A novel function for the conserved glutamate residue in the walker B motif of replication factor C.
A novel function for the conserved glutamate residue in the walker B motif of replication factor C.
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DOI：
10.3390/genes4020134
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发表时间：
2013-03-26
期刊：
影响因子：
3.5
通讯作者：
Bloom LB
中科院分区：
文献类型：
--
作者：
Chiraniya A;Finkelstein J;O'Donnell M;Bloom LB
在生命的所有领域，滑动夹将DNA聚合酶拴在DNA上以增加合成的持续性。夹子加载器通过需要ATP结合和水解的多步过程将夹子加载到DNA上。与其他AAA+蛋白质一样，夹装载物含有保守的步行者A和步行者B序列基序，其分别参与ATP结合和水解。AAA+蛋白中步行者B基序（或DExx盒）中谷氨酸残基的突变通常可使ATP水解减少几个数量级，但对ATP结合没有影响。在这里，步行者B Glu的四个活性ATP位点的真核细胞的钳加载器，RFC，分别突变为谷氨酰胺和丙氨酸，和ATP结合和水解依赖性活动的四重突变钳加载器的特点。使用基于荧光的测定来测量夹加载所需的各个反应步骤，包括夹结合、夹打开、DNA结合和ATP水解。我们的研究结果表明，步行者B突变影响ATP结合依赖的相互作用RFC的钳和DNA，除了减少配体依赖的ATP水解活性。在这里，我们表明，步行者B谷氨酸是必需的ATP依赖性配体结合活性，一个以前未知的功能，这个保守的Glu残基RFC。
In all domains of life, sliding clamps tether DNA polymerases to DNA to increase the processivity of synthesis. Clamp loaders load clamps onto DNA in a multi-step process that requires ATP binding and hydrolysis. Like other AAA+ proteins, clamp loaders contain conserved Walker A and Walker B sequence motifs, which participate in ATP binding and hydrolysis, respectively. Mutation of the glutamate residue in Walker B motifs (or DExx-boxes) in AAA+ proteins typically reduces ATP hydrolysis by as much as a couple orders of magnitude, but has no effect on ATP binding. Here, the Walker B Glu in each of the four active ATP sites of the eukaryotic clamp loader, RFC, was mutated to Gln and Ala separately, and ATP binding- and hydrolysis-dependent activities of the quadruple mutant clamp loaders were characterized. Fluorescence-based assays were used to measure individual reaction steps required for clamp loading including clamp binding, clamp opening, DNA binding and ATP hydrolysis. Our results show that the Walker B mutations affect ATP-binding-dependent interactions of RFC with the clamp and DNA in addition to reducing ligand-dependent ATP hydrolysis activity. Here, we show that the Walker B glutamate is required for ATP-dependent ligand binding activity, a previously unknown function for this conserved Glu residue in RFC.
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