Biochemical interactions within a ternary complex of the bacteriophage T4 recombination proteins uvsY and gp32 bound to single-stranded DNA.
Biochemical interactions within a ternary complex of the bacteriophage T4 recombination proteins uvsY and gp32 bound to single-stranded DNA.
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DOI：
10.1021/bi9817055
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发表时间：
1999-01
期刊：
影响因子：
2.9
通讯作者：
M. Sweezy;S. Morrical
中科院分区：
文献类型：
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作者：
M. Sweezy;S. Morrical
同源重组的突触前阶段需要形成包裹有重组酶的单链DNA （ssDNA）丝。在T4噬菌体中，至少需要三种蛋白质来组装突触前丝。除T4重组酶外，还需要uvsX蛋白、T4 ssDNA结合蛋白（gp32）和uvsY重组辅助蛋白。在这里，我们报告了一个包含ssDNA的三方丝的详细分析，这些丝由化学计量量的uvsY和gp32结合，这似乎是T4突触前丝组装的重要中间体。我们证明了uvsY和gp32同时以非竞争的方式共同占用ssDNA。此外，我们发现uvsY和gp32之间的蛋白-蛋白相互作用不是这种三元配合物的组装所必需的，也不会影响uvsY对ssDNA晶格的亲和力。最后，我们证明了gp32与ssDNA的相互作用在这个复合物内是不稳定的，以一种独立于gp32- uvsy相互作用的方式。这些数据表明，uvsY蛋白通过uvsY- ssdna相互作用重塑gp32-ssDNA复合体。这些发现对T4重组系统突触前机制的影响进行了讨论。
The presynaptic phase of homologous recombination requires the formation of a filament of single-stranded DNA (ssDNA) coated with a recombinase enzyme. In bacteriophage T4, at least three proteins are required for the assembly of this presynaptic filament. In addition to the T4 recombinase, uvsX protein, the T4 ssDNA binding protein (gp32), and the uvsY recombination accessory protein are also required. Here we report on a detailed analysis of a tripartite filament containing ssDNA bound by stoichiometric quantities of both uvsY and gp32, which appears to be an important intermediate in the assembly of the T4 presynaptic filament. We demonstrate that uvsY and gp32 simultaneously co-occupy the ssDNA in a noncompetitive fashion. In addition, we show that protein-protein interactions between uvsY and gp32 are not required for the assembly of this ternary complex and do not affect the affinity of uvsY for the ssDNA lattice. Finally, we demonstrate that the interaction of gp32 with the ssDNA is destabilized within this complex, in a manner which is independent of gp32-uvsY interactions. The data suggest that the uvsY protein acts to remodel the gp32-ssDNA complex via uvsY-ssDNA interactions. The implications of these findings for the mechanism of presynapsis in the T4 recombination system are discussed.