvement in ATP binding and ATP hydrolysis of the invariant Lys(68) and Thr(69) residues of Walker motif A of RuvB and suggest that these residues play key roles in interrelating these activities with the conformational change of RuvB, which is required for the branch migration activity.