DISSOCIATION OF SYNTHETIC HOLLIDAY JUNCTIONS BY ESCHERICHIA-COLI RECG PROTEIN
DISSOCIATION OF SYNTHETIC HOLLIDAY JUNCTIONS BY ESCHERICHIA-COLI RECG PROTEIN
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DOI：
10.1002/j.1460-2075.1993.tb05627.x
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发表时间：
1993-01-01
期刊：
影响因子：
11.4
通讯作者：
SHARPLES, GJ
中科院分区：
文献类型：
--
作者：
LLOYD, RG;SHARPLES, GJ
正常水平的重组和修复被紫外线、丝裂霉素C和电离辐射损伤的DNA都需要大肠杆菌的RecG蛋白。它参与这些过程的真实程度在很大程度上被似乎与三个RUV基因的产物功能重叠所掩盖。RuVA和RuvB共同作用促进Holliday连接的分支迁移，而RuvC通过内切酶切割催化这些重组中间产物分解为可行的产物。在这篇文章中，我们描述了RecG的过量生产和纯化，并证明了这种重叠延伸到生物化学。我们发现76 kDa的RecG蛋白是一个DNA依赖的ATPase，就像RuvB一样。利用凝胶滞留分析，我们证明了它与合成的Holliday连接，如Ruva和RuvC特异结合。最后，我们证明了在ATP和Mg2+存在的情况下，RecG将这些连接解离为双链产物，如RuvAB。我们认为，RecG和RuvAB在重组和DNA修复过程中提供了促进Holliday连接分支迁移的替代活性。
The RecG protein of Escherichia coli is needed for normal levels of recombination and for repair of DNA damaged by ultraviolet light, mitomycin C and ionizing radiation. The true extent of its involvement in these processes is masked to a large degree by what appears to be a functional overlap with the products of the three ruv genes. RuvA and RuvB act together to promote branch migration of Holliday junctions, while RuvC catalyses the resolution of these recombination intermediates into viable products by endonuclease cleavage. In this paper, we describe the overproduction and purification of RecG and demonstrate that the overlap extends to the biochemistry. We show that the 76 kDa RecG protein is a DNA-dependent ATPase, like RuvB. Using gel retardation assays we demonstrate that it binds specifically to a synthetic Holliday junction, like RuvA and RuvC. Finally, we show that in the presence of ATP and Mg2+, RecG dissociates these junctions to duplex products, like RuvAB. We suggest that RecG and RuvAB provide alternative activities that can promote branch migration of Holliday junctions in recombination and DNA repair.