Sweezy M A, Morrical S W
Department of Biochemistry, Vermont Cancer Center, University of Vermont College of Medicine, Burlington 05405, USA.
Biochemistry. 1999 Jan 19;38(3):936-44. doi: 10.1021/bi9817055.
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.
同源重组的突触前阶段需要形成由重组酶包被的单链DNA（ssDNA）细丝。在噬菌体T4中，组装这种突触前细丝至少需要三种蛋白质。除了T4重组酶uvsX蛋白外，还需要T4 ssDNA结合蛋白（gp32）和uvsY重组辅助蛋白。在此，我们报告了对一种三方细丝的详细分析，该细丝含有由化学计量的uvsY和gp32结合的ssDNA，它似乎是T4突触前细丝组装中的一个重要中间体。我们证明uvsY和gp32以非竞争性方式同时占据ssDNA。此外，我们表明uvsY和gp32之间的蛋白质 - 蛋白质相互作用对于这种三元复合物的组装不是必需的，并且不影响uvsY对ssDNA晶格的亲和力。最后，我们证明gp32与ssDNA的相互作用在该复合物中不稳定，其方式独立于gp32 - uvsY相互作用。数据表明uvsY蛋白通过uvsY - ssDNA相互作用来重塑gp32 - ssDNA复合物。讨论了这些发现对T4重组系统中突触前机制的影响。
