Helicase assembly protein Gp59 of bacteriophage T4: fluorescence anisotropy and sedimentation studies of complexes formed with derivatives of Gp32, the phage ssDNA binding protein.
Helicase assembly protein Gp59 of bacteriophage T4: fluorescence anisotropy and sedimentation studies of complexes formed with derivatives of Gp32, the phage ssDNA binding protein.
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噬菌体 T4 的解旋酶组装蛋白 Gp59：与噬菌体 ssDNA 结合蛋白 Gp32 衍生物形成的复合物的荧光各向异性和沉降研究。
DOI：
10.1021/bi010116n
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发表时间：
2001
期刊：
影响因子：
2.9
通讯作者：
Morrical,SW
中科院分区：
文献类型：
--
作者：
Xu,H;Wang,Y;Bleuit,JS;Morrical,SW
T4噬菌体的基因59蛋白（gp59）在噬菌体DNA复制中发挥着至关重要的作用，它指导T4原体的DNA解旋酶成分gp41在新生复制叉的后链ssDNA上的组装。在链置换DNA合成过程中，gp59的解旋酶组装活性是复制叉获得解旋酶的最佳效率所必需的，也是T4重组依赖DNA复制交易中解旋酶和原体组装所必需的。最重要的是gp59将gp41解旋酶装载到先前被gp32 （T4 ssDNA结合蛋白）协同结合分子包裹的ssDNA上的能力。Gp59与ssDNA、gp32和gp41的异相关似乎都是这种装载反应所必需的。先前的研究表明，含有gp59和gp32的三方复合体同时占用ssDNA是gp59依赖性解旋酶装载的必要中间体；然而，gp59−gp32复合物的生化和结构参数是否含有ssDNA目前尚不清楚。为了更好地了解gp59 - gp32的相互作用，我们进行了荧光各向异性和分析性超离心实验，使用天然或罗丹明标记的gp59物种与改变形式的gp32结合，使我们能够确定它们的结合参数，形状参数和其他流体动力学性质。gp32的两种截断形式：gp32- b，缺乏与ssDNA合作结合和稳定自结合所需的n端b结构域，以及gp32的c端肽a结构域片段，缺乏与ssDNA结合的能力。结果表明gp59与gp32衍生物具有高亲和力，形成1:1的异源二聚体。在这两种情况下，异源二聚体的形成都伴随着gp59的构象变化，这与gp59 - DNA结合亲和力的降低有关。流体力学模型表明gp59为不对称的长椭球形状，与其x射线晶体结构一致，并且这种不对称性随着gp32衍生物的结合而增加。我们的研究结果对gp59和gp59 - gp32复合物在T4复制中的结构和功能的影响进行了讨论。
The gene 59 protein (gp59) of bacteriophage T4 performs a vital function in phage DNA replication by directing the assembly of gp41, the DNA helicase component of the T4 primosome, onto lagging strand ssDNA at nascent replication forks. The helicase assembly activity of gp59 is required for optimum efficiency of helicase acquisition by the replication fork during strand displacement DNA synthesis and is essential for helicase and primosome assembly during T4 recombination-dependent DNA replication transactions. Of central importance is the ability of gp59 to load the gp41 helicase onto ssDNA previously coated with cooperatively bound molecules of gp32, the T4 ssDNA binding protein. Gp59 heteroassociations with ssDNA, gp32, and gp41 all appear to be essential for this loading reaction. Previous studies demonstrated that a tripartite complex containing gp59 and gp32 simultaneously cooccupying ssDNA is an essential intermediate in gp59-dependent helicase loading; however, the biochemical and structural parameters of gp59−gp32 complexes with or without ssDNA are currently unknown. To better understand gp59−gp32 interactions, we performed fluorescence anisotropy and analytical ultracentrifugation experiments employing native or rhodamine-labeled gp59 species in combination with altered forms of gp32, allowing us to determine their binding parameters, shape parameters, and other hydrodynamic properties. Two truncated forms of gp32 were used: gp32-B, which lacks the N-terminal B-domain required for cooperative binding to ssDNA and for stable self-association, and A-domain fragment, which is the C-terminal peptide of gp32 lacking ssDNA binding ability. Results indicate that gp59 binds with high affinity to either gp32 derivative to form a 1:1 heterodimer. In both cases, heterodimer formation is accompanied by a conformational change in gp59 which correlates with decreased gp59−DNA binding affinity. Hydrodynamic modeling suggests an asymmetric prolate ellipsoid shape for gp59, consistent with its X-ray crystallographic structure, and this asymmetry appears to increase upon binding of gp32 derivatives. Implications of our findings for the structure and function of gp59 and gp59−gp32 complexes in T4 replication are discussed.