Liu Jie, Bond Jeffrey P, Morrical Scott W
Department of Biochemistry, University of Vermont College of Medicine, Burlington, Vermont 05405, USA.
Biochemistry. 2006 May 2;45(17):5493-502. doi: 10.1021/bi0525167.
UvsY is the recombination mediator protein (RMP) of bacteriophage T4, which promotes homologous recombination by facilitating presynaptic filament assembly. The results of previous studies suggest that UvsY promotes the assembly of presynaptic filaments in part by stabilizing interactions between T4 UvsX recombinase and single-stranded DNA (ssDNA). To test this hypothesis, we studied the interactions of UvsX and UvsY with a fluorescein-derivatized oligonucleotide. This assay distinguishes between bipartite UvsX- or UvsY-ssDNA and tripartite UvsX-UvsY-ssDNA complex formation via differential fluorescence quenching effects. Salt stabilities of the three complexes were measured at equilibrium in the presence and absence of various nucleotide ligands of the UvsX protein and also under steady-state conditions for UvsX-catalyzed ssDNA-dependent ATP hydrolysis. The results demonstrate that UvsY globally stabilizes UvsX-ssDNA complexes, consistent with an increase in the apparent equilibrium binding affinity, K(ss)omega, of the UvsX-ssDNA interactions. The UvsY-mediated affinity increase is observed at equilibrium in the presence of ADP, ATPgammaS, or in the absence of the nucleotide and also at steady-state in the presence of ATP. Intriguingly, the stabilizing effects of UvsY and ATPgammaS on UvsX-ssDNA interactions are synergistic, indicating nonredundant mechanisms for UvsX-ssDNA complex stabilization by RMP versus nucleoside triphosphate effectors. Experiments with UvsY missense mutants defective in ssDNA binding demonstrate that UvsY-ssDNA interactions are of major importance in stabilizing UvsX-ssDNA complexes, whereas UvsY-UvsX protein-protein interactions provide residual stabilization energy. Together, the data is consistent with a mechanism in which UvsY stabilizes presynaptic filaments by organizing the ssDNA lattice into a structure that is favorable for UvsX-ssDNA interactions.
UvsY是噬菌体T4的重组介导蛋白（RMP），它通过促进突触前细丝组装来促进同源重组。先前的研究结果表明，UvsY部分通过稳定T4 UvsX重组酶与单链DNA（ssDNA）之间的相互作用来促进突触前细丝的组装。为了验证这一假设，我们研究了UvsX和UvsY与荧光素衍生化寡核苷酸的相互作用。该测定通过差异荧光猝灭效应区分二分体UvsX-或UvsY-ssDNA和三分体UvsX-UvsY-ssDNA复合物的形成。在存在和不存在UvsX蛋白的各种核苷酸配体的情况下，在平衡状态下测量了这三种复合物的盐稳定性，并且还在UvsX催化的ssDNA依赖性ATP水解的稳态条件下进行了测量。结果表明，UvsY全局稳定UvsX-ssDNA复合物，这与UvsX-ssDNA相互作用的表观平衡结合亲和力K(ss)omega的增加一致。在存在ADP、ATPγS的情况下，或在不存在核苷酸的平衡状态下，以及在存在ATP的稳态下，均观察到UvsY介导的亲和力增加。有趣的是，UvsY和ATPγS对UvsX-ssDNA相互作用的稳定作用是协同的，表明RMP与核苷三磷酸效应物对UvsX-ssDNA复合物稳定的机制是非冗余的。对ssDNA结合有缺陷的UvsY错义突变体进行的实验表明，UvsY-ssDNA相互作用在稳定UvsX-ssDNA复合物中至关重要，而UvsY-UvsX蛋白-蛋白相互作用提供残余的稳定能量。总之，这些数据与一种机制一致，即UvsY通过将ssDNA晶格组织成有利于UvsX-ssDNA相互作用的结构来稳定突触前细丝。
