Menetski J P, Kowalczykowski S C
Department of Molecular Biology, Northwestern University Medical School, Chicago, IL 60611.
J Mol Biol. 1990 Feb 20;211(4):845-55. doi: 10.1016/0022-2836(90)90078-Z.
The biochemical properties of the recA430 protein have been examined and compared to those of wild-type recA protein. We find that, while the recA430 protein possesses ssDNA-dependent rATP activity, this activity is inhibited by the Escherichia coli single-stranded DNA binding protein (SSB protein) under many conditions that enhance wild-type recA protein rATPase hydrolysis. Stimulation of rATPase activity by SSB protein is observed only at high concentrations of both rATP (greater than 1 mM) and recA430 protein (greater than 5 microM). In contrast, stimulation of ssDNA-dependent dATPase activity by SSB protein is less sensitive to protein and nucleotide concentration. Consistent with the nucleotide hydrolysis data, recA430 protein can carry out DNA strand exchange in the presence of either rATP or dATP. However, in the presence of rATP, both the rate and the extent of DNA strand exchange by recA430 protein are greatly reduced compared to wild-type recA protein and are sensitive to recA430 protein concentration. This reduction is presumably due to the inability of recA430 protein to compete with SSB protein for ssDNA binding sites under these conditions. The cleavage of lexA repressor protein by recA430 protein is also sensitive to the nucleotide cofactor present and is completely inhibited by SSB protein when rATP is the cofactor but not when dATP is used. Finally, the steady-state affinity and the rate of association of the recA430 protein-ssDNA complex are reduced, suggesting that the mutation affects the interaction of the ATP-bound form of recA protein with ssDNA. This alteration is the likely molecular defect responsible for inhibition of recA430 protein rATP-dependent function by SSB protein. The biochemical properties observed in the presence of dATP and SSB protein, i.e. the reduced levels of both DNA strand exchange activity and cleavage of lexA repressor protein, are consistent with the phenotypic behavior of recA430 mutations.
已对recA430蛋白的生化特性进行了检测，并与野生型recA蛋白的特性进行了比较。我们发现，虽然recA430蛋白具有依赖于单链DNA的rATP活性，但在许多增强野生型recA蛋白rATP酶水解的条件下，这种活性会受到大肠杆菌单链DNA结合蛋白（SSB蛋白）的抑制。仅在高浓度的rATP（大于1 mM）和recA430蛋白（大于5 μM）时，才观察到SSB蛋白对rATP酶活性的刺激作用。相比之下，SSB蛋白对依赖于单链DNA的dATP酶活性的刺激作用对蛋白和核苷酸浓度不太敏感。与核苷酸水解数据一致，recA430蛋白在rATP或dATP存在的情况下都能进行DNA链交换。然而，在rATP存在的情况下，与野生型recA蛋白相比，recA430蛋白进行DNA链交换的速率和程度都大大降低，并且对recA430蛋白浓度敏感。这种降低可能是由于在这些条件下recA430蛋白无法与SSB蛋白竞争单链DNA结合位点。recA430蛋白对lexA阻遏蛋白的切割也对存在的核苷酸辅因子敏感，当rATP作为辅因子时，会被SSB蛋白完全抑制，而当使用dATP时则不会。最后，recA430蛋白-单链DNA复合物的稳态亲和力和结合速率降低，这表明该突变影响了结合ATP形式的recA蛋白与单链DNA的相互作用。这种改变可能是导致SSB蛋白抑制recA430蛋白依赖rATP功能的分子缺陷。在dATP和SSB蛋白存在的情况下观察到的生化特性，即DNA链交换活性和lexA阻遏蛋白切割水平的降低，与recA430突变的表型行为一致。
