Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.
J Mol Biol. 2011 Jan 7;405(1):65-76. doi: 10.1016/j.jmb.2010.10.004. Epub 2010 Oct 28.
Bacteriophage T4 provides an important model system for studying the mechanism of homologous recombination. We have determined the crystal structure of the T4 UvsX recombinase, and the overall architecture and fold closely resemble those of RecA, including a highly conserved ATP binding site. Based on this new structure, we reanalyzed electron microscopy reconstructions of UvsX-DNA filaments and docked the UvsX crystal structure into two different filament forms: a compressed filament generated in the presence of ADP and an elongated filament generated in the presence of ATP and aluminum fluoride. In these reconstructions, the ATP binding site sits at the protomer interface, as in the RecA filament crystal structure. However, the environment of the ATP binding site is altered in the two filament reconstructions, suggesting that nucleotide cannot be as easily accommodated at the protomer interface of the compressed filament. Finally, we show that the phage helicase UvsW completes the UvsX-promoted strand-exchange reaction, allowing the generation of a simple nicked circular product rather than complex networks of partially exchanged substrates.
噬菌体 T4 为研究同源重组机制提供了一个重要的模型系统。我们已经确定了 T4 UvsX 重组酶的晶体结构,其整体结构和折叠与 RecA 非常相似,包括一个高度保守的 ATP 结合位点。基于这个新结构,我们重新分析了 UvsX-DNA 丝的电子显微镜重构,并将 UvsX 晶体结构对接入两种不同的丝形式:在 ADP 存在下生成的压缩丝和在 ATP 和氟化铝存在下生成的伸长丝。在这些重构中,ATP 结合位点位于单体界面,与 RecA 丝晶体结构相同。然而,在两种丝重构中,ATP 结合位点的环境发生了改变,这表明在压缩丝的单体界面上核苷酸不能轻易容纳。最后,我们表明噬菌体解旋酶 UvsW 完成了 UvsX 促进的链交换反应,生成了简单的缺口环状产物,而不是部分交换底物的复杂网络。