Reiss B, Kosak H, Klemm M, Schell J
Max-Planck-Institut für Züchtungsforschung, Koln, Germany.
Mol Gen Genet. 1997 Feb 27;253(6):695-702. doi: 10.1007/s004380050373.
We have characterised a RecA protein fused to the simian virus 40 large T nuclear-localisation signal. The fusion protein was targeted to the nucleus in transgenic tobacco plants with high efficiency. By contrast, authentic RecA was not enriched in the nuclei of plant cells expressing comparable amounts of protein. For detailed characterisation of the strand-exchange activity of the nuclear-targeted RecA protein, a nearly identical protein was expressed in Escherichia coli and purified to homogeneity. This protein was found to bind to single-stranded DNA with the same stoichiometry and to promote the exchange of homologous DNA strands with the same kinetics as authentic RecA. It was concluded that the amino-terminal modification did not alter any of the essential properties of RecA and that the fusion protein is a fully functional strand-exchange protein. However, the ATPase activity of this protein was 20 times greater than that of RecA in the absence of single-stranded DNA. As with RecA, this activity was further stimulated by the addition of single-stranded DNA. Since ATPase activity is correlated with the ability of RecA to assume its high affinity state for DNA, the nuclear-targeted RecA protein might be regarded as a constitutively stimulated RecA variant, fully functional in promoting homologous recombination.
我们已对一种与猿猴病毒40大T抗原核定位信号融合的RecA蛋白进行了表征。该融合蛋白在转基因烟草植株中高效定位于细胞核。相比之下，在表达等量蛋白质的植物细胞核中，天然RecA并未富集。为了详细表征核定位RecA蛋白的链交换活性，在大肠杆菌中表达了一种几乎相同的蛋白并纯化至同质。发现该蛋白以相同的化学计量比与单链DNA结合，并以与天然RecA相同的动力学促进同源DNA链的交换。得出的结论是，氨基末端修饰并未改变RecA的任何基本特性，且融合蛋白是一种功能完全正常的链交换蛋白。然而，在不存在单链DNA的情况下，该蛋白的ATP酶活性比RecA高20倍。与RecA一样，添加单链DNA可进一步刺激该活性。由于ATP酶活性与RecA对DNA呈现高亲和力状态的能力相关，核定位RecA蛋白可被视为一种持续受刺激的RecA变体，在促进同源重组方面功能完全正常。
